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Sample records for biotin

  1. 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...

  2. 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...

  3. Biotin dependency due to a defect in biotin transport

    OpenAIRE

    Mardach, Rebecca; Zempleni, Janos; Wolf, Barry; Martin J. Cannon; 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...

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

    OpenAIRE

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

    2010-01-01

    Abstract Background 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 ...

  5. Biotin requirements for DNA damage prevention

    OpenAIRE

    Zempleni, Janos; Teixeira, Daniel Camara; Kuroishi, Toshinobu; Cordonier, Elizabeth L; Baier, Scott

    2011-01-01

    Biotin serves as a covalently bound coenzyme in five human carboxylases; biotin is also attached to histones H2A, H3, and H4, although the abundance of biotinylated histones is low. Biotinylation of both carboxylases and histones is catalyzed by holocarboxylase synthetase. Human biotin requirements are unknown. Recommendations for adequate intake of biotin are based on the typical intake of biotin in an apparently healthy population, which is only a crude estimate of the true intake due to an...

  6. Smoking accelerates biotin catabolism in women123

    OpenAIRE

    Sealey, Wendy M.; April M. Teague; Stratton, Shawna L.; Mock, Donald M.

    2004-01-01

    Background: Smoking accelerates the degradation of many nutrients, including lipids, antioxidants, and certain B vitamins. Accelerated biotin catabolism is of concern in women because marginal biotin deficiency is teratogenic in mammals.

  7. 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.

  8. 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...

  9. 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...

  10. Impaired biotin status in anticonvulsant therapy

    OpenAIRE

    Krause, Klaus-Henning; Berlit, Peter; Bonjour, Jean-Pierre

    1982-01-01

    In 264 epileptics undergoing long-term therapy with anticonvulsants, significantly reduced plasma biotin levels were found compared with a normal control group: 74% of the epileptics had biotin levels for those treated with sodium valproate were higher than for those treated with phenytion, primidone, or carbamazepine. The observed reduction in biotin levels might be a factor influencing the efficacy of these three antionvulsants.

  11. Determination of Biotin: Antibody Molar Ratio

    International Nuclear Information System (INIS)

    The determination of the biotinylation yield (number of biotin molecules per molecule of antibody) is important to ensure that the MAb has maintained its immunoreactivity. If the biotinylation of the MAb is carried out with a molar ratio of biotin:antibody ~10:1, then the number of biotins per MAb usually ranges between 6 and 8

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

    International Nuclear Information System (INIS)

    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

  13. Intestinal absorption of biotin in the rat

    International Nuclear Information System (INIS)

    We examined the absorption of biotin using the in vivo intestinal loop technique. Jejunal segments from male rats were filled with solutions containing [3H]biotin and [14C]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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-04-01

    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.

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

    International Nuclear Information System (INIS)

    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

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

    OpenAIRE

    Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

    2013-01-01

    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 ...

  17. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    A radioligand assay for biotin in liver tissue is described. 3H-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.)

  18. Genetics Home Reference: biotin-thiamine-responsive basal ganglia disease

    Science.gov (United States)

    ... Health Conditions biotin-thiamine-responsive basal ganglia disease biotin-thiamine-responsive basal ganglia disease Enable Javascript to ... boxes. Print All Open All Close All Description Biotin-thiamine-responsive basal ganglia disease is a disorder ...

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

    OpenAIRE

    Ikeda, Masato; 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,...

  20. Biotin absorption by distal rat intestine

    International Nuclear Information System (INIS)

    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

  1. Epigenetic regulation of chromatin structure and gene function by biotin: are biotin requirements being met?

    OpenAIRE

    Zempleni, Janos; Chew, Yap Ching; Hassan, Yousef I.; Wijeratne, Subhashinee SK

    2008-01-01

    Histones H2A, H3, and H4 are modified by covalent binding of the vitamin biotin to distinct lysine residues. Binding of biotin to histones is mediated by holocarboxylase synthetase (HCS) and perhaps biotinidase. Biotinylation of lysine- 12 in histone H4 (K12BioH4) plays roles in gene repression, stability of repeat regions and transposable elements, and regulation of biotin transporter expression in eukaryotes. Decreased biotinylation of histones in biotin-deficient and HCS-deficient human ce...

  2. Determination of biotin content in beet molasses by Lactobacillus plantarum

    OpenAIRE

    Lončar Eva S.; Došenović Irena S.; Markov Siniša L.; Malbaša Radomir V.; Kolarov Ljiljana A.

    2005-01-01

    D-biotin content in beet molasses was determined by microbiological method using Lactobacillus plantarum, based on the comparison of the growth of this microorganism in molasses solutions with those in standard solutions of biotin. Incubation of the microorganism was performed on original Vitamin Biotin Testbouillon and laboratory prepared liquid culture medias. The amount of "real" biotin in molasses is low. The results depend upon the sample and volume of molasses solutions. Biotin contents...

  3. Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention

    OpenAIRE

    Salaemae, Wanisa; Azhar, Al; Booker, Grant W.; Polyak, Steven W

    2011-01-01

    Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in b...

  4. Biotin Sensing at the Molecular Level1–3

    OpenAIRE

    Beckett, Dorothy

    2009-01-01

    Biotin influences transcription in organisms from bacteria to humans. The enzyme, biotin protein ligase, which catalyzes post-transcriptional biotin addition to biotin-dependent carboxylases, plays a central roll in transmitting the demand for biotin to gene expression. The molecular mechanism of this communication in bacteria is well understood and involves competing protein:protein interactions. Biochemical measurements indicate that this competition is kinetically controlled. In humans, th...

  5. Recombinant Rhizobium meliloti strains with extra biotin synthesis capability.

    OpenAIRE

    Streit, W. R.; Phillips, D. A.

    1996-01-01

    The growth of Rhizobium meliloti 1021 in an experimental alfalfa (Medicago sativa L.) rhizosphere was stimulated by adding nanomolar amounts of biotin. To overcome this biotin limitation, R. meliloti strains were constructed by conjugating the Escherichia coli biotin synthesis operon into biotin auxotroph R. meliloti 1021-B3. Transconjugant strains Rm1021-WS10 and Rm1021-WS11 grew faster in vitro and achieved a higher cell density than did R. meliloti 1021 and overproduced biotin on a defined...

  6. Biotin Deficiency Reduces Expression of SLC19A3, a Potential Biotin Transporter, in Leukocytes from Human Blood12

    OpenAIRE

    Vlasova, Tatyana I; Stratton, Shawna L.; Wells, Amanda M.; Mock, Nell I.; Mock, Donald M.

    2005-01-01

    In evaluating potential indicators of biotin status, we quantitated the expression of biotin-related genes in leukocytes from human blood of normal subjects before and after inducing marginal biotin deficiency. Biotin deficiency was induced experimentally by feeding an egg-white diet for 28 d. Gene expression was quantitated for the following biotin-related proteins: methylcrotonyl-CoA carboxylase chains A (MCCA) and B (MCCB); propionyl-CoA carboxylase chains A (PCCA) and B (PCCB); pyruvate c...

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

    Directory of Open Access Journals (Sweden)

    Schneider Jens

    2012-01-01

    Full Text Available Abstract 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.

  8. Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention.

    Science.gov (United States)

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

    2011-09-01

    Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents. PMID:21976058

  9. Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention

    Institute of Scientific and Technical Information of China (English)

    Wanisa Salaemae; Al Azhar; Grant W. Booker; Steven W. Polyak

    2011-01-01

    Biotin is an important micronutrient that serves as an essential enzyme cofactor.Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources.Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis.Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria.Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth,infection and survival during the latency phase.These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.

  10. Instability of the biotin-protein bond in human plasma.

    Science.gov (United States)

    Bogusiewicz, Anna; Mock, Nell I; Mock, Donald M

    2004-04-15

    Labeling proteins with biotin offers an alternative to labeling with radioisotopes for pharmacokinetic studies in humans. However, stability of the biotin-protein bond is a critical tacit assumption. Using release of biotin from immunoglobulin G as the outcome, we individually evaluated stability of the biotin label produced by six biotinylation agents: biotin PEO-amine, 5-(biotinamido)-pentylamine, iodoacetyl-LC-biotin, NHS-LC-biotin, sulfo-NHS-LC-biotin, and biotin-LC-hydrazide. Each of the six biotinylated proteins was incubated at room temperature for 4h in human plasma or in phosphate-buffered saline (control). Free biotin was separated from the biotinylated protein by ultrafiltration and quantitated by avidin-binding assay. For each biotinylation reagent, biotin release was significantly increased by plasma (p europium-streptavidin by the immobilized biotinylated immunoglobulin G. Consistent with biotin release data, streptavidin capture was reduced by plasma to 8% of control. We conclude that all of the biotinylating agents produce biotin-protein bonds that are susceptible to hydrolysis by factors present in human plasma; five of six are stable in buffer. PMID:15051531

  11. Effects of biotin supplementation on serum biotin levels and physical properties of samples of solar horn of Holstein cows

    OpenAIRE

    Higuchi, H; T. Maeda; Nakamura, M.; Kuwano, A; Kawai, K.; Kasamatsu, M.; Nagahata, H

    2004-01-01

    Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 ± 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 ± 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control...

  12. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    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)

  13. Human herpes virus 6 and endogenous biotin in salivary glands.

    OpenAIRE

    Green, M.; Sviland, L; Taylor, C. E.; Peiris, M.; McCarthy, A. L.; Pearson, A. D.; Malcolm, A. J.

    1992-01-01

    AIMS: To detect the presence of human herpes virus 6 (HHV6) and endogenous biotin in paraffin wax embedded and frozen salivary glands. METHODS: Two stage indirect and streptavidin-biotin immunoperoxidase techniques were used to visualise the antigens. RESULTS: HHV6 could not be shown in any of the tissues. However, considerable endogenous biotin antigenicity was detected in the glandular elements of the paraffin wax embedded material. CONCLUSIONS: Results obtained with avidin-biotin detection...

  14. Scientific Opinion on Dietary Reference Values for biotin

    OpenAIRE

    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA)

    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 of fatty acids, the catabolism of branched-chain amino acids and gluconeogenesis. Dietary biotin deficiency is rare. Data on biomarkers of biotin intake or status are insufficient to be used in determinin...

  15. 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. PMID:27601860

  16. Radioimmunocytochemistry with [3H]biotin

    International Nuclear Information System (INIS)

    The authors exploit the high affinity of biotin for avidin in the design of radioimmunocytochemical methods using [3H]biotin. [3H]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 [3H]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 [3H]biotin have proved successful in quantification, electron microscopy and double labelling studies. (Auth.)

  17. Protein labelling with avidin-biotin systems

    International Nuclear Information System (INIS)

    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)

  18. 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

    -CoA (or 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...

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

    International Nuclear Information System (INIS)

    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 18O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5'-([α,β-18O,β,β-18O2]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 18O label directly from [18O]bicarbonate into the product Pi. 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

  20. Yttrium-90 Radiolabelling of Biotin DOTA

    International Nuclear Information System (INIS)

    Yttrium-90 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid (DOTA) biotin was prepared by adding an equal volume of sodium acetate (1.0M, pH5.0) to the 90YCl3 vial (concentration 37–74 GBq/mL in 0.05M HCl) containing 2.96–4.44 GBq of 90Y followed by 1.0 mg of biotin DOTA dissolved in 0.5 mL of saline (recommended specific activity of 3.7 GBq/mg). After mixing, the reaction vial was incubated for 30 min at 95°C. Aliquots of the mixture were drawn at defined time intervals to determine the radiochemical purity (RCP)

  1. 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...

  2. Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding

    OpenAIRE

    Staggs, C.G.; Sealey, W.M.; McCabe, B J; Teague, A.M.; Mock, D.M.

    2004-01-01

    Assessing dietary biotin content, biotin bioavailability, and resulting biotin status are crucial in determining whether biotin deficiency is teratogenic in humans. Accuracy in estimating dietary biotin is limited both by data gaps in food composition tables and by inaccuracies in published data. The present study applied sensitive and specific analytical techniques to determine values for biotin content in a select group of foods. Total biotin content of 87 foods was determined using acid hy...

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

    International Nuclear Information System (INIS)

    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 (3H)-biotin and (14C)-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

  4. Scientific Opinion on Dietary Reference Values for biotin

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA

    2014-02-01

    Full Text Available 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 of fatty acids, the catabolism of branched-chain amino acids and gluconeogenesis. Dietary biotin deficiency is rare. Data on biomarkers of biotin intake or status are insufficient to be used in determining the requirement for biotin. Data available on biotin intakes and health consequences are very limited and cannot be used for deriving DRVs for biotin. As there is insufficient evidence available to derive an Average Requirement and a Population Reference Intake, an Adequate Intake (AI is proposed. The setting of AIs is based on observed biotin intakes with a mixed diet and the apparent absence of signs of deficiency in the EU, suggesting that current intake levels are adequate. The AI for adults is set at 40 µg/day. The AI for adults also applies to pregnant women. For lactating women, an additional 5 µg biotin/day over and above the AI for adults is proposed, to compensate for biotin losses through breast milk. For infants over six months, an AI of 6 µg/day is proposed by extrapolating from the biotin intake of exclusively breastfed infants aged zero to six months, using allometric scaling and reference body weight for each age group, in order to account for the role of biotin in energy metabolism. The AIs for children aged 1–3 and 4–10 years are set at 20 and 25 µg/day, respectively, and for adolescents at 35 µg/day, based on observed intakes in the EU.

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

    International Nuclear Information System (INIS)

    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)

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

    International Nuclear Information System (INIS)

    The extraordinary 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, streptavin (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)

  7. Construction of a Biotin-Overproducing Strain of Serratia marcescens

    OpenAIRE

    Sakurai, Naoki; Imai, Yuji; Masuda, Makoto; Komatsubara, Saburo; Tosa, Tetsuya

    1993-01-01

    We have isolated mutants resistant to acidomycin, a biotin analog, from Serratia marcescens Sr41. Strain SB304, resistant to 0.5 mg of acidomycin (frequently called actithiazic acid) per ml, produced 5 mg of d-biotin per liter of a medium containing sucrose and urea. Strain SB412, which was isolated from SB304 on a minimal agar plate containing 2 mg of acidomycin per ml and 0.1 mg of 5-(2-thienyl)-valeric acid per ml, produced 20 mg of d-biotin per ml. The two enzymes related to biotin synthe...

  8. 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...... 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....

  9. Functional Loop Dynamics of the Streptavidin-Biotin Complex

    Science.gov (United States)

    Song, Jianing; Li, Yongle; Ji, Changge; Zhang, John Z. H.

    2015-01-01

    Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop3-4 in the strong-binding streptavidin-biotin complex system. Conventional molecular (cMD) simulation is also performed for comparison. The present study reveals the following important properties of the loop dynamics: (1) The transition of loop3-4 from open to closed state is observed in 200 ns aMD simulation. (2) In the absence of biotin binding, the open-state streptavidin is more stable, which is consistent with experimental evidences. The free energy (ΔG) difference is about 5 kcal/mol between two states. But with biotin binding, the closed state is more stable due to electrostatic and hydrophobic interactions between the loop3-4 and biotin. (3) The closure of loop3-4 is concerted to the stable binding of biotin to streptavidin. When the loop3-4 is in its open-state, biotin moves out of the binding pocket, indicating that the interactions between the loop3-4 and biotin are essential in trapping biotin in the binding pocket. (4) In the tetrameric streptavidin system, the conformational change of the loop3-4 in each monomer is independent of each other. That is, there is no cooperative binding for biotin bound to the four subunits of the tetramer.

  10. Functional Analysis of Sinorhizobium meliloti Genes Involved in Biotin Synthesis and Transport

    OpenAIRE

    Entcheva, Plamena; Phillips, Donald A.; Streit, Wolfgang R.

    2002-01-01

    External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis ...

  11. Conservation of the Biotin Regulon and the BirA Regulatory Signal in Eubacteria and Archaea

    OpenAIRE

    Rodionov, Dmitry A.; Mironov, Andrei A.; Gelfand, Mikhail S.

    2002-01-01

    Biotin is a necessary cofactor of numerous biotin-dependent carboxylases in a variety of microorganisms. The strict control of biotin biosynthesis in Escherichia coli is mediated by the bifunctional BirA protein, which acts both as a biotin–protein ligase and as a transcriptional repressor of the biotin operon. Little is known about regulation of biotin biosynthesis in other bacteria. Using comparative genomics and phylogenetic analysis, we describe the biotin biosynthetic pathway and the Bir...

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

    DEFF Research Database (Denmark)

    Lisbjerg, Micke; Nielsen, Bjarne Enrico; Milhøj, Birgitte Olai; Sauer, Stephan P. A.; Pittelkow, Michael

    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...... calorimetry (ITC), computational calculations and single crystal X-ray crystallography....

  13. 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.

  14. An Improved Synthesis of Arsenic-Biotin Conjugates

    OpenAIRE

    Heredia-Moya, Jorge; KIRK, KENNETH L.

    2008-01-01

    An amide linked conjugate of p-aminophenylarsine oxide and biotin is conveniently prepared in a one-pot procedure by reaction of biotinyl chloride, formed in situ, with p-aminophenyldichloroarsine. Reaction of the arsine oxide-biotin conjugate with 1,2-ethanedithiol produces the stabilized dithiarsolane. These reagents are now readily available for a variety of applications.

  15. Chemical and enzymatic biotin-labeling of oligodeoxyribonucleotides.

    OpenAIRE

    Kempe, T; Sundquist, W I; Chow, F; Hu, S L

    1985-01-01

    Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis. The 5'-biotinylated oligonucleotides were enzymatically coupled to other DNA fragments by T4 DNA ligase or T4 RNA ligase. The hybridization properties of such biotin-labeled oligonucleotide probes were studied.

  16. 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...

  17. Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin.

    OpenAIRE

    Sano, T; Cantor, C R

    1995-01-01

    In natural streptavidin, tryptophan 120 of each subunit makes contacts with the biotin bound by an adjacent subunit through the dimer-dimer interface. To understand quantitatively the role of tryptophan 120 and its intersubunit communication in the properties of streptavidin, a streptavidin mutant in which tryptophan 120 is converted to phenylalanine was produced and characterized. The streptavidin mutant forms a tetrameric molecule and binds one biotin per subunit, as does natural streptavid...

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

    OpenAIRE

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

    2012-01-01

    Abstract 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 m...

  19. Biotin-specific synthetic receptors prepared using molecular imprinting

    International Nuclear Information System (INIS)

    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

  20. Biotin Synthesis Begins by Hijacking the Fatty Acid Synthetic Pathway

    OpenAIRE

    Lin, Steven; Hanson, Ryan E.; Cronan, John E.

    2010-01-01

    Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven carbon dicarboxylic acid, but the mechanism whereby Escherichia coli assembles this intermediate remains unknown. Genetic analysis identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl m...

  1. A newly discovered function of peroxisomes: Involvement in biotin biosynthesis

    OpenAIRE

    Maruyama, Jun-ichi; Yamaoka, Shohei; Matsuo, Ichiro; Tsutsumi, Nobuhiro; Kitamoto, Katsuhiko

    2012-01-01

    In plants, peroxisomes are the organelles involved in various metabolic processes and physiological functions including β-oxidation, mobilization of seed storage lipids, photorespiration, and hormone biosynthesis. We have recently shown that, in fungi and plants, peroxisomes play a vital role in biosynthesis of biotin, an essential cofactor required for various carboxylation and decarboxylation reactions. In fungi, the mutants defective in peroxisomal protein import exhibit biotin auxotrophy....

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

    OpenAIRE

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

    2013-01-01

    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...

  3. The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

    OpenAIRE

    P. Marini(GANIL); Li, S J; Gardiol, D; Cronan, J E; De Mendoza, D

    1995-01-01

    The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

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

    OpenAIRE

    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 bioti...

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

    International Nuclear Information System (INIS)

    The labeling method for 177Lu-DTPA-BIS-BIOTIN was established, and the biodistribution of 177Lu-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 177Lu-DTPA-BIS-BIOTIN is more than 99.0%. 177Lu-DTPA-BIS-BIOTIN shows pretty good in vitro stability. The biodistribution of 177Lu-DTPA-BIS-BIOTIN in normal mice shows a rapid blood clearance. The uptake of 177Lu-DTPA-BIS-BIOTIN is mainly accumulated in liver, spleen and kidney. 177Lu-DTPA-BIS-BIOTIN is excreted by kidney. The results provide the basis for further study on 177Lu-DTPA-BIS-BIOTIN used in pretargeted radioimage and radiotherapy of cancer. (authors)

  6. Studies on biotin dependent carboxylases and the properties of carboxybiotin

    International Nuclear Information System (INIS)

    Biotin dependent carboxyl-transfer reactions have been studied using biotin and pyruvate carboxylases. The pH profile for the Mg2+ and MgATP dependent carboxylation of biotin by bicarbonate shows that an enzymic base with a pK of 6.5 must be unprotonated for catalysis to occur. The pH profiles for the carboxyl-transfer reaction of pyruvate carboxylase have been obtained by studying the decarboxylation of oxalacetate stimulated by the presence of oxamate. Similarly, 13C and 2H isotope effects have been measured for the decarboxylation of oxalacetate by both enzymic and nonenzymic means. From these studies the authors can conclude that carboxyl-transfer between biotin and oxalacetate is at least partially rate-limiting and is not concerted with proton-transfer. The lack of any apparent enzymic acid-base catalyst (the V/K profile for oxalacetate is pH independent) suggests that proton transfer may occur directly between biotin and the carbanion formed when oxalacetate is decarboxylated. The pH profile for the nonenzymatic decarboxylation of carboxybiotin shows a plateau below pH 4 (k = 0.012 min-1 at 20C), and a lower plateau above pH 8 (k = 0.005 min-1 at 250C). A proton inventory at low pH is linear, while at high pH it is curved. These data suggest that two different mechanisms operate at high and low pH

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

    International Nuclear Information System (INIS)

    Introduction: The natural occurring vitamin biotin, also known as vitamin H or vitamin B7, 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 [18F]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. [18F]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 [18F]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). [18F]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 [18F]4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination. Conclusion: An efficient synthesis for [18F]4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [18F]4.

  8. Gamma-ray inactivation of biotin in dilute aqueous solution

    International Nuclear Information System (INIS)

    The relative roles of the radicals produced by water radiolysis in the inactivation of biotin in aqueous solution were investigated. The effects of nitrous oxide and isopropanol used as selective free radical scavengers allowed the inactivation efficiencies per unit G-value of OH, H, and esub(aq)- to be estimated; these efficiencies were 0.73, 0.10, and 0.02 in neutral solution, respectively. Hydrogen gas and hydrogen peroxide unaffected the activity of biotin. G0-Value for biotin inactivation in oxygen-free neutral solution was 2.08. Under these conditions the hydroxyl radical attack was found to be responsible for the large part of inactivation. On the other hand, in oxygenated neutral solution, G0-value was 4.16. This large increase of inactivation in oxygenated solution suggested that, although hydrated electrons were considerably ineffective as an inactivating species in oxygen-free solution, superoxide ions would be much more effective in causing inactivation of biotin in oxygenated solution. A rate constant for the reaction of biotin with hydroxyl radical was 1.34 x 1010M-1 sec-1 as determined by the PNDA method. (auth.)

  9. Chemiluminescent immunoassay for TSH using biotin-streptavidin system

    International Nuclear Information System (INIS)

    TSH chemiluminescent immunoassay using biotin-streptavidin system was established and studied. Two monoclonal antibodies of TSH were used in the assay, with one coated to 96 well plate and the other labeled with biotin. Streptavidin was labeled by DMAE·NHS. Assay sensitivity was 0.007 mIU/L. Intraassay coefficient of variation was 1.47%-5.65%. Interassay coefficient of variation was 2.45%-8.72%. Average recovery was 101.7%. Correlation coefficient and correlation equation of the assay with TSH IRMA were 0.989 and Y= -0.015+1.02X, respectively. The assay sensitivity and CPS of this assay were higher than those of TSH-CLIA, in which no biotin-streptavidin system was used

  10. Design of Biotin-Functionalized Luminescent Quantum Dots

    OpenAIRE

    Hedi Mattoussi; Igor L. Medintz; H. Tetsuo Uyeda; Kimihiro Susumu

    2007-01-01

    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...

  11. Streptavidin and its biotin complex at atomic resolution

    International Nuclear Information System (INIS)

    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

  12. Labelling of Biotin with 188Re. Chapter 8

    International Nuclear Information System (INIS)

    Different chemical strategies have been employed for labelling biotin using 188Re with the aim to develop a sterile and pyrogen free kit formulation that is suitable for clinical use. A number of biotin conjugated 188Re complexes were prepared and evaluated to determine their affinity for avidin. The most difficult challenge was to devise an efficient reaction pathway that was able to obtain the final radiocompounds in a high radiochemical yield. This work describes the molecular design and the chemical strategy that were followed to obtain reliable preparation of the new radiopharmaceuticals starting from generator produced [188ReO4]–. (author)

  13. Functional Loop Dynamics of the Streptavidin-Biotin Complex

    OpenAIRE

    Jianing Song; Yongle Li; Changge Ji; Zhang, John Z.H.

    2015-01-01

    Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop3-4 in the strong-binding streptavidin-biotin complex system. Conventional molecular (cMD) simulation is also performed for comparison. The present study reveals the following important properties of the loop dynamics: (1) The transition of loop3-4 from open to closed state is observed in 200 ns aMD simulation. (2) In the absence of biotin binding, the open-state streptavidin is mo...

  14. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    OpenAIRE

    C. Du; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing pro...

  15. The Reacquisition of Biotin Prototrophy in Saccharomyces cerevisiae Involved Horizontal Gene Transfer, Gene Duplication and Gene Clustering

    OpenAIRE

    Hall, Charles; Dietrich, Fred S

    2007-01-01

    The synthesis of biotin, a vitamin required for many carboxylation reactions, is a variable trait in Saccharomyces cerevisiae. Many S. cerevisiae strains, including common laboratory strains, contain only a partial biotin synthesis pathway. We here report the identification of the first step necessary for the biotin synthesis pathway in S. cerevisiae. The biotin auxotroph strain S288c was able to grow on media lacking biotin when BIO1 and the known biotin synthesis gene BIO6 were introduced t...

  16. Biotin Uptake into Human Peripheral Blood Mononuclear Cells Increases Early in the Cell Cycle, Increasing Carboxylase Activities1,2

    OpenAIRE

    Stanley, J. Steven; Mock, Donald M.; Griffin, Jacob B.; Zempleni, Janos

    2002-01-01

    Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle...

  17. Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay (ELISA) method.

    Science.gov (United States)

    Chang, Y S; Wu, C H; Chang, R J; Shiuan, D

    1994-12-01

    A method based on competitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of biotin concentrations which takes advantage of the extraordinarily high affinity between biotin and avidin. The biotin assay consisted of two steps, (i) a competition reaction between excess streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and solutions of known biotin concentrations or sample solutions and (ii) the measurement of the residual activities of the free form streptavidin-HRP which were correlated with the initial biotin concentrations. The procedure was modified by including an extra step of antibody-antigen interaction to assay biotin concentration unambiguously in more complex media. The entire assay was completed within 6 with sensitivities of approximately 1 pg/ml for biotin in a simple aqueous medium and 5 pg/ml in complex media. The method offers significant advantages in time, sensitivity and simplicity for determinations of biotin concentrations in various solutions. PMID:7699208

  18. Biochemical Properties and Biological Function of a Monofunctional Microbial Biotin Protein Ligase

    OpenAIRE

    Daniels, Kyle G.; Beckett, Dorothy

    2010-01-01

    Biotin protein ligases constitute a family of enzymes that catalyze biotin linkage to biotin-dependent carboxylases. In bacteria these enzymes are functionally divided into two classes; the monofunctional enzymes that only catalyze biotin addition and the bifunctional enzymes that also bind to DNA to regulate transcription initiation. Biochemical and biophysical studies of the bifunctional Escherichia coli ligase suggest that several properties of the enzyme have evolved to support its additi...

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

    OpenAIRE

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

    2013-01-01

    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...

  20. Modulation of the Rat Hepatic Cytochrome P4501A Subfamily Using Biotin Supplementation

    OpenAIRE

    Ronquillo-Sánchez, M. D.; Camacho-Carranza, R.; C. Fernandez-Mejia; S. Hernández-Ojeda; Elinos-Baez, M.; Espinosa-Aguirre, J. J.

    2013-01-01

    Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated dail...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  7. 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 ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    OpenAIRE

    Xianwei Zhu; Hiroaki Shinohara

    2014-01-01

    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-(AC5)2-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-(AC5)2-hydrazide with the concentrat...

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

    International Nuclear Information System (INIS)

    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

  17. 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.

  18. A Sensitive Competitive ELISA for Determination of Biotin in Transformed Yeast Culture Media

    Institute of Scientific and Technical Information of China (English)

    YANGHong

    2003-01-01

    Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit ami-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L-1. Results The detection limit for biotin was found to be 83 ng·L-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations of 200 ng·L-1, 500 ng·L-1 , and 1000 ng·L-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.

  19. 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. PMID:24486440

  20. Modulation of the Rat Hepatic Cytochrome P4501A Subfamily Using Biotin Supplementation

    Directory of Open Access Journals (Sweden)

    M. D. Ronquillo-Sánchez

    2013-01-01

    Full Text Available Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated daily with biotin (2 mg/kg, i.p., while the control groups were treated with saline. All of the rats were sacrificed by cervical dislocation after 1, 3, 5, or 7 days of treatment. CYP1A1 and CYP1A2 mRNAs were modified by biotin while enzyme activity and protein concentration were not affected. The lack of an effect of biotin on CYP1A activity was confirmed using other experimental strategies, including (i cotreatment of the animals with biotin and a known CYP1A inducer; (ii the addition of biotin to the reaction mixtures for the measurement of CYP1A1 and CYP1A2 activities; and (iii the use of an S9 mixture that was prepared from control and biotin-treated rats to analyze the activation of benzo[a]pyrene (BaP into mutagenic metabolites using the Ames test. The results suggest that biotin does not influence the CYP1A-mediated metabolism of xenobiotics.

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

    International Nuclear Information System (INIS)

    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. Immunoradiometric assay for carcinoembryonic antigenusing avidin-biotin separation technique

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    A sensitive, specific, noncompetitive, sandwich-typeradioimmunoassay for carcinoembryonic antigen (CEA) has been developedin our laboratory, which can be performedconveniently. The assay involves two monoclonal antibodies, selected for highaffinity and specificity and also for reaction against antigenic sites on CEA that aredistal from each other. One of these antibodies was labeled with125I and the other wasconjugated covalently to biotin. Polystyrene tubes were conjugated covalently toavidin. These tubes represent a rapid, simple method for separating the CEA-boundantibody from the free antibody. The biotin-antibody-CEA-125I-labeled antibodycomplexes bind to the tubes and CEA concentration is directly related to counts perminute. This assay can detect the CEA at a concentration of 0.22 μg/L in serum.

  3. Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability.

    Science.gov (United States)

    Takakura, Yoshimitsu; Sofuku, Kozue; Tsunashima, Masako

    2013-03-10

    A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8-4.4×10(-7)M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules. PMID:23333918

  4. 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. PMID:27117847

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

    International Nuclear Information System (INIS)

    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. 188Re from 188W-188Re 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 MAG2GABA-Biocytin (MGB), labeled with 188Re and evaluated biological behavior of 188Re-MGB. biotinyl MAG2GABA bind the therapeutic radiometal 188Re with excellent in vitro stability and have the required physiological properties for pretargetted therapy. In normal mice, 188Re-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. 188Re-MGB may have a role in pretargetted radioimmunotherapy

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

    International Nuclear Information System (INIS)

    To find a method for determining the immunoreactivity of monoclonal antibodies after radiolabeling avidin is unlabeled and labeled with Rodamine, 131I and 188Re, 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, 188Re and 131I 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

  7. [99mTc(CO)3] - BiotinNTA derivative for tumor pretargeting

    International Nuclear Information System (INIS)

    Tumor pretargeting using monoclonal antibodies in combination with biotin, labeled with radionuclides, could be useful for cancer diagnosis and treatment. Biotin nitrilrotriacetic acid (BiotinNTA) having functional groups of three -COOH was used as biotin derivative for antibody pretargeting strategies. In this investigation, 99mTc(CO)3-BiotinNTA was prepared to determine its potential use in antibody pretargeting strategies for tumor diagnosis. 99mTc is considered to be ideal radionuclides for the development of radioimaging of tumor. Radiolabeling of the BiotinNTA was performed in PBS buffer (pH=7.4) at 75 .deg. C, 30 min. The radiolabeling efficiency and radiochemical purity were analyzed by a reverse-phase high performance liquid chromatography (RP-HPLC). The radiolabeled complex was tested in vitro for stability in plasma and biological affinity against avidin using magnetic particle seperator in PBS buffer (pH7.4)

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

    OpenAIRE

    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 col...

  9. Ligand Specificity of Group I Biotin Protein Ligase of Mycobacterium tuberculosis

    OpenAIRE

    Purushothaman, Sudha; Gupta, Garima; Srivastava, Richa; Ramu, Vasanthakumar Ganga; Surolia, Avadhesha

    2008-01-01

    Background: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC. Methodology/Principal Findings: BPL/BirA (Biotin Protein L...

  10. Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition*

    OpenAIRE

    Ingaramo, Maria; Beckett, Dorothy

    2009-01-01

    The human holocarboxylase synthetase (HCS) catalyzes transfer of biotin to biotin-dependent carboxylases, and the enzyme is therefore of fundamental importance for many physiological processes, including fatty acid synthesis, gluconeogenesis, and amino acid catabolism. In addition, the enzyme functions in regulating transcription initiation at several genes that code for proteins involved in biotin metabolism. Two major forms of HCS exist in humans, which differ at the amino terminus by 57 am...

  11. Mode of action of the biotin antimetabolites actithiazic acid and alpha-methyldethiobiotin.

    OpenAIRE

    Eisenberg, M. A.; Hsiung, S C

    1982-01-01

    Actithiazic acid and alpha-methyldethiobiotin inhibited the conversion of dethiobiotin to biotin resting-cell suspensions of Escherichia coli. The concentrations which effected 50% inhibition were 0.45 and 1.1 microM for actithiazic acid and alpha-methyldethiobiotin, respectively. Cells grown in low concentrations of the two biotin antimetabolites showed derepression of the biotin A operon, as evidenced by the enhanced levels of the enzymes 7,8-diaminopelargonic acid aminotransferase and deth...

  12. Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency.

    OpenAIRE

    Burri, B J; Sweetman, L.; Nyhan, W L

    1985-01-01

    Holocarboxylase synthetase activity has been determined in fibroblasts of seven patients with the neonatal form of biotin-responsive multiple carboxylase deficiency. The normal Km for biotin was 15 +/- 3 nmol/l, while in the patients the values ranged from 48 to 1,062 nmol/l. The mean maximum velocity was 27% of normal. Differences among the values obtained for the Km for biotin and the heat stability of holocarboxylase synthetase suggested that the patients studied represented at least four ...

  13. Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58.

    OpenAIRE

    Desai, J. D.; Desai, A J; Patel, H C

    1983-01-01

    Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.

  14. Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

    OpenAIRE

    Barat, Bhaswati; Anna M. Wu

    2007-01-01

    Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the E. coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In th...

  15. Biotin affects the immune response of piglets inoculated with porcine circovirus type 2

    OpenAIRE

    HONG, Chen; Keying, Zhang; XUEMEI, Ding; DAIWEN, Chen

    2012-01-01

    Weanling crossbred pigs were used in a 5-week trial to evaluate the effects of biotin and porcine circovirus type 2 (PCV2) challenge on serum cytokine concentrations and humoral immunoresponse. Although not significant, there was a trend towards reduced interferon-g concentration in the challenged pigs compared to the controls, especially at 14 and 21 days postinoculation (dpi). Biotin supplementation with 50 or 200 µg/kg biotin improved these levels, with higher doses producing an earlier an...

  16. Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus⋆

    OpenAIRE

    Gralla, Michael; Camporeale, Gabriela; Zempleni, Janos

    2007-01-01

    The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in respo...

  17. Identification and Characterization of a Novel Biotin Biosynthesis Gene in Saccharomyces cerevisiae

    OpenAIRE

    Wu, Hong; Ito, Kiyoshi; Shimoi, Hitoshi

    2005-01-01

    Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake ye...

  18. A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments.

    Science.gov (United States)

    Strassberger, Verena; Trüssel, Sabrina; Fugmann, Tim; Neri, Dario; Roesli, Christoph

    2010-10-01

    The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-β-Ala-(L-Asp)(3)-biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-β-Ala-(L-Asp)(3)-biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature. PMID:20821733

  19. Nitric Oxide Signaling Depends on Biotin in Jurkat Human Lymphoma Cells12

    OpenAIRE

    Rodriguez-Melendez, Rocio; Zempleni, Janos

    2009-01-01

    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 pharmac...

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

    International Nuclear Information System (INIS)

    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 Ef0=−1.6to−1.8 vs. (Fc/Fc+)/V (Ef0=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. 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

    2016-01-01

    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. PMID:27161258

  2. The rat as a model for evaluation of biotin bioavailability from feed ingredients for poultry and swine.

    Science.gov (United States)

    Misir, R

    1987-01-01

    Biotin bioavailability from poultry and swine feed ingredients was determined in an experiment involving growing rats (55-60 g body weight, initially), housed individually in stainless steel cages with raised metal floors. The rats were fed a biotin-free diet fortified with egg-white powder for 7 d prior to being put on test. Thereafter, 5 rats were randomly assigned to each of the experimental diets, as follows: a basal egg white-free diet (A) without added biotin, or supplemented with graded levels of d-biotin, i.e., 0.05, 0.10, 0.25, 0.50 and 1.00 microgram/g, and also 12 test diets prepared by incorporating various cereal grains and/or two protein supplements into diet A by partial replacement of casein and carbohydrates. The experimental diets were fed ad libitum for 21 d, and met or exceeded recommended levels for all nutrients except biotin. Results showed significant correlations between pairs of parameters, including plasma biotin vs biotin intake (P less than 0.01), liver biotin vs biotin intake (P less than 0.05) and plasma biotin vs liver biotin (P less than 0.01). The bioavailable biotin from test ingredients was estimated using the derived regression equation, Y = 0.54X + 1.05, (r = 0.85), where X = biotin intake (microgram/d) and Y = plasma biotin (ng/ml). In most cases, these values were greater than the corresponding biotin intakes, indicating that intestinal biotin synthesis and coprophagy might be increasing the supply of bioavailable biotin to the rats. Therefore, the rat might not be a good model animal for routine evaluation of biotin bioavailability from feed ingredients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3583596

  3. Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains.

    OpenAIRE

    Song, J.; Wurtele, E S; Nikolau, B J

    1994-01-01

    Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes. The corresponding cDNA was isolated and expressed in Escherichia coli through fusion to the bacterial trpE gene. The resulting chimeric protein was biotinylated in E. coli. Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (...

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

    OpenAIRE

    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 P21 and diffract X-rays to 2.7 and 2.0 Å resolution, respectively.

  5. Identification of the biotin transporter in Escherichia coli, biotinylation of histones in Saccharomyces cerevisiae and analysis of biotin sensing in Saccharomyces cerevisiae

    OpenAIRE

    Ringlstetter, Stefan Ludwig

    2011-01-01

    Biotin transport in E. coli was already observed in 1972. Results in this work demonstrate yigM is the gene encoding the E. coli biotin transporter. The protein has 10 predicited transmembrane domains and shows homology to members of the subfamiliy of carboxylate/amino acid/amine transporters. Deletion of yigM led to complete loss, overexpression with different expression systems to a dose dependent increase of biotin uptake. The function of YigM was shown in whole cells and in reconstituted ...

  6. Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Sudha Purushothaman

    Full Text Available BACKGROUND: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP provides the co-factor for catalytic activity of ACC. METHODOLOGY/PRINCIPAL FINDINGS: BPL/BirA (Biotin Protein Ligase, and its substrate, biotin carboxyl carrier protein (BCCP of Mycobacterium tuberculosis (Mt were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the approximately 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS. Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved 'GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K(m for BCCP was approximately 5.2 microM and approximately 420 nM for biotin. MtBPL has low affinity (K(b = 1.06x10(-6 M for biotin relative to EcBirA but their K(m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity. CONCLUSIONS/SIGNIFICANCE: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

  7. 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. PMID:22159614

  8. Biotin synthesis begins by hijacking the fatty acid synthetic pathway.

    Science.gov (United States)

    Lin, Steven; Hanson, Ryan E; Cronan, John E

    2010-09-01

    Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven-carbon dicarboxylic acid, but the mechanism whereby this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-carboxyl group of a malonyl-thioester is methylated by BioC, which allows recognition of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl-acyl carrier protein (ACP) methyl ester, which is hydrolyzed to pimeloyl-ACP and methanol by BioH. PMID:20693992

  9. 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

    2016-02-01

    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. PMID:26750716

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

    International Nuclear Information System (INIS)

    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

  11. Measurement of Acylcarnitine Substrate to Product Ratios Specific to Biotin-Dependent Carboxylases Offers a Combination of Indicators of Biotin Status in Humans12

    OpenAIRE

    Bogusiewicz, Anna; Horvath, Thomas D; Stratton, Shawna L.; Mock, Donald M; Boysen, Gunnar

    2012-01-01

    This work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine : 3-methylglutarylcarnitine (3HIAc : MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio of propionylc...

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

    International Nuclear Information System (INIS)

    Due to the high affinity of biotin to Av or SA. The authors labelled a biotin derivative (DTPA-biotin) with 153Sm and then bound this 153Sm labelled DTPA-biotin to Av or SA. The in vivo kinetics and biodistribution of 153Sm labelled Av, SA and DTPA-biotin were studied in the rat and mice. The results demonstrated that 153Sm-Av cleared from the blood rapidly with high liver and renal uptake; 153Sm-SA cleared from blood slowly with high retention in liver, spleen and kidney, whereas 153Sm 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

  13. Electrophoretic behavior of streptavidin complexed to a biotinylated probe : A functional screening assay for biotin-binding proteins

    OpenAIRE

    Humbert, Nicolas; Zocchi, Andrea; Ward, Thomas R.

    2006-01-01

    The biotin-binding protein streptavidin exhibits a high stability against thermal denaturation, especially when complexed to biotin. Herein we show that, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), streptavidin is stabilized at high temperature in the presence of biotinylated fluorescent probes, such as biotin-4-fluorescein, which is incorporated within the binding pocket. In nondenaturing SDS-PAGE, streptavidin is detectable when complexed with biotin-4-fluoresce...

  14. Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography

    OpenAIRE

    Hayakawa, Kou; Katsumata, Noriyuki; Abe, Kiyomi; Hirano, Masahiko; Yoshikawa, Kazuyuki; Ogata, Tsutomu; Horikawa, Reiko; Nagamine, Takeaki

    2009-01-01

    The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs a...

  15. Marginal Biotin Deficiency is Common in Normal Human Pregnancy and Is Highly Teratogenic in Mice1–3

    OpenAIRE

    Mock, Donald M.

    2009-01-01

    In studies of marginal biotin deficiency induced experimentally in adults, increased urinary excretion of 3-hydroxyisovaleric acid (3HIA), which likely reflects decreased activity of the biotin-dependent enzyme β-methylcrotonyl-CoA carboxylase, and decreased activity of the biotin-dependent enzyme propionyl-CoA carboxylase (PCC) in peripheral blood lymphocytes have been validated as indices of biotin status. About half of pregnant women excrete increased amounts of urinary 3HIA. However, inte...

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

    International Nuclear Information System (INIS)

    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 99Tcm to substitute 111In to label DTPA-biotin to evaluate the value of the 99Tcm-DTPA-biotin in BAS. Methods: DTPA-biotin solution was mixed with SnCl2 and then fresh eluted 99Tcm. 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 99Tcm-DTPA-biotin was respectively given 1 day, 2 days later. Directly labelled C50 with 99Tcm was used as control agent. Results: The labelling yield of 99Tcm-DTPA-biotin was over 90%. The amount of SnCl2 was the key feature in labelling. The tumor could be seen at 2 h after injection of 99Tcm-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 99Tcm-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 99Tcm, and the BAS can image the tumor as early as 2 h after injection

  17. Biotin Responsive Multiple Carboxylase Deficiency Presenting as Diabetic Ketoacidosis.

    Directory of Open Access Journals (Sweden)

    Jia-Woei Hou

    2004-02-01

    Full Text Available Multiple carboxylase deficiency (MCD is a rare inherited metabolic disease of biotindependency due to deficiency of holocarboxylase synthetase (HCS or biotinidase deficiency.A 30-month-old female patient who presented with the initial features of diabeticketoacidosis (severe metabolic acidosis, ketosis, and hyperglycemia, lactic acidemia, moderatehyperammonemia, and generalized organic aciduria is described. Associated symptomsand signs included erythematous skin rashes, alopecia and developmental delay. The patientresponded dramatically to treatment with biotin (10 mg/day showing normalization of clinicalsymptoms and most biochemical abnormalities. Based on the urine organic profile bygas chromatography/ mass spectrometry (GC/MS, the diagnosis of MCD was made. A plasmatandem mass study confirmed this diagnosis. The biotinase activity in serum was normal,indicating that this was a rare case of late-onset HCS deficiency.

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

    Directory of Open Access Journals (Sweden)

    Gaby Tiemi Suzuki

    2011-09-01

    Full Text Available 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.

  19. A Francisella Virulence Factor Catalyzes an Essential Reaction of Biotin Synthesis

    Science.gov (United States)

    Feng, Youjun; Napier, Brooke A.; Manandhar, Miglena; Henke, Sarah K; Weiss, David S.; Cronan, John E.

    2014-01-01

    Summary We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side chain. Expression of bioJ allows growth of an E. coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel sub-clade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence. PMID:24313380

  20. 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. PMID:26924180

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

    International Nuclear Information System (INIS)

    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.

  2. Three-step tumor imaging with biotinylated monoclonal antibody, streptavidin and 111In-DTPA-biotin

    International Nuclear Information System (INIS)

    The purpose of this study was to test the three-step targeting of tumors in mice using biotinylated antibody, streptavidin and radiolabeled biotin. Nude mice bearing subcutaneous LS180 human colon cancer xenografts were intravenously administered with 200 μg of the biotinylated anti-Tn monoclonal antibody MLS128, and 2 days later they got intravenous injection of 50 μg of streptavidin. They were intravenously injected 1, 4 or 7 days later with 0.5 μg of 111In-diethylenetriamine pentaacetic acid (DTPA)-biotin. The tumor uptake, determined 2 h later, was 1.4, 0.5 and 0.6% injected dose/gram of tissue (ID/g), respectively, and the blood radioactivity was 1.0, 0.2 and 0.2% ID/g, respectively. When the interval between the streptavidin and radiolabeled biotin injections was prolonged from 1 day to 7 days, the tumor-to-blood ratio 2 h after injection of 111In-labeled biotin increased from 1.5 to 4.0. Clear tumor images were obtained as early as 2 h after injection of radiolabeled biotin. In conclusion, these preliminary data suggested that the three-step method using the streptavidin-biotin system would be applicable in an experimental mouse tumor model and provides images of tumors rapidly and clearly after injection of radiolabeled biotin

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

    International Nuclear Information System (INIS)

    We synthesized the ZnS:Mn2+ 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:Mn2+ 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:Mn2+ nanoparticles shows that the Mn2+ ions are incorporated within ZnS host matrix. We found that biotin capping will enhance the luminescence from ZnS:Mn2+ 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

  4. Determination of Biotin in Pharmaceutical Formulations by Potassium Permanganate-luminol-CdTe Nanoparticles Chemiluminescence System

    Institute of Scientific and Technical Information of China (English)

    TRAORE Zoumana Sékou; SU Xing-guang

    2012-01-01

    A sensitive flow-injection chemiluminescence method was developed for the determination of biotin in the pharmaceutical formulations.The affinity between avidin and biotin was used to adsorb biotin on the polystyrene,with subsequent quantification of biotin based on its ability to enhance the chemiluminescence(CL) signal generated by the redox reaction of potassium permanganate-luminol-CdTe nanoparticles CL system.The investigations prove that apart from 3-aminophthalate,the CdTe quantum dots(QDs) play both catalytic and emitter roles.Under optimum conditions,the linear range for the determination of biotin was 0.01-25 ng/mL with a detection limit of 7.3×10-3ng/mL(S/N=3).The relative standard deviation of 5 ng/L biotin was 2.06%(n=7).The proposed method was used to determine the biotin concentration in the pharmaceutical formulations and the recovery was between 96.4% and 104%.The proposed method is simple,convenient,rapid and sensitive.

  5. Intake and Urinary Amounts of Biotin in Japanese Elementary School Children, College Students, and Elderly Persons

    OpenAIRE

    Katsumi Shibata; Tomiko Tsuji; Tsutomu Fukuwatari

    2013-01-01

    Biotin enzymes such as pyruvate carboxylase and acetyl-CoA carboxylase are involved with the most basic metabolism. Thus, it is very important to monitor the biotin nutritional status for maintaining good health. We examined urinary excretion and the intake of biotin in a Japanese sample population of 60 boys and 36 girls (10–12 y), 37 male and 135 female college students (18–27 y), and 35 female elderly persons (70–84 y) living freely. All food consumed, and the corresponding weighing, for 4...

  6. Development of a System for Clinical Evaluation of the Biotin Status of Sows

    OpenAIRE

    Misir, R.; Blair, R.; Doige, C.E.

    1986-01-01

    Sixteen test gilts were fed an egg white-fortified practical diet and four control gilts an egg white-free diet over a two-parity period in order to monitor changes in the serum biotin levels as induced biotin deficiency progressed. Gilts were individually housed in metal crates with slatted floors. Serum biotin (ng/L) of test animals declined from 1490 (initially) to 610 (month 3), remained stable (months 4-8) and thereafter approached 400, 135 and 30 after months 9, 13 and 15, respectively....

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

    International Nuclear Information System (INIS)

    This paper will describe the preparation of ''no-carrieradded'' 18F-labeled biotin where the radiolabel bound to an aromatic moiety is described. This has been accomplished by preparation of [18F]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)

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

    International Nuclear Information System (INIS)

    This paper will describe the preparation of ''no-carrieradded'' 18F-labeled biotin where the radiolabel bound to an aromatic moiety is described. This has been accomplished by preparation of [18F]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)

  9. An improved smaller biotin ligase for BioID proximity labeling.

    Science.gov (United States)

    Kim, Dae In; Jensen, Samuel C; Noble, Kyle A; Kc, Birendra; Roux, Kenneth H; Motamedchaboki, Khatereh; Roux, Kyle J

    2016-04-15

    The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. PMID:26912792

  10. 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)

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

    OpenAIRE

    1997-01-01

    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-s...

  12. Methods to determine biotin-binding capacity of streptavidin-coated magnetic particles

    Science.gov (United States)

    Dorgan, Lonnie; Magnotti, Ralph; Hou, Janming; Engle, Terri; Ruley, Kevin; Shull, Bruce

    1999-04-01

    Two assays to determine the biotin-binding capacity of streptavidin magnetic particles are described and compared. The two assays are based on the use of biotinylated alkaline phosphatase and biotinylated fluorescein, respectively. Also, an assay for bound protein is presented. When the biotin-binding methods are combined with the protein assay, the specific activity can be determined. The fluorescent version is used to compare the streptavidin magnetic particles from several manufacturers.

  13. Synthesis of Biotin Tagged Chemical Cross-linkers and Their Applications for Mass Spectrometry

    OpenAIRE

    Kang, Sebyung; Mou, Liyuan; Brouillette, Wayne J.; Prevelige, Peter E.

    2009-01-01

    Chemical cross-linking combined with mass spectrometry has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them for enriching cross-linked peptides. The reaction of biotin tagged cros...

  14. Biotin Limitation in Sinorhizobium meliloti Strain 1021 Alters Transcription and Translation

    OpenAIRE

    Heinz, Elke B.; Streit, Wolfgang R.

    2003-01-01

    Most Sinorhizobium meliloti strains lack several key genes involved in microbial biotin biosynthesis, and it is assumed that this may be a special adaptation which allows the microbe to down-regulate metabolic activities in the absence of a host plant. To further explore this hypothesis, we employed two different strategies. (i) Searches of the S. meliloti genome database in combination with the construction of nine different gusA reporter fusions identified three genes involved in a biotin s...

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

    International Nuclear Information System (INIS)

    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

  16. Determination of pantothenic acid, biotin, and vitamin B12 in nutritional products.

    Science.gov (United States)

    Hudson, T S; Subramanian, S; Allen, R J

    1984-01-01

    Until recently, liquid chromatographic (LC) methodology for pantothenic acid, biotin, and B12 (cyanocobalamin) has been only marginally successful. These vitamins are difficult to determine by conventional LC techniques and UV detection at 254 or 280 nm, because either the chromophore is inadequate for detection or interference from co-eluting vitamins is overwhelming. Biotin and B12 are usually present in pharmaceutical products at concentrations 100-1000 times lower than other commonly occurring water-soluble vitamins. Co-extraction of all water-soluble vitamins results in gross interferences, especially in LC when the interfering vitamins co-elute with biotin or B12. In addition, pantothenic acid and biotin are colorless in solution and do not exhibit strong UV absorption above 240 nm. As a result, they must be quantitated either by using a low UV wavelength for detection or by derivatizing the vitamin to obtain an adequate chromophore. A description of procedures for LC determination of pantothenic acid, panthenol, cyanocobalamin, and biotin in pharmaceutical products is presented. Pantothenic acid has been measured by using both a derivatization technique and low UV wavelength detection. Biotin has been quantitated by using low UV wavelength detection. The limitations of these techniques are also discussed. Chromatographic separation of cyanocobalamin is complicated by co-eluting vitamins such as riboflavin. It is detected by using the 546 nm wavelength where riboflavin does not interfere. PMID:6501166

  17. Carbon nanofiber-based luminol-biotin probe for sensitive chemiluminescence detection of protein.

    Science.gov (United States)

    Baj, Stefan; Krawczyk, Tomasz; Pradel, Natalia; Azam, Md Golam; Shibata, Takayuki; Dragusha, Shpend; Skutil, Krzysztof; Pawlyta, Miroslawa; Kai, Masaaki

    2014-01-01

    A carbon nanofiber-based luminol-biotin probe was synthesized for the sensitive chemiluminescence (CL) detection of a target protein by grafting luminol and biotin onto an oxidized carbon nanofiber. This carbon nanofiber was prepared by chemical vapor-deposition with methane in the presence of the Ni-Cu-MgO catalyst, which was followed by oxidization with HNO3-H2SO4 to produce a carboxyl group on the surface of the nanofiber. The material was grafted with luminol and biotin by means of a standard carbodiimide activation of COOH groups to produce corresponding amides. The substance was water-soluble and thus could be utilized as a sensitive CL probe for a protein assay. The probe showed highly specific affinity towards the biotin-labeled antibody via a streptavidin-biotin interaction. The detection limit for this model assay was approximately 0.2 pmol of the biotinized IgG spotted on a polyvinylidene fluoride (PVDF) membrane. Nonspecific binding to other proteins was not observed. Therefore, the synthesized carbon nanofiber-based CL probe may be useful for a sensitive and specific analysis of the target protein. PMID:25382040

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

    International Nuclear Information System (INIS)

    The synthesis of N-(4-[127/125/123I]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 CF3COOH is described. Iodinated in this way biotin formed a stable complex with avidin (Kd = 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)

  19. Total synthesis of amiclenomycin, an inhibitor of biotin biosynthesis.

    Science.gov (United States)

    Mann, Stéphane; Carillon, Sophie; Breyne, Olivier; Marquet, Andrée

    2002-01-18

    We describe the first synthesis of amiclenomycin, a natural product that has been found to inhibit biotin biosynthesis and, as a consequence, to exhibit antibiotic properties. Structure 1, with a trans relationship between the ring substituents. had previously been proposed for amiclenomycin on the basis of its 1H NMR spectrum. We have prepared the trans and cis isomers 1 and 2 by unequivocal routes and we conclude that the natural product is in fact the cis isomer 2. The properly substituted cyclohexadienyl rings were constructed first. A cycloaddition reaction between 1,2-di(phenylsulfonyl)ethylene and the N-allyloxycarbonyl diene 13, followed by reductive elimination of the phenylsulfinyl groups, gave the cis isomer 15. To obtain the trans isomer, the O-trimethylsilyl diene was used to give the cis hydroxylated Diels-Alder adduct 33, which was transformed into the corresponding trans amino derivative by means of a Mitsunobu reaction. The L-alpha-amino acid functionality was introduced by means of a Strecker reaction on the aldehydes 16 and 42, followed by enzymatic hydrolysis with immobilised pronase. PMID:11843156

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

    Directory of Open Access Journals (Sweden)

    Taheri A

    2011-09-01

    Full Text Available Azade Taheri1, Rassoul Dinarvand1,2, Faranak Salman Nouri1, Mohammad Reza Khorramizadeh3, Atefeh Taheri Borougeni4, Pooria Mansoori5, Fatemeh Atyabi1,21Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical sciences, Tehran, Iran; 3Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran; 5Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IranAbstract: 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

  1. Effect of supplementing zinc oxide and biotin with or without carbadox on nursery pig performance.

    Science.gov (United States)

    Wilt, H D; Carlson, M S

    2009-10-01

    A 28-d nursery experiment was conducted to evaluate the effects of supplementing zinc oxide and biotin with or without a feed-grade antimicrobial agent (carbadox) on nursery pig performance, and plasma and fecal Zn concentrations. One hundred ninety-two crossbred pigs (initial BW = 5.94 +/- 0.03 kg; age = 17 +/- 2 d) were weaned and allotted to 1 of 8 dietary treatments based on BW, sex, and ancestry in a randomized complete block design (3 pigs/pen and 8 replications). Dietary treatments consisted of supplementation of ZnO at 0 or 3,000 mg/kg, d-biotin at 0 or 440 microg/kg, and carbadox at 0 or 55 mg/kg of diets in a 2 x 2 x 2 factorial arrangement of treatments. Phase 1 (d 0 to 14) and phase 2 (d 14 to 28) nursery diets were fed in meal form. Fecal samples were collected weekly, and blood samples were collected at d 0, 14, and 28 to determine fecal and plasma Zn concentrations, respectively. The basal diet contained 165 mg/kg of Zn as ZnSO(4) and 220 microg/kg biotin as d-biotin. Pigs supplemented with 440 microg/kg of d-biotin, independent of antibiotic and ZnO additions, had greater overall ADG (P = 0.02) than pigs fed no supplemental d-biotin postweaning. Overall ADG, ADFI, and G:F were not affected when pigs were supplemented with 3,000 mg/kg of Zn as ZnO or 55 mg/kg of carbadox. When pigs were fed 55 mg/kg of carbadox without supplemental biotin, plasma Zn concentration was less, whereas when biotin and carbadox were supplemented to nursery pig diets, plasma Zn concentrations did not decrease as with feeding carbadox alone (biotin x carbadox, P pigs fed 3,000 mg/kg of Zn as ZnO and 440 microg/kg of d-biotin had greater fecal Zn concentrations than pigs fed diets with only 3,000 mg/kg of Zn as ZnO (Zn x biotin, P = 0.04). In addition, pigs supplemented with 3,000 mg/kg of Zn as ZnO in combination with carbadox and d-biotin had greater fecal Zn concentrations compared with pigs fed diets containing no additional Zn during wk 2 (Zn x biotin x carbadox, P = 0

  2. 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

    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 of...... and cannot be used for deriving DRVs for biotin. As there is insufficient evidence available to derive an Average Requirement and a Population Reference Intake, an Adequate Intake (AI) is proposed. The setting of AIs is based on observed biotin intakes with a mixed diet and the apparent absence of...... through breast milk. For infants over six months, an AI of 6 µg/day is proposed by extrapolating from the biotin intake of exclusively breastfed infants aged zero to six months, using allometric scaling and reference body weight for each age group, in order to account for the role of biotin in energy...

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

    International Nuclear Information System (INIS)

    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) 99mTc-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 99mTc-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

  4. 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)

    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 P21 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 P21, 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 VM value of 2.45 Å3 Da−1 and a solvent content of 50%

  5. Effect of biotin limitation on the conversion of xylose to ethanol and xylitol by Pachysolen tannophilus and Candida guilliermondii

    Energy Technology Data Exchange (ETDEWEB)

    Hung Lee; Atkin, A.L.; Barbosa, M.F.S.; Dorscheid, D.R.; Schneider, Henry

    1988-02-01

    The relative amount of ethanol and xylitol accumulated in aerobic batch cultures of Pachysolen tannophilus and Candida guilliermondii on D-xylose depended on the extent of limitation by biotin. In high biotin media P. tannophilus favored ethanol production over that of xylitol while C. guilliermondii favoured xylitol formation. However, as the extent of biotin limitation increased, the ratio of ethanol to xylitol produced by both organisms increased. The results are of interest in efforts to control such ratios.

  6. Biotin derivatives carrying two chelating DOTA units. Synthesis, in vitro evaluation of biotinidases resistance, avidin binding, and radiolabeling tests

    OpenAIRE

    Pratesi, Alessandro; Bucelli, Francesca; Mori, Ilaria; Chinol, Marco; Verdoliva, Antonio; Paganelli, Giovanni; Rivieccio, Vincenzo; Gariboldi, Lucia; Ginanneschi, Mauro

    2010-01-01

    The synthesis of four biotin derivatives carrying two DOTA moieties for each ligand (BisDOTA set) is reported, for increasing radiation/dose ratio and improving efficiency in the pretargeted avidin-biotin radioimmunotherapy. The biotin-containing scaffold of two BisDOTA was similar to the mono-DOTA derivative previously described. Then the scaffold was elongated by trifunctionalized spacers of different length and conjugated with one of the COOH groups of two DOTA. Two others were prepared st...

  7. Plasma concentration of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans1234

    OpenAIRE

    Stratton, Shawna L.; Horvath, Thomas D.; Bogusiewicz, Anna; Matthews, Nell I.; Henrich, Cindy L; Spencer, Horace J.; Moran, Jeffery H.; Mock, Donald M.

    2010-01-01

    Background: Blood-based indicators of biotin status in humans were shown to be useful tools in several clinical situations, including pregnancy. We previously validated the activity of the biotin-dependent enzyme propionyl-coenzyme A carboxylase (PCC) in lymphocytes as a sensitive and specific blood-based indicator of marginal degrees of biotin deficiency. However, the measurement of PCC activity in population studies presents substantial analytic challenges. 3-Hydroxyisovaleryl carnitine (3H...

  8. 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; Larsen, Sara Charlotte; Poulsen, Jon Wriedt; Andersen, Marianne Agerholm; Palmqvist, Eva A; Hey-Mogensen, Martin; Jensen, Per B.; Treebak, Jonas Thue; Lisby, Michael; Nielsen, Michael Lund

    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...

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

    International Nuclear Information System (INIS)

    Four 99mTc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99mTc-MAG3-biotin conjugates as model compounds for 186Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99Tc were added as carrier to 99mTc. 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 99mTc/99Tc-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 99mTc-MAG3-biotin conjugates might have potential

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

    International Nuclear Information System (INIS)

    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 166Dy/166Ho radionuclide pair can be considered as an in vivo generator system. The aim of this work was to synthesize 166Dy/166Ho-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 164Dy2O3 in a TRIGA Mark III reactor at a flux in the central thimble of 3.1013 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 900C. 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. 166Dy/166Ho-DTPA-bisBiotin traveled with the solvent front Rf 0.9-1.0 and the Dy+3/Ho+3 species remained at the origin (Rf 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 in an agarose column

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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 166Dy/166Ho-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 Dy2O3 (164Dy, 99 %, from Oak Ridge NL) in a Triga Mark III reactor at a flux in the central thimble of 3.1013 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 166Dy2Cl3 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. 166Dy/166Ho-DTPA-bisBiotin travelled with the solvent front Rf 0.9-1.0 and the Dy+3/Ho+3 species remained at the origin (Rf = 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 166Dy/166Ho complex solution was determined by TLC. The complex 166Dy/166Ho-DTPA-bisBiotin was

  13. 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.

  14. 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.

  15. 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

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

    International Nuclear Information System (INIS)

    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, Rct, 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 Rct 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

  17. [Construction of biotin-modified polymeric micelles for pancreatic cancer targeted photodynamic therapy].

    Science.gov (United States)

    Deng, Chun-yue; Long, Ying-ying; Liu, Sha; Chen, Zhang-bao; Li, Chong

    2015-08-01

    In this study, we explored the feasibility of biotin-mediated modified polymeric micelles for pancreatic cancer targeted photodynamic therapy. Poly (ethylene glycol)-distearoyl phosphatidyl ethanolamine (mPEG2000-DSPE) served as the drug-loaded material, biotin-poly(ethylene glycol)-distearoyl phosphatidyl ethanolamine (Biotin-PEG3400-DSPE) as the functional material and the polymeric micelles were prepared by a thin-film hydration method. The targeting capability of micelles was investigated by cell uptake assay in vitro and fluorescence imaging in vivo and the amounts of Biotin-PEG-DSPE were optimized accordingly. Hypocrellin B (HB), a novel photosensitizer was then encapsulated in biotinylated polymeric micelles and the anti-tumor efficacy was evaluated systemically in vitro and in vivo. The results showed that micelles with 5 mol % Biotin-PEG-DSPE demonstrated the best targeting capability than those with 20 mol % or 0.5 mol % of corresponding materials. This formulation has a small particle size [mean diameter of (36.74 ± 2.16) nm] with a homogeneous distribution and high encapsulation efficiency (80.06 ± 0.19) %. The following pharmacodynamics assays showed that the biotinylated micelles significantly enhanced the cytotoxicity of HB against tumor cells in vitro and inhibited tumor growth in vivo, suggesting a promising potential of this formulation for treatment of pancreatic cancer, especially those poorly permeable, or insensitive to radiotherapy and chemotherapy. PMID:26669006

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

    International Nuclear Information System (INIS)

    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 (Ka) of biotin imprinted Au/Ag nanoclusters has been determined using the Scatchard method and found to be 3.89 × 105 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.

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

    International Nuclear Information System (INIS)

    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 1012 M-1, 3.2 x 1012 M-1 and 4.0 x 1012 M-1, respectively

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

    International Nuclear Information System (INIS)

    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 P42212, with unit-cell parameters a = b = 93.665, c = 131.95

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

    International Nuclear Information System (INIS)

    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 300C, 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 360C, 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.)

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

    International Nuclear Information System (INIS)

    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)

  3. 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. © 2014 the Partner Organisations....

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

    OpenAIRE

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

    2011-01-01

    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 ribof...

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

    OpenAIRE

    Gaby Tiemi Suzuki; Juliana Alves Macedo; Gabriela Alves Macedo

    2011-01-01

    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 ribof...

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

    International Nuclear Information System (INIS)

    Biotinyl-m-[125I]iodoanilide (BIA) was synthesized by coupling biotin to m-[125I]iodoaniline via a mixed anhydride reaction. m-[125I]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)

  7. 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

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

    International Nuclear Information System (INIS)

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

    OpenAIRE

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

    2014-01-01

    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.

  10. 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)

    In terms of nuclear decay 18F is the most ideal PET nuclide but its short t1/2 precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as NeutravidinTM-biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with NeutravidinTM-conjugated antibodies. Methods: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with 18F 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 NeutravidinTM-conjugated trastuzumab, washed, and then incubated with 18F-PEG-biotin. Results: The 18F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel 18F-PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with NeutravidinTM-conjugated trastuzumab. Conclusion: Biotin can be functionalised with boroaryl and readily 18F-radiolabelled in aqueous solution and will bind to cells pre-incubated with NeutravidinTM-antibody conjugates. - Highlights: → Boroaryl-biotin precursor is prepared. → Rapid 18F-fluorination is demonstrated. → HER-2 expressing breast cancer cells pre-treated with trastuzumab-NeutravidinTM. → 18F-PEG-biotin binding to pre-treated cells corresponds with HER-2 expression.

  11. 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.

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

    Science.gov (United States)

    Xu, Hao; Sabit, Hairat; Amidon, Gordon L; Showalter, H D Hollis

    2013-01-01

    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

  13. 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

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

    International Nuclear Information System (INIS)

    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 Dy203 in the TRIGA Mark III reactor. The labelled was carried out in aqueous solution to p H 8.0 for addition of 166 Dy Cl3 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 Ho3+ 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)

  15. Electrochemical monitoring of biotin binding to avidin. Electroactivity of avidin and streptavidin

    Czech Academy of Sciences Publication Activity Database

    Havran, Luděk; Billová, Sabina; Paleček, Emil

    Galway : European Society for ElectroAnalytical Chemistry, 2004. s. 135. [International Conference on Electroanalysis /10./. 06.06.2004-10.06.2004, Galway] R&D Projects: GA ČR GA204/03/0566; GA AV ČR KJB4004302 Keywords : avidin- biotin binding * electrochemical methods * interfacial behavior Subject RIV: BO - Biophysics

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

    International Nuclear Information System (INIS)

    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

  17. 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…

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

    International Nuclear Information System (INIS)

    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 111In-labeled anti-CEA antibody (C110) or 111In-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 111In-labeled biotin (EB1) 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)

  19. 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 v

  20. 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. PMID:26212997

  1. Three-step radioimmunotherapy with yttrium-90 biotin: dosimetry and pharmacokinetics in cancer patients

    International Nuclear Information System (INIS)

    A three-step avidin-biotin approach has been applied as a pretargeting system in radioimmunotherapy (RIT) as an alternative to conventional RIT with directly labelled monoclonal antibodies (MoAbs). Although dosimetric and toxicity studies following conventional RIT have been reported, these aspects have not previously been evaluated in a three-step RIT protocol. This report presents the results of pharmacokinetic and dosimetric studies performed in 24 patients with different tumours. Special consideration was given to the dose delivered to the red marrow and to the haematological toxicity. The possible additive dose to red marrow due to the release of unbound yttrium-90 was investigated. The protocol consisted in the injection of biotinylated MoAbs (first step) followed 1 day later by the combined administration of avidin and streptavidin (second step). After 24 h, biotin radiolabelled with 1.85-2.97 GBq/m2 of 90Y was injected (third step). Two different chelating agents, DTPA and DOTA, coupled to biotin, were used in these studies. Indium-111 biotin was used as a tracer of 90Y to follow the biodistribution during therapy. Serial blood samples and complete urine collection were obtained over 3 days. Whole-body and single-photon emission tomography images were acquired at 1, 16, 24 and 40 h after injection. The sequence of images was used to extrapolate 90Y-biotin time-activity curves. Numerical fitting and compartmental modelling were used to calculate the residence time values (τ) for critical organs and tumour, and results were compared; the absorbed doses were estimated using the MIRDOSE3.1 software. The residence times obtained by the numerical and compartmental models showed no relevant differences (90Y activities capable of delivering a high dose to the tumour and sparing red marrow and other normal organs. Although 90Y-biotin clears rapidly from circulation, the use of DOTA-biotin conjugate for a stable chelation of 90Y is strongly recommended, considering

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

    International Nuclear Information System (INIS)

    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 111In-labeled anti-CEA antibody (C110) or 111In-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 111In-labeled biotin (EB1) 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)

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

    International Nuclear Information System (INIS)

    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)

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

    International Nuclear Information System (INIS)

    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 [68Ga]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 [68Ga]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 [68Ga]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 [68Ga]Ga-DOTA-(PEG)2-biotin and PET.

  5. High-throughput Immunoblotting Identifies Biotin-dependent Signaling Proteins in HepG2 Hepatocarcinoma Cells1

    OpenAIRE

    Rodriguez-Melendez, Rocio; Griffin, Jacob B.; Sarath, Gautam; Zempleni, Janos

    2005-01-01

    Biotin affects the abundance of mRNA coding for approximately 10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.025 nmol/L (denoted “deficient”), 0.25 nmol/L (“physiological” = control), and 10 nmol/L (“pharmacological”) for...

  6. 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. PMID:26722835

  7. Biotin-avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

    International Nuclear Information System (INIS)

    Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11)

  8. 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'.

  9. Immunoradiometric assay for carcinoembryonic antigen using avidin-biotin separation technique

    International Nuclear Information System (INIS)

    A sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for carcinoembryonic antigen (CEA) has been developed in authors' laboratory, which can be performed conveniently. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on CEA that are distal from each other. One of these antibodies was labelled with 125I and the other was conjugated covalently to biotin. Polystyrene tubes were conjugated covalently to avidin. These tubes represent a rapid, simple method for separating the CEA-bound antibody from the free antibody. The biotin-antibody-CEA-12'5I-labelled antibody complexes bind to the tubes and CEA concentration is directly related to counts per minute. This assay can detect the CEA at a concentration of 0.22 μg/L in serum

  10. Scintigraphic imaging of tumors by in vivo application of biotin-conjugated antibodies and radiolabelled streptavidin

    International Nuclear Information System (INIS)

    Scintigraphic tumor imaging by in vivo application of the streptavidin-biotin system was studied. Binding of anti-cytokeratin antibodies to HeLa carcinoma cells was demonstrated in vitro by immunohistochemical staining and in vivo by scintigraphic imaging using 125I labeled antibodies in nude rats bearing HeLa cell carcinomas. For imaging of tumors with radiolabeled streptavidin, 'cold' biotin-conjugated antiboties were preinjected i.v. In this case, the tumor could be localized after 15 to 60 min whereas it took at least 3 days with the directly labeled antibody. The application of radiolabeled streptavidin was limited by an antibody-independent radioactivity uptake in kidney, liver and spleen. Nevertheless, the short time needed for imaging gives the opportunity of using high energy/short half life isotopes in radioimmunodetection. Furthermore, one single tracer can be used for a wide spectrum of antibodies. (orig.)

  11. Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

    OpenAIRE

    Wu, Sau-Ching; Wong, Sui-Lam

    2002-01-01

    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. Th...

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

    International Nuclear Information System (INIS)

    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 111In-biotin; imaging was acquired 30 min and 16-18 h after administration of 111In-biotin. Technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO)-labelled leucocyte scintigraphy was performed in 39/54 patients. The overall sensitivity of the avidin/111In-biotin scan was 97.7% (versus 88.9% for 99mTc-HMPAO leucocyte scintigraphy). While the diagnostic performance of avidin/111In-biotin scintigraphy was similar to that of 99mTc-HMPAO leucocyte scintigraphy in patients with prosthetic joint replacements or osteomyelitis of appendicular bones, the avidin/111In-biotin approach clearly performed better than 99mTc-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 99mTc-HMPAO-leucocyte scintigraphy). These results demonstrate the feasibility of the avidin/111In-biotin approach for imaging sites of infection/inflammation in the clinical setting. Although no systematic advantages of avidin/111In-biotin scintigraphy were found versus 99mTc-HMPAO leucocyte scintigraphy, the newer scintigraphic method is more practicable and involves lower biological risk for the operators. (orig.)

  13. The quantitation of biotinylated compounds by a solid-phase assay using a 125I-labelled biotin derivative

    International Nuclear Information System (INIS)

    The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with 125I. 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.)

  14. Marginal Biotin Deficiency Can Be Induced Experimentally in Humans Using a Cost-Effective Outpatient Design123

    OpenAIRE

    Stratton, Shawna L.; Henrich, Cindy L; Matthews, Nell I.; Bogusiewicz, Anna; Dawson, Amanda M.; Horvath, Thomas D.; Owen, Suzanne N.; Boysen, Gunnar; Moran, Jeffery H.; Mock, Donald M.

    2011-01-01

    To date, marginal, asymptomatic biotin deficiency has been successfully induced experimentally by the use of labor-intensive inpatient designs requiring rigorous dietary control. We sought to determine if marginal biotin deficiency could be induced in humans in a less expensive outpatient design incorporating a self-selected, mixed general diet. We sought to examine the efficacy of three outpatient study designs: two based on oral avidin dosing and one based on a diet high in undenatured egg ...

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

    International Nuclear Information System (INIS)

    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/111In-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 111In-chloride. Two-step scintigraphy was performed by first infusing 3 mg streptavidin intravenously, followed 4 h later by 111In-biotin. Imaging was begun 60 min later. Streptavidin/111In-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/111In-biotin scintigraphy than for either MRI or CT. Streptavidin/111In-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.)

  16. Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.

    OpenAIRE

    Barker, D F; Campbell, A M

    1980-01-01

    The technique developed by Casadaban (M. J. Casadaban, J. Mol. Biol. 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon. Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated. When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium. Repressing conditio...

  17. Sinorhizobium meliloti Cells Require Biotin and either Cobalt or Methionine for Growth

    OpenAIRE

    Watson, Robert J.; Heys, Roselyn; Martin, Teresa; Savard, Marc

    2001-01-01

    Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growt...

  18. Introduction of biotin or folic acid into polypyrrole magnetite core-shell nanoparticles

    International Nuclear Information System (INIS)

    In order to contribute to the trend in contemporary research to develop magnetic core shell nanoparticles with better properties (reduced toxicity, high colloidal and chemical stability, wide scope of application) in straightforward and reproducible methods new core shell magnetic nanoparticles were developed based on polypyrrole shells functionalized with biotin and folic acid. Magnetite nanoparticles stabilized by sebacic acid were used as magnetic cores. The morphology of magnetite was determined by transmission electron microscopy TEM, while the chemical structure investigated by FT-IR

  19. Biological and chemical decoration of peptide nanostructures via biotin-avidin interactions.

    Science.gov (United States)

    Reches, Meital; Gazit, Ehud

    2007-07-01

    Novel architectures with nanometric dimensions hold an immense promise as building blocks for future nanotechnological applications. Biological nanostructures are of special interest due to their biocompatibility and because they allow the utilization of biochemical recognition interfaces. The ability to decorate bio-nanostructures with functional groups is highly important in order to utilize them in several applications including ultrasensitive sensors, drug delivery systems, and tissue engineering. Peptide-based nanostructures have a distinct advantage over other assemblies because they can be easily modified with chemical and biological elements. Aromatic dipeptide nanotubes (ADNT) are formed by the self-assembly of a very simple building block, the diphenylalanine peptide. These nanotubes have remarkable chemical and mechanical properties and their utilization in various applications has previously been demonstrated. Here we report on the chemical modification of ADNT with biotin moieties, in order to enable the selective decoration of the tubes with avidin-labeled species. First, ADNT were prepared in aqueous solution by self-assembly of the dipeptide building blocks. Next, they were modified using N-hydroxysuccinimido-biotin. The level of biotinylation was assessed by the interaction of the tubes with gold-labeled strepavidin and ultrastructural analysis by electron microscopy. The ability of the modified assemblies to serve as a generic functional platform was demonstrated by avidin-mediated conjugation. Avidin was added as a molecular linker to allow the decoration with biotin-labeled quantum dots. The efficient decoration was again probed by the imaging of the modified tubes using laser confocal microscopy. Taken together, we demonstrated the ability to decorate ADNT using a generic avidin-biotin adaptor. This decoration should lead to the integration and utilization of the tubes in various applications. PMID:17663236

  20. 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.

  1. Red Cell Volume Can Be Accurately Determined in Sheep Using a Non-radioactive Biotin Label

    OpenAIRE

    Mock, Donald M.; Mock, Nell I.; Lankford, Gary L.; Burmeister, Leon F.; Strauss, Ronald G.; Widness, John A.

    2008-01-01

    The sheep has served as an informative animal model for investigation of human fetal and newborn erythropoiesis and red blood cell (RBC) kinetics. We previously validated the permanent label (14C)cyanate for measuring red cell volume (RCV) in sheep. Here we validate biotin labeling of RBCs as a nonradioactive method for measuring RCV in sheep with the anticipation that it can be applied in studies of human infants. The RCV was determined simultaneously using two techniques for quantitation of...

  2. Introduction of biotin or folic acid into polypyrrole magnetite core-shell nanoparticles

    Science.gov (United States)

    Nan, Alexandrina; Turcu, Rodica; Liebscher, Jürgen

    2013-11-01

    In order to contribute to the trend in contemporary research to develop magnetic core shell nanoparticles with better properties (reduced toxicity, high colloidal and chemical stability, wide scope of application) in straightforward and reproducible methods new core shell magnetic nanoparticles were developed based on polypyrrole shells functionalized with biotin and folic acid. Magnetite nanoparticles stabilized by sebacic acid were used as magnetic cores. The morphology of magnetite was determined by transmission electron microscopy TEM, while the chemical structure investigated by FT-IR.

  3. Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

    International Nuclear Information System (INIS)

    13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. /sup D/(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the βγ-bridge position to the β-nonbridge position in [18O4]ATP or washout of 18O from the γ-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate is formed by nucleophilic attack of bicarbonate on ATP. Decarboxylation of carboxyphosphate in the active site generates CO2, which serves to carboxylate the isourea tautomer of biotin that is generated by the removal of the proton on N1' by the ionized sulfhydryl

  4. Experimental study of biotin-avidin pretargeting technique for inflammation imaging

    International Nuclear Information System (INIS)

    The usefulness of biotin-avidin pretargeting technique in inflammation imaging was evaluated. A two-step method was used. 6 hours after a preinjection of biotin-hIgG(B-hIgG) or streptavidin (SA), 131I-SA or 113mIn-DTPA-biotin (113mIn-DB) was injected. 2 h, 6 h or 24 h later, γ-image was performed and biodistribution was studied. 99mTc-hIgG, a one-step method using 99mTc-SA, was used as control. An animal model was caused by staphlococci. For B-hIgG and 131I-SA two-step method, T/NT was only 0.39 at 2h and reached 2.70 at 24 h. In γ-imaging, no concentration of radioactivity was found in inflammatory site at 2 h, 6 h and 24 h. Because it took a long time to clear hIgG in blood, the blood background was higher. For SA and 113mIn-DB two-step method, in γ-imaging, concentration of radioactivity was observed in inflammatory site as early as 2h, T/NT was 1.83 at 2h and 4.33 at 6h. For 99mTc-hIgG, 99mTc-SA one-step method, SA, as a kind of protein, could concentrate in inflammatory site, but high concentration of radioactivity was also visualized in liver and spleen. Owing to high molecule weight, labelled hIgG not only maintained a high level in blood, but also accumulated in liver, hence the imaging quality was not satisfied. The biotin-avidin pretargeting technique can attain a higher T/NT ratio than the one-step method and a low blood background so as early imaging can be got with better imaging quality

  5. Introduction of biotin or folic acid into polypyrrole magnetite core-shell nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Nan, Alexandrina; Turcu, Rodica [National Institute of Research and Development for Isotopic and Molecular Technologies, Donath 65-103, Cluj-Napoca (Romania); Liebscher, Jürgen [National Institute of Research and Development for Isotopic and Molecular Technologies, Donath 65-103, Cluj-Napoca, Romania and Institute of Chemistry, Humboldt-University Berlin, Brook-Taylor 2, D-12489 Berlin (Germany)

    2013-11-13

    In order to contribute to the trend in contemporary research to develop magnetic core shell nanoparticles with better properties (reduced toxicity, high colloidal and chemical stability, wide scope of application) in straightforward and reproducible methods new core shell magnetic nanoparticles were developed based on polypyrrole shells functionalized with biotin and folic acid. Magnetite nanoparticles stabilized by sebacic acid were used as magnetic cores. The morphology of magnetite was determined by transmission electron microscopy TEM, while the chemical structure investigated by FT-IR.

  6. Elektrochemická analýza vazby streptavidin-biotin

    Czech Academy of Sciences Publication Activity Database

    Masařík, Michal; Brázdová, Marie; Vacek, Jan; Kizek, René; Billová, Sabina; Havran, Luděk; Fojta, Miroslav; Paleček, Emil

    Brno : Masarykova univerzita, 2002. s. 24. ISBN 80-210-2777-0. [Pracovní setkání biochemiků a molekulárních biologů /6./. 07.02.2002, Brno] R&D Projects: GA AV ČR IAA4004108; GA AV ČR IAA4004901; GA ČR GA204/00/D049 Keywords : streptavidin * biotin * electrochemical analysis Subject RIV: BO - Biophysics

  7. Antibody-guided three-step therapy for high grade glioma with yttrium-90 biotin

    International Nuclear Information System (INIS)

    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/m2 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). 90Y doses of 2.22-2.96 GBq/m2 were administered; maximum tolerated dose (MTD) was determined at 2.96 GBq/m2. 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 90Y 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/m2, allowing the injection of 90Y-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.)

  8. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Chi-Yuan; Tong, Liang (Columbia)

    2012-06-19

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  9. Elektrochemická studie komplexu streptavidin-biotin a jeho osmiem-modifikovaného analogu

    Czech Academy of Sciences Publication Activity Database

    Billová, Sabina; Masařík, Michal; Brázdová, Marie; Kizek, René; Havran, Luděk; Vacek, Jan; Fojta, Miroslav; Paleček, Emil

    Brno : Masarykova univerzita, 2002. s. 11. [Pracovní setkání fyzikální chemiků a elektrochemiků /3./. 06.02.2002, Brno] R&D Projects: GA AV ČR IAA4004108; GA AV ČR IAA4004901; GA ČR GA204/00/D049 Keywords : streptavidin * biotin * electrochemical detection Subject RIV: BO - Biophysics

  10. Amalgam electrodes covered by thiol monolayers as basis for (strept)avidin-biotin interactions

    Czech Academy of Sciences Publication Activity Database

    Josypčuk, Bohdan; Mareček, Vladimír; Yosypchuk, O.

    Ljubljana : National Institute of Chemistry, 2012. s. 140. ISBN 978-961-6104-20-3. [International Conference on Electroanalysis /14./. 03.06.2012-07.06.2012, Portorož] R&D Projects: GA ČR GAP206/11/1638; GA ČR GAP208/12/1645 Institutional support: RVO:61388955 Keywords : amalgam electrodes * (strept)avidin- biotin interastions Subject RIV: CG - Electrochemistry

  11. Development of Robust and Standardized Cantilever Sensors Based on Biotin/Neutravidin Coupling for Antibody Detection

    Science.gov (United States)

    Zhang, Jiayun; Lang, Hans Peter; Battiston, Felice; Backmann, Natalija; Huber, Francois; Gerber, Christoph

    2013-01-01

    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. PMID:23604028

  12. Electroactivity of avidin and streptavidin. Avidin signals at mercury and carbon electrodes respond to biotin binding

    Czech Academy of Sciences Publication Activity Database

    Havran, Luděk; Billová, Sabina; Paleček, Emil

    2004-01-01

    Roč. 16, 13-14 (2004), s. 1139-1148. ISSN 1040-0397 R&D Projects: GA ČR GA204/03/0566; GA MŠk OC D21.002; GA AV ČR KJB4004302 Institutional research plan: CEZ:AV0Z5004920 Keywords : streptavidin * avidin-biotin interaction * electrochemical methods Subject RIV: BO - Biophysics Impact factor: 2.038, year: 2004

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

    International Nuclear Information System (INIS)

    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)63-/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

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyun Kyu; Chung, Bong Hyun [BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-806 (Korea, Republic of); Gokarna, Anisha [Department of Physics, Chungbuk National University, Gaesin-dong, Heungduk-gu, Cheongju 361-763 (Korea, Republic of); Hulme, John P [Gachon Bio-Nano Institute, Kyungwon University, Seongnam-Si 461-701 (Korea, Republic of); Park, Hyun Gyu [Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of)], E-mail: chungbh@kribb.re.kr, E-mail: hgpark@kaist.ac.kr

    2008-06-11

    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{sub 2}) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH{sub 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{sub 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 {approx}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.

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

    International Nuclear Information System (INIS)

    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-NH2) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH2 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-NH2 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. Studies on radiolabeled streptavidin-biotin and its use as an indirect immunoimaging system

    International Nuclear Information System (INIS)

    The purpose of this research was to study the biotin-streptavidin system coupled to an antigen-antibody system to amplify the sensitivity of tumor detection. Human serum albumin (HSA) was used as an antigen and conjugated to Sepharose 4B beads. The beads-HSA were used as a tumor model. Rabbit-anti human serum albumin antibody (Ab) was used as antibody and derivatized with biotin (AbXB). Streptavidin (STA) radiolabeled with I-125 and In-111 was used to detect the antigenic sites. The three areas of research included chemical studies, in vitro binding studies, and biodistribution studies. The conjugation reactions of biotin to Ab, diethylenetriaminepentaacetic acid (DTPA) to STA, and the radiolabeling of STA conjugates with In-111 were investigated. The in vitro binding assay was performed in two steps. The AbXB was bound first to beads-HSA in excess. The unbound AbXB was removed and a molar excess of beads-HSA/AbXB was then incubated with I-125 or In-111 labeled STA. For the biodistribution studies, AbXB was injected into a tail vein of rats with beads-HSA localized in lungs. The In-111 or I-125 labeled STA was injected 24 and 48 hours after the administration of the AbXB. These biodistribution studies indicated no amplification of the target to normal organ ratios as compared to In-111 labeled antibody

  17. The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

    International Nuclear Information System (INIS)

    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 NaBH4 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ding, S.-J. [Institute of Oral Materials Science, Chung-Shan Medical University, Taichung 402, Taiwan (China); Chang, B.-W. [Department of Healthcare Administration, Hung-Kuang University, Taichung 433, Taiwan (China); Wu, C.-C. [Institute of Biomedical Engineering, National Cheng-Kung University, No. 1, Ta-Hseuh Road, Tainan 701, Taiwan (China); Department of Bio-Industiral Mechatronics Engineering, National Chung Hsing University, Taichung 402, Taiwan (China); Lai, M.-F. [Institute of Biomedical Engineering, National Cheng-Kung University, No. 1, Ta-Hseuh Road, Tainan 701, Taiwan (China); Chang, H.-C. [Institute of Biomedical Engineering, National Cheng-Kung University, No. 1, Ta-Hseuh Road, Tainan 701, Taiwan (China)]. E-mail: hcchang@mail.ncku.edu.tw

    2005-06-10

    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){sub 6} {sup 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 {mu}g/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 k{omega} 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.

  19. Functional definition of BirA suggests a biotin utilization pathway in the zoonotic pathogen Streptococcus suis.

    Science.gov (United States)

    Ye, Huiyan; Cai, Mingzhu; Zhang, Huimin; Li, Zhencui; Wen, Ronghui; Feng, Youjun

    2016-01-01

    Biotin protein ligase is universal in three domains of life. The paradigm version of BPL is the Escherichia coli BirA that is also a repressor for the biotin biosynthesis pathway. Streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. Unlike the scenario in E. coli, S. suis lacks the de novo biotin biosynthesis pathway. In contrast, it retains a bioY, a biotin transporter-encoding gene, indicating an alternative survival strategy for S. suis to scavenge biotin from its inhabiting niche. Here we report functional definition of S. suis birA homologue. The in vivo functions of the birA paralogue with only 23.6% identity to the counterpart of E. coli, was judged by its ability to complement the conditional lethal mutants of E. coli birA. The recombinant BirA protein of S. suis was overexpressed in E. coli, purified to homogeneity and verified with MS. Both cellulose TLC and MALDI-TOFF-MS assays demonstrated that the S. suis BirA protein catalyzed the biotinylation reaction of its acceptor biotin carboxyl carrier protein. EMSA assays confirmed binding of the bioY gene to the S. suis BirA. The data defined the first example of the bifunctional BirA ligase/repressor in Streptococcus. PMID:27217336

  20. Diversity in functional organization of class I and class II biotin protein ligase.

    Directory of Open Access Journals (Sweden)

    Sudha Purushothaman

    Full Text Available The cell envelope of Mycobacterium tuberculosis (M. tuberculosis is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA activates apo-biotin carboxyl carrier protein (BCCP by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz, an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL. MtBPL specifically biotinylates homologous BCCP domain, MtBCCP(87, but not EcBCCP(87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the self-biotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over self-biotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-5' AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

  1. 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

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

    International Nuclear Information System (INIS)

    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) -0014CCH2COCOO- + 1.3S biotin pyruvate + 1.3S biotin-COO- catalyzed by the 5S subunit (2) 14CH3CH(14COO-)COSCoA + 1.3S biotin CH3CH2COSCoA + 1.3S biotin-14COO-, 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

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

    OpenAIRE

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

    2009-01-01

    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 β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal dom...

  4. Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-activated K+ Channel Expressed in a Human Cell Line

    OpenAIRE

    Bingham, Jon-Paul; Bian, Shumin; Tan, Zhi-Yong; Takacs, Zoltan; Moczydlowski, Edward

    2006-01-01

    Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca2+-activated K+ channels with high affinity and specificity. Automated solid phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-l-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biot...

  5. 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. PMID:27181349

  6. 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. PMID:26132538

  7. Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

    Science.gov (United States)

    Shaw; Lehner; Fuhrmann; Kulla; Brass; Birch; Tinschert; Venetz; Venetz; Sanchez; Tonella; Hochstrasser

    1999-06-01

    The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions. PMID:10455485

  8. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    Science.gov (United States)

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-06-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  9. Direct effects of radiation on the avidin-biotin system. Absence of energy transfer

    International Nuclear Information System (INIS)

    Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation. Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed. The loss of function of each compound was analyzed according to target theory. Target analysis revealed that the radiation-sensitive mass associated with the enzymatic activity and that associated with the fluorescence were unchanged by irradiation in the strongly coupled state. Therefore the noncovalent bonds between biotin and avidin do not permit the transfer of radiation-deposited energy in amounts sufficient to destroy the activity of apposing molecule

  10. Two-dimensional gel electrophoretic detection of protein carbonyls derivatized with biotin-hydrazide.

    Science.gov (United States)

    Wu, Jinzi; Luo, Xiaoting; Jing, Siqun; Yan, Liang-Jun

    2016-04-15

    Protein carbonyls are protein oxidation products that are often used to measure the magnitude of protein oxidative damage induced by reactive oxygen or reactive nitrogen species. Protein carbonyls have been found to be elevated during aging and in age-related diseases such as stroke, diabetes, and neurodegenerative diseases. In the present article, we provide detailed protocols for detection of mitochondrial protein carbonyls labeled with biotin-hydrazide followed by 2-dimensional isoelectric focusing (IEF)/SDS-PAGE and Western blotting probed with horse-radish peroxidase-conjugated streptavidin. The presented procedures can also be modified for detection of carbonylation of non-mitochondrial proteins. PMID:26590475

  11. Experimental study of biotin-avidin pretargeting technique for anti-CEA McAb radioimmunoimaging

    International Nuclear Information System (INIS)

    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 131I (131I-SA) and DTPA-biotin with 111In(111In-DTPA-B). Experimental human colonic tumor bearing nude mice were used. Two step method: B-McAb was preinjected, followed by 131I 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 111In-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

  12. Development of Therapeutic Radiopharmaceuticals Based on 90Y Biotin. Chapter 7

    International Nuclear Information System (INIS)

    The preparation of a biotin derivative labelled with 90Y to be employed as a breast cancer therapeutic radiopharmaceutical in the application of the new IART approach is described in this chapter. The effects of pH on the labelling yield and in vitro affinity for avidin of the resulting radiolabelled conjugate are evaluated. Radiolabelling was performed using a manual and an automated procedure, and radiation exposure was measured for each operational condition. Microbiological tests were conducted on each final batch after decay of activity. Results obtained from a first clinical study are also described. (author)

  13. Y2O3: Eu,Zn nanocrystals as a fluorescent probe for the detection of biotin

    International Nuclear Information System (INIS)

    We report on the application of nanocrystals (NCs) of the type Y2O3: 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 103 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)

  14. Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique

    International Nuclear Information System (INIS)

    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 125I; 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-125I-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

  15. Enhanced hypoglycemic effect of biotin-modified liposomes loading insulin: effect of formulation variables, intracellular trafficking, and cytotoxicity

    Science.gov (United States)

    Zhang, Xingwang; Qi, Jianping; Lu, Yi; Hu, Xiongwei; He, Wei; Wu, Wei

    2014-04-01

    Peroral protein/peptide delivery has been one of the most challenging, but encouraging topics in pharmaceutics. This article was intended to explore the potential of biotin-modified liposomes (BLPs) as oral insulin delivery carriers. By incorporating biotin-DSPE into the lipid bilayer, we prepared BLPs using reverse evaporation/sonication method. We investigated hypoglycemic effects in normal rats after oral administration of BLPs, and the possible absorption mechanism by a series of in vitro tests. The relative pharmacological bioavailability of BLPs was up to 11.04% that was as much as 5.28 folds of conventional liposomes (CLPs). The results showed that the enhanced oral absorption of insulin mainly attributed to biotin ligand-mediated endocytosis. The results provided proof of BLPs as effective carriers for oral insulin delivery.

  16. Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin.

    Science.gov (United States)

    Laitinen, Olli H; Hytönen, Vesa P; Ahlroth, Mervi K; Pentikäinen, Olli T; Gallagher, Ciara; Nordlund, Henri R; Ovod, Vladimir; Marttila, Ari T; Porkka, Eevaleena; Heino, Sanna; Johnson, Mark S; Airenne, Kari J; Kulomaa, Markku S

    2002-01-01

    Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications. PMID:11964162

  17. Highly Sensitive Two-Dimensional Paper Network Incorporating Biotin-Streptavidin for the Detection of Malaria.

    Science.gov (United States)

    Grant, Benjamin D; Smith, Chelsey A; Karvonen, Kristine; Richards-Kortum, Rebecca

    2016-03-01

    Recently, two-dimensional paper networks have been developed to enable multistep assays to be performed in a lateral flow format. These devices have been used to perform simple enzyme linked immunoassays on paper. However, these devices have yet to incorporate more complex immunoassays, including the use of streptavidin-biotin detection strategies. Here we present a modified two-dimensional paper network capable of consecutively delivering six reagents. The device requires only a single user step and delivers (i) the sample, (ii) the biotinylated detection antibody, (iii) streptavidin horseradish peroxidase, (iv) a wash buffer, (v) a colorimetric substrate, and (vi) a final wash buffer. To demonstrate the utility of this approach we designed an assay to detect the malaria protein Pf HRP2. Using this platform, we were able to achieve a limit-of-detection equivalent to that of a traditional 96-well plate sandwich ELISA. In addition to improvements in the limit-of-detection, the inclusion of streptavidin-biotin simplifies the development of similar tests for other targets. PMID:26824718

  18. Construction of pegylated multilayer architectures via (strept)avidin/biotin interactions

    International Nuclear Information System (INIS)

    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

  19. Modification of monoclonal antibodies with enzymes, biotin, and fluorochromes and their applications

    International Nuclear Information System (INIS)

    A number of reagents have been used for enzyme-labeling of antibodies during the past two decades. However most of them suffer from serious disadvantages, and only a few reagents are in current use, including glutaraldehyde, periodate, maleimides, and pyridyldisulfide compounds. Antibodies and enzymes can be indirectly and noncovalently cross-linked using the strong binding ability of biotin and avidin instead of the direct and covalent cross-link as just described. A feature of this method is amplification of signals, which can be achieved by introduction of many biotin residues into antibody molecules and subsequent binding of avidin molecules linked to enzyme molecules. However the nonspecific binding or the background is also amplified. Enzymes as labels of antibodies can be replaced by fluorochromes. Labeling with fluorochromes is simple and easy, and fluorochrome-labeled antibodies have been widely used to demonstrate antigens in tissue sections. However immunofluorescence of tissue sections decreases rapidly on exposure to light, and sensitive immunoassays have been developed using enzymes rather than fluorochromes

  20. 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.

  1. Use of avidin-biotin-peroxidase complex for measurement of UV lesions in human DNA by microELISA

    International Nuclear Information System (INIS)

    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/m2 UVC or a suberythermal dose of UVB light could be detected. (Auth.)

  2. Remarkable Diversity in the Enzymes Catalyzing the Last Step in Synthesis of the Pimelate Moiety of Biotin

    OpenAIRE

    Shapiro, Madelyn M.; Chakravartty, Vandana; Cronan, John E.

    2012-01-01

    Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG prot...

  3. In vivo evaluation of an anti-PSMA antibody conjugated with varying numbers of biotin molecules in a pretargeting protocol

    International Nuclear Information System (INIS)

    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 [125I]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 [131I]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

  4. 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...

  5. Dermatose responsiva à biotina em cão Dermatosis responsive to Biotin in a dog

    Directory of Open Access Journals (Sweden)

    Sandra Prudente Nogueira

    2010-03-01

    Full Text Available Os transtornos da pele e dos pelos são parte importante na prática clínica de pequenos animais. Numerosos fatores nutricionais afetam a homeostase, a qualidade e o aspecto da pelagem. As vitaminas do complexo B incluem compostos hidrossolúveis necessários como coenzimas em diversas funções celulares envolvidas no metabolismo energético e na síntese tecidual. A biotina, em especial, é necessária nas reações de carboxilação, participando da síntese de ácidos graxos, aminoácidos e purinas pelo tecido epitelial. Uma cadela com quadro de cistite recorrente e tumor venéreo transmissível foi tratada com antibioticoterapia prolongada e quimioterapia. Após alguns meses de tratamento, foram observadas lesões no plano nasal e nos coxins plantar e palmar, caracterizadas por hiperceratose, espessamento, fissuras, sangramento e inflamação. O paciente recebeu suplementação de 15mg de biotina por via oral (equivalente a 1,4mg kg-1 de peso corporal, uma vez por dia, durante 60 dias, havendo importante regressão das lesões. Sugere-se que, sob antibioticoterapia e doença, a síntese intestinal de biotina possa não ter sido suficiente, sendo necessária sua suplementação.Skin and hair diseases are an important part in small animal's clinical practice. Many nutritional factors can affect the quality and the aspect of the coat. B complex vitamins are water-soluble compounds used as coenzymes in several cellular functions that are involved in energy metabolism and tissue synthesis. Biotin, in particular, is necessary for carboxylation reactions, fatty acids synthesis, and incorporation of essential amino acids and purines in the epithelial tissue. A female canine with recurrent cystitis and sticker tumor was treated chemotherapy and prolonged antibiotic therapy. After a few months of medications, lesions were observed in nasal plan and palmar and plantar pads, characterized by hyperkeratosis, skin thickness, bleeding fissures, and

  6. 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…

  7. Labelling of a biotin-derivative by 99mTc, its purification, and binding-capacity to avidin

    International Nuclear Information System (INIS)

    The paper summarizes the procedure of 99mTc-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.)

  8. 177Lu-DTPA-BIS-BIOTIN Binding of Octreotide-dextran-avidinated PANC-1 Cell Lines in Vitro

    International Nuclear Information System (INIS)

    Tyr3-octreotide, dextran-40 and avidin were used to prepare octreotide-dextran-avidin (TOC-Dx40-Av). DTPA-BIS-BIOTIN was labelled with 177Lu. The in vitro somatostatin receptor binding study was carried out by pretargeted method using TOC-Dx40-Av and 177Lu-DTPA-BIS-BIOTIN. The 24 well cell culture plates were prepared with PANC-1 cell monolayer and then incubated with TOC-Dx40-Av. After two washed with PBS, the cells were incubated with different concentration of 177Lu-DTPA-BIS-BIOTIN (48.8 ∼ 391 pmol). Cells uptake was evaluated with γ counter. The results showed that the chemical purity of TOC-Dx40-Av was over 99%. The results also showed that TOC-Dx40-Av remained high receptor binding affinity to somatostatin receptor which indicated that TOC- Dx40-Av could bind to 177Lu-DTPA-BIS-BIOTIN with the molar ratio of 1 : 1 on the cell surface. (authors)

  9. Quantum dot-fluorescence in situ hybridisation for Ectromelia virus detection based on biotin-streptavidin interactions.

    Science.gov (United States)

    Wang, Ting; Zheng, Zhenhua; Zhang, Xian-En; Wang, Hanzhong

    2016-09-01

    Ectromelia virus (ECTV) is an pathogen that can lead to a lethal, acute toxic disease known as mousepox in mice. Prevention and control of ECTV infection requires the establishment of a rapid and sensitive diagnostic system for detecting the virus. In the present study, we developed a method of quantum-dot-fluorescence based in situ hybridisation for detecting ECTV genome DNA. Using biotin-dUTP to replace dTTP, biotin was incorporated into a DNA probe during polymerase chain reaction. High sensitivity and specificity of ECTV DNA detection were displayed by fluorescent quantum dots based on biotin-streptavidin interactions. ECTV DNA was then detected by streptavidin-conjugated quantum dots that bound the biotin-labelled probe. Results indicated that the established method can visualise ECTV genomic DNA in both infected cells and mouse tissues. To our knowledge, this is the first study reporting quantum-dot-fluorescence based in situ hybridisation for the detection of viral nucleic acids, providing a reference for the identification and detection of other viruses. PMID:27343592

  10. 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.

  11. False Immunohistochemical Results for Herpesviridae and Other Clusters of Differentiation Due To Biotin Intranuclear Inclusions in the Gestational Endometrium

    Directory of Open Access Journals (Sweden)

    Francesco Rivasi

    2014-02-01

    Conclusions. Immunohistochemical investigations of the gestational endometrium (particularly in pregnancies near to term may yield false results for several herpes viruses, as well as for other immunohistochemical reactions obtained using the ABC method without prior biotin inactivation. [J Interdiscipl Histopathol 2014; 2(1.000: 32-37

  12. Evaluation of a new biotin-DOTA conjugate for pretargeted antibody-guided radioimmunotherapy (PAGRIT registered)

    International Nuclear Information System (INIS)

    A novel biotin-DOTA conjugate (r-BHD: reduced biotinamidohexylamine-DOTA) was investigated in order to provide an efficient pretargeted antibody-guided radioimmunotherapy (PAGRIT registered) application. Preclinical and clinical results are described. 90Y and 177Lu were used to label r-BHD. The effect of pH and a wide range of specific activities were studied. Radiolabelled r-BHD was tested for affinity towards avidin and for stability in saline or in human serum with and without ascorbic acid. Pharmacokinetic data were collected and organ biodistribution evaluated in a tumour-bearing pretargeted animal model. A pilot study was performed in a metastatic melanoma patient and dosimetry was estimated. High radiochemical purity (>99%) was routinely achieved with 90Y or 177Lu in sodium acetate buffer (1.0 M, pH 5.0) at a specific activity of 2.6 MBq/nmol. Both 90Y- and 177Lu-r-BHD were also prepared at higher specific activities. Radiolabelled r-BHD was stable up to 96 h in human serum and saline with the addition of ascorbic acid. The structural modifications proposed for the r-BHD stabilised it against enzymatic degradation while retaining high binding affinity for avidin. Renal clearance appeared to be the main route of excretion in animals, and high tumour uptake was observed in the pretargeted animals. The patient study showed a total body clearance of ∝85% in 24 h, with a kidney absorbed dose of 1.5 mGy/MBq. Tumour uptake was rapid and the calculated dose to a 10-mm tumour lesion was ∝12 mGy/MBq. These results indicate that the new biotin-DOTA conjugate may be a suitable candidate for pretargeting trials. (orig.)

  13. 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

  14. Pharmacokinetics and biodistribution of [111In]-avidin and [99mTc]-biotin-liposomes injected in the pleural space for the targeting of mediastinal nodes

    International Nuclear Information System (INIS)

    Pharmacokinetics and mediastinal node uptake of [111In]-avidin and [99mTc]-biotin-liposomes following either intrapleural (pleural) or intraperitoneal (ip) injection were determined using scintigraphic imaging. Biodistribution results of [111In]-avidin at 44 h showed 3.3% uptake in mediastinal nodes by pleural injection vs 1.3% with ip injection. Mediastinal node accumulation with [99mTc]-biotin-liposomes was not different between injections (0.6% ip vs 0.5% pleural). This study demonstrates the potential of the pleural route as a technique for mediastinal node targeting using the avidin/biotin-liposome system

  15. Detection of viral genomes in the liver by in situ hybridization using 35S-, bromodeoxyuridine-, and biotin-labeled probes

    International Nuclear Information System (INIS)

    Methods employing 35S-, biotin-, and bromodeoxyuridine (BrdUrd)-labeled DNA probes were compared for the detection of hepatitis B virus (HBV) and cytomegalovirus (CMV) in the liver. The results demonstrate that: 1) HBV can be detected reliably only by the use of radiolabeled probes, whereas methods employing nonradioactive probes obviously are not sensitive enough for this virus. The use of 35S-labeled probes shortens the exposure times considerably in comparison to tritiated probes. 2) Biotin-labeled probes are of limited value for in situ hybridization on liver tissues because the presence of endogenous avidin-binding activity often leads to false positive results. 3) Brd-Urd-labeled probes are a useful alternative to biotinylated probes for the detection of CMV. In comparison with biotinylated probes, BrdUrd-labeled probes produce a specific signal of similar staining intensity in the absence of background staining in the liver

  16. Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin-Avidin Interactions.

    Science.gov (United States)

    Singh, Serena; Kluger, Ronald

    2016-05-24

    Hypertension resulting from vasoconstriction in clinical trials of cross-linked tetrameric (α2β2) human hemoglobins implicates the extravasation of the hemoglobins into endothelia where they scavenge nitric oxide (NO), which is the signal for relaxation of the surrounding smooth muscle. Thus, we sought an efficient route to create a larger species that avoids extravasation while maintaining the oxygenation function of hemoglobin. Selectively formed cysteine-linked biotin conjugates of hemoglobin undergo self-assembly with avidin into a stable triple protein, hemoglobin-avidin-hemoglobin (HbAvHb), which binds and releases oxygen with moderate affinity and cooperativity. The triple protein is likely to be stabilized by interactions of each constituent hemoglobin (pI 6.9) with the oppositely charged avidin (pI 10.5) as well as the strong association of the biotin moieties on hemoglobin with avidin. PMID:27126305

  17. Biotin-decorated silica coated PbS nanocrystals emitting in the second biological near infrared window for bioimaging

    Science.gov (United States)

    Corricelli, M.; Depalo, N.; di Carlo, E.; Fanizza, E.; Laquintana, V.; Denora, N.; Agostiano, A.; Striccoli, M.; Curri, M. L.

    2014-06-01

    Nanoparticles (NPs) emitting in the second biological near infrared (NIR) window of the electromagnetic spectrum have been successfully synthesized by growing a silica shell on the hydrophobic surface of OLEA/TOP PbS nanocrystals (NCs), by means of a reverse microemulsion approach, and subsequently decorated with biotin molecules. The fabrication of very uniform and monodisperse NPs, formed of SiO2 shell coated single core PbS NCs, has been demonstrated by means of a set of complementary optical and structural techniques (Vis-NIR absorption and photoluminescence spectroscopy, transmission electron microscopy) that have highlighted how experimental parameters, such as PbS NC and silica precursor concentration, are crucial to direct the morphology and optical properties of silica coated PbS NPs. Subsequently, the silica surface of the core-shell NPs has been grafted with amino groups, in order to achieve covalent binding of biotin to NIR emitting silica coated NPs. Finally the successful reaction with a green-fluorescent labelled streptavidin has verified the molecular recognition response of the biotin molecules decorating the PbS@SiO2 NP surface. Dynamic light scattering (DLS) and ζ-potential techniques have been used to monitor the hydrodynamic diameter and colloidal stability of both PbS@SiO2 and biotin decorated NPs, showing their high colloidal stability in physiological media, as needed for biomedical applications. Remarkably the obtained biotinylated PbS@SiO2 NPs have been found to retain emission properties in the `second optical window' of the NIR region of the electromagnetic spectrum, thus representing attractive receptor-targeted NIR fluorescent probes for in vivo tumour imaging.Nanoparticles (NPs) emitting in the second biological near infrared (NIR) window of the electromagnetic spectrum have been successfully synthesized by growing a silica shell on the hydrophobic surface of OLEA/TOP PbS nanocrystals (NCs), by means of a reverse microemulsion

  18. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylamino)toluic Acid] (PATA) for Efficient Biotinylation of Peptides and Oligonucleotides

    OpenAIRE

    Martina Jezowska; Joanna Romanowska; Burcu Bestas; Ulf Tedebark; Malgorzata Honcharenko

    2012-01-01

    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 chemist...

  19. Quantitative Measurement of Urinary Excretion of 3-Hydroxyisovaleryl Carnitine by LC–MS/MS as an Indicator of Biotin Status in Humans

    OpenAIRE

    Horvath, Thomas D.; Stratton, Shawna L; Bogusiewicz, Anna; Owen, Suzanne N.; Mock, Donald M; Moran, Jeffery H.

    2010-01-01

    Abnormally increased urinary excretion of 3-hydroxyisovaleryl carnitine (3HIA-carnitine) results from impairment in leucine catabolism caused by reduced activity of the biotin-dependent enzyme 3-methylcrotonyl-CoA carboxylase. Accordingly, urinary 3HIA-carnitine might reflect biotin status. Here, we describe an LC–MS/MS method for accurately quantitating the urinary concentration of 3HIA-carnitine at concentrations that are typical for excretion rates that are normal or only modestly increase...

  20. Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns 1,2

    OpenAIRE

    Smith, Erin M; Hoi, Jia Tse; Eissenberg, Joel C.; Shoemaker, James D.; Neckameyer, Wendi S; Ilvarsonn, Anne M.; Harshman, Lawrence G.; Schlegel, Vicki L.; Zempleni, Janos

    2007-01-01

    Caloric restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e., biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient medium increases s...

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

    Directory of Open Access Journals (Sweden)

    Liu L

    2014-05-01

    Full Text Available Lin Liu,1,2 Yun Xing,1 Hui Zhang,1 Ruili Liu,1 Huijing Liu,1 Ning Xia1,21College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People’s Republic of China; 2College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People’s Republic of ChinaAbstract: 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.Keywords: electrochemical biosensor, boronic acid, signal amplification, alkaline phosphatase

  2. A synthetic host-guest system achieves avidin-biotin affinity by overcoming enthalpy–entropy compensation

    OpenAIRE

    Rekharsky, Mikhail V.; Mori, Tadashi; Yang, Cheng; Ko, Young Ho; Selvapalam, N.; Kim, Hyunuk; Sobransingh, David; Kaifer, Angel E.; Liu, Simin; Isaacs, Lyle; Chen, Wei; Moghaddam, Sarvin; Gilson, Michael K.; Kim, Kimoon; Inoue, Yoshihisa

    2007-01-01

    The molecular host cucurbit[7]uril forms an extremely stable inclusion complex with the dicationic ferrocene derivative bis(trimethylammoniomethyl)ferrocene in aqueous solution. The equilibrium association constant for this host-guest pair is 3 × 1015 M−1 (Kd = 3 × 10−16 M), equivalent to that exhibited by the avidin–biotin pair. Although purely synthetic systems with larger association constants have been reported, the present one is unique because it does not rely on polyvalency. Instead, i...

  3. Detection antigen virus den on monocyts by streptavidin biotin test as early diagnostic for dengue fever hemorrhagic

    Directory of Open Access Journals (Sweden)

    Y NINING SRI WURYANINGSIH

    2007-07-01

    Full Text Available Dengue virus infection is the main cause of morbidity and mortality in the tropical and sub-tropical countries of the world. Clinically it may manifest as asymtomastic,undifferentiated fever,dengue ever,dengue haemorrhagic fever and dengue shock syndrome cases. The mechanism underlying the disease with severe complication is not clear yet,however it has been previosus reported that primary and secondary infections of dengue virus play an important role in the patogenesis of this diseases. Early diagnosis of dengue virus infection has a great contribution for appropriate management of the disease, especialy for the prognosis of the patient. Laboratory investigations for such cases will be methods on serological investigation as well as virus isolation and identification.of dengue virus infection could be made by detection of specific virus ,viral antigen,genomic sequence and or detection of antibodies. These methods are sensitive and precise for detecting dengue virus infection,but there need special equipment,costly and detection of IgM and IgG often positive or negative false the dengue virus in the blood stream There for, this study was performed in order to develop a method to detect dengue virus antigen on the monocytes using Streptavidin biotin technique. The result of Streptavidin biotin study demonstrated that 32 sera from patient suspected with DHF 78,1% were positive DHF,and 21,9% were negative DHF. These results are consistent with the result from WHO criteria as standard .The Chi Square analysis showed that the presentage of sensitivity and specificity of Streptavidin biotin methode were 88% and 87,7% respectively. In conclusions, immunocytochemistry method using streptavidin biotin technique could be used as a method to detect antigen dengue virus on monocytes in the serum patient suspected with DHF. This technique has high sensitivity and specivicity and consistent with the clinical WHO criteria for DHF.

  4. Avidin-biotin-based approach to forming heterotypic cell clusters and cell sheets on a gas-permeable membrane

    International Nuclear Information System (INIS)

    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. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    Directory of Open Access Journals (Sweden)

    Shuji Ueda

    Full Text Available Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.

  7. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    Science.gov (United States)

    Ueda, Shuji; Blee, Alexandra M; Macway, Katherine G; Renner, Derrick J; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  8. Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Magwamba, Clement Chedza; Rukseree, Kamolchanok; Palittapongarnpim, Prasit

    2016-05-01

    The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 μM, IC50 = 267.4 μM; Ki = 0.84 μM, IC50 = 9.28 μM; and Ki = 0.592 μM, IC50 = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS. PMID:27156617

  9. Influences of dietary biotin and avidin on growth, survival, deficiency syndrome and hepatic gene expression of juvenile Nile tilapia Oreochromis niloticus.

    Science.gov (United States)

    Sarker, Pallab Kumer; Yossa, Rodrigue; Karanth, Santhosh; Ekker, Marc; Vandenberg, Grant W

    2012-08-01

    This study was undertaken to assess the interactive effects of dietary biotin and avidin on growth, feed conversion, survival and deficiency syndrome of tilapia and to determine the influence of dietary biotin deficiency on the expression of key genes related to biotin metabolism in tilapia. Six iso-nitrogenous and iso-energetic diets based on a common purified basal diet (vitamin-free casein as the protein source) were prepared for this study. The six dietary groups were 0 g avidin with 0 mg biotin (A0B0), 0 g avidin with 0.06 mg biotin/kg diet (A0B1), four avidin-supplemented diets incorporating at a incremental concentrations 0.25, 0.5, 1.0 and 2.0 g/kg diet with 0.06 mg biotin/kg diet (A15B1, A30B1, A60B1 and A120B1). Fish were hand-fed three times a day to apparent satiation for 12 weeks. Each diet was fed to three replicate groups of fish. Fish were kept in glass aquaria in a recirculating aquaculture system under standardized environmental conditions. Growth was significantly higher in fish that received the biotin-supplemented diet (A0B1), compared to diets lacking biotin or supplemented with avidin. Tilapia fed higher concentration of avidin-supplemented diets (A60B1 and A120B1) showed significant growth depression and displayed severe deficiency syndromes such as lethargy, anorexia, circular swimming and convulsions, which ultimately lead to death. There was a strong proportional linear relationship between the avidin content of the diet and feed conversion ratio, FCR (y = 0.43x + 0.135; r = 0.960; P < 0.001) and strong inverse relationship with protein efficiency ratio, PER (y = -0.309x + 2.195; r = 0.961; P < 0.0001). Elevated levels of biotinidase, pyruvate carboxylase, propionyl-CoA carboxylase-A and propionyl-CoA carboxylase-B transcripts were noted in fish fed all graded level of avidin-supplemented diets. A broken-line analysis indicated that feeding tilapia a diet with 44.5 times more avidin than the dietary biotin

  10. Antibody-Mediated Targeting of siRNA Via the Human Insulin Receptor Using Avidin-Biotin Technology

    Science.gov (United States)

    Xia, Chun-Fang; Boado, Ruben J.; Pardridge, William M.

    2013-01-01

    Delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (MAb) and avidin-biotin technology. In the present studies, the luciferase gene is transiently expressed in human 293 epithelial cells. The siRNA delivery system is comprised of the siRNA, mono-biotinylated on the 3′-terminus of the sense strand, and a conjugate of streptavidin (SA) and a MAb to the human insulin receptor (HIR). Exposure of cells to 3′-biotinyl-siRNA bound to the HIRMAb/SA conjugate, but not to unconjugated SA, avidin, or the HIRMAb, causes a >90% reduction in luciferase gene expression. The receptor-targeted siRNA effect is maximal at 48 hours after delivery of the siRNA to the cells, and the effect is lost by 7 days after a single application of the targeted siRNA in culture. The KI of the receptor-targeted siRNA inhibition of gene expresssion is 30.5 ± 11.7 nM, and significant inhibition is observed with siRNA concentrations as low as 3 nM. In conclusion, the combination of a receptor-specific targeting ligand, such as the HIRMAb, and avidin-biotin technology, allows for high affinity capture of the mono-biotinylated siRNA by the targeting MAb. The siRNA is effectively delivered to the cytosol of cells and knockdown of gene expression with the HIRMAb/SA delivery system is comparable to RNA interference effects obtained with cationic polyplexes. Whereas the use of cationic polyplexes in vivo is problematic, the bond between the targeting MAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA. PMID:19093871

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

    International Nuclear Information System (INIS)

    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

  12. 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)

    The biotinylated monoclonal antibody (MoAb) ior cea1 and its F(ab')2 fragments were labeled with Re-188 by combination of avidin-biotin strategy. 188Re-MoAb, 188Re-MoAb-biotin, 188Re-F(ab')2, and 188Re-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 188Re-labeled immunoconjugates. When avidin was injected as a chase after injection of 188Re-MoAb-biotin or 188Re-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 188Re-labeled biotinylated MoAb ior cea1 and its F(ab')2 fragments prepared by this method are stable complexes in vivo

  13. 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. PMID:26946492

  14. Immunoradiometric assay of human proinsulin and partially processed proinsulin with use of monoclonal antibody and streptavidin-biotin labeling.

    OpenAIRE

    Kim, J Q; Cho, H. I.; Kim, S. I.; Lee, H. K.; Hales, C. N.

    1989-01-01

    The sensitive and specific immunoradiometric assay is described for human proinsulin and its intermediate peptides (65-66 split and 32-33 split proinsulin). We developed a monoclonal antibody-based two-site immunoradiometric assay with use of streptavidin-biotin labeling. The detection limits of the assays lie in the range of 0.5-2.0 pM. In the proinsulin assay proinsulin cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. In the assay of 65-66 split ...

  15. 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.

  16. 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.

  17. 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.

  18. One-electron redox reactions of water-soluble vitamins. IV. Thiamin (vitamin B1), biotin, and pantothenic acid

    International Nuclear Information System (INIS)

    The technique of pulse radiolysis and kinetic absorption spectrophotometry was used to study the one-electron reduction of thiamin, thiazole, 4-aminopyrimidine, biotin, and pantothenic acid in aqueous solution. The acetone ketyl radical and e/sub aq/- were used as the one-electron reducing agents. The reaction rate constants of e/sub aq/- and (CH3)2COH with these compounds were determined at different pH values, taking into account the dissociation constants of the substrates. The transient optical absorption spectra of the intermediates produced, their extinction coefficients, decay kinetics, and ionization constants were determined. One-electron reduction of the thiazolium ring of thiamin is suggested, based on the formation of dihydrothiamin as a final product. Other assignments for these radicals are suggested and discussed. The reaction of OH radicals with biotin and pantothenic acid leads, primarily, to H atom abstraction at various sites in the molecule. The formation and ionization of the -C(OH)CONH- radical from pantothenic acid, pK/sub a/ = 6.0 +- 0.3, is proposed

  19. Use of 99mTc-MAG3-biocytin in Avidin/Biotin based Three Step Targeting

    International Nuclear Information System (INIS)

    A general point of concern in clinical radioimmunoscintigraphy(RIS) and radioimmunotherapy(RIT) studies is the long residence time of radioimmunoconjugates in the bloodstream, resulting in limited sensitivity and specificity in RIS and in myelotoxicity in RIT. A solution to this problem might be the use of so-called pretargeting strategies. The concept of pretargeting is based on the separation, in time, of antibody localization and radionuclide targeting. Several ligand-receptor systems have been evaluated of which the biotin-(strept)avidin system is the most widely studied. The attractiveness of this system lies in the very high affinity of avidin and streptavidin for biotin. The 3E8 antibody was produced from humanized anti- TAG-72 monoclonal antibody (AKA) by amino acid change in 95-99 residues of heavy chain complementary determinant regions (HCDRs) 3 using phage displayed library technology.In this study, we are investigating the feasibility of pretargeting strategy using biontinylated 3E8, avidin and 99mTc-MAG3-biocytin in LS174T tumor bearing mice model. We prepare biotinylated 3E8 antibody conjugates, perform radiolabeling and in vitro targeting of 99mTc-MAG3-biocytin and evaluate tumor pretargeting in tumor bearing nude mice model

  20. Active removal of radioactivity in the blood circulation using biotin-bearing liposomes and avidin for rapid tumour imaging

    International Nuclear Information System (INIS)

    In order to shorten the delay between the administration of tumour-imaging agents and obtainment of scintigrams, rapid delivery of radionuclide to tumours followed by rapid clearance from the blood is required. We used liposomes with biotin bound on their surface (B-liposomes) as carriers for rapid delivery. For rapid blood clearance, we employed avidin in the expectation that the strong affinity between biotin and avidin would result in the aggregation of B-liposomes in the blood circulation, and that these aggregates would be taken up rapidly by the reticuloendothelial system, resulting in the rapid elimination of liposomes and the radionuclide encapsulated in them. When B-liposomes encapsulating gallium-67 deferoxamine were intravenously administered to mice bearing sarcoma 180, large amounts of 67Ga were delivered to tumours by 4 h after injection, though the 67Ga blood level remained high. On the other hand, administration of avidin 4 h after administration of the B-liposomes dramatically reduced the blood level so that it was only 5% of that in the non-treated group 1 h later. As a result, the tumour-to-blood ratio reached nearly 14 and 5 h after radionuclide administration, suggesting that rapid tumour-imaging will be feasible by means of this method. (orig.)

  1. Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum and S. gallinarum

    International Nuclear Information System (INIS)

    A novel electrochemical enzyme immunosensor based on the electrodeposited gold nanoparticles and the multistage amplification of streptavidin-biotin affinity system for detection of Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) was investigated in this study. The electrochemical characteristics of the stepwise modified electrodes were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), whereas the determinations of the targets of S. pullorum and S. gallinarum were carried out by CV. As shown in the results of this study, the electron transfer was promoted by the electrodeposition of gold nanoparticles, thus the communication of electrons was enhanced and the conductivity of the electrode was strengthened too. Moreover, the number of the conjugated bio-molecules was elevated greatly by the electrodeposited gold nanoparticles and the streptavidin-biotin, which contributed to the integration of the following modifications and amplification of the current response signal. Under the optimized working conditions, the sensor showed a good performance with a linear response range from 102 CFU/ml to109 CFU/ml, the detection limit for S. pullorum and S. gallinarum determination was 1.95 × 102 CFU/ml (S/N = 3). The proposed enzyme immunosensor with high sensitivity, good specificity, acceptable accuracy and reproducibility, and low detection limit characteristics could be a promising analytical tool in detection of S. pullorum and S. gallinarum in practical samples and a model for the development of immunosensor of other bacterium of interests

  2. 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; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

    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...

  3. Biotin Supplementation Decreases the Expression of the SERCA3 Gene (ATP2A3) in Jurkat Cells, thus Triggering Unfolded Protein Response*

    OpenAIRE

    Griffin, Jacob B.; Rodriguez-Melendez, Rocio; Dode, Leonard; Wuytack, Frank; Zempleni, Janos

    2005-01-01

    Protein folding in the endoplasmic reticulum (ER) depends on Ca2+; uptake of Ca2+ into the ER is mediated by SERCA3. The 5′-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells w...

  4. Investigation of McAb labelling with 99mTc by avidin-biotin system and its biodistribution in mice

    International Nuclear Information System (INIS)

    Anti-melanoma McAb Ng76 was labelled with 99mTc by avidin-biotin system. The labelling conditions and methods, and biodistribution of McAb Ng76 in tumor bearing nude mice had been studied. The results showed that the labelling rate was the highest in pH8 and optimal quantity of SnCl2; T/NT and ID%/g of the two-step method B were higher than the two-step method A. Thereby, labelling McAb with 99mTc by avidin-biotin system could be used in RII

  5. Optimization of biotin labeling of antibodies using mouse IgG and goat anti-mouse IgG-conjugated fluorescent beads and their application as capture probes on protein chip.

    Science.gov (United States)

    Lee, Jin Hyung; Choi, Hong Kyung; Chang, Jeong Ho

    2010-10-31

    This study shows the optimization of biotin labeling to antibodies using mouse IgG. Several parameters of the biotin labeling, including the molar ratio of biotin to antibody, the coupling time and the dialysis time, were studied to optimum conditions. The biotin-tagged mouse IgGs were immobilized on avidin-coated PMMA (Polymethyl Methacrylate) plates via a biotin-avidin linkage. The immobilization of the IgG to the chip was quantified using goat anti-mouse IgG bound fluorescent beads. It was found that the binding of the fluorescent beads saturated when a 10-fold or higher molar ratio of biotin to antibody was used. In biotin coupling time tests, sixty minutes was sufficient for the capture probes to bind to the surface. However, the results from the dialysis experiments showed no difference, indicating that 2 hours was sufficient to remove any unbound biotin. Finally, to prove the universality of this protocol using mouse antibodies, the optimum conditions were successfully applied in sandwich immunoassays designed to detect troponin I (TnI) and N-terminal probrain natriuretic peptide (NT-proBNP). PMID:20804762

  6. 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)

    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

  7. DEMONSTRATION OF H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA VIRAL ANTIGEN IN FORMALIN-FIXED AVIAN TISSUE SPECIMENS BY AN AVIDIN-BIOTIN IMMUNOHISTOCHEMISTRY PROCEDURE

    Science.gov (United States)

    The avidin-biotin immunohistochemistry (IHC) procedure has been used successfully to identify a variety of bacterial, viral and cellular antigens in formalin-fixed tissues. The procedure is rapid, reproducible, and specific making it an extremely useful method for screening diagnostic specimens. T...

  8. 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

  9. Development of a formulation for the preparation of sup 9 sup 9 sup m Tc-Ida-bis-Biotin complex

    CERN Document Server

    Gutíerrez, L C

    2000-01-01

    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 sup 9 sup 9 sup m Tc-Ida-bis-biotin complex were realized in healthy laboratory animals, showing stability 'In vivo' with renal purification. (Author) 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 sup 9 sup 9 sup m 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 ...

  10. Advantages of detecting monoclonal antibody binding to tissue sections with biotin and avidin reagents in Coplin jars.

    Science.gov (United States)

    Bindl, J M; Warnke, R A

    1986-04-01

    We describe a method of biotin/avidin-peroxidase detection using second and third stage reagents in Coplin jars. This method allows a large quantity of sections to be stained simultaneously with a minimal amount of technical time involved. A wide range of mouse monoclonal antibodies of varying specificities and isotypes were used to stain both frozen and paraffin-embedded sections of various normal and neoplastic tissues. Three different biotinylated anti-mouse antibodies were tested, including F(ab')2 antibody fragments of one, followed by horseradish peroxidase conjugated avidin. All monoclonal antibodies employed gave good staining, using incubation times of 30-50 minutes. The staining was done during a mean period of 25 to 27 days with an average staining load of 500 sections per Coplin jar. PMID:2420169

  11. Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach.

    Science.gov (United States)

    Reolon, Luciano Antonio; Martello, Carolina Lumertz; Schrank, Irene Silveira; Ferreira, Henrique Bunselmeyer

    2014-01-01

    The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae), the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia. PMID:25386928

  12. Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach.

    Directory of Open Access Journals (Sweden)

    Luciano Antonio Reolon

    Full Text Available The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae, the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.

  13. Pre-targeted immunodetection in glioma patients: tumour localization and single-photon emission tomography imaging of [99mTc ]PnAO-biotin

    International Nuclear Information System (INIS)

    We have developed a three-step pre-targeting method using the avidin-biotin system. The rationale of this technique consists in vivo labelling of biotinylated MoAbs targeted onto tumour deposits, when most of the unbound antibodies have been cleared from the bloodstream as avidin-bound complexes. The anti-tenascin MoAb BC2, specific for the majority of gliomas, was biotinylated and 1 mg was administered i.v. in 20 patients with histologically documented cerebral lesions. After 24-36 h, 5 mg avidin was injected i.v. followed 24 h later by a third i.v. injection of 0.2 mg PnAO-biotin labelled with 15-20 mCi technetium-99m. No evidence of toxicity was observed. Whole-body biodistribution was measured at 20 min, 3 h and 5 h post-injection. [99mTc]PnAO-biotin had a fast blood clearance and was primarily excreted through the biliary system. A dedicated single-photon emission tomography system was used to acquire brain tomographic images 1-2 h after the administration of [99mTc]PnAO-biotin. Tumours were detected in 15/18 glioma patients with a tumour to non-tumour ratio of up 14:1. This three-step method, based on the sequential adminsitration of anti-tenascin MoAb BC2, avidin and [99mTc]PnAO-biotin, can support computed tomography or magnetic resonance imaging for the diagnosis and follow-up of patients with glioma. (orig./MG)

  14. MR immuno-imaging study using avidin-biotin pre-targeting system on nude mice grafted with human colorectal carcinoma

    International Nuclear Information System (INIS)

    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 GdCl3 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

  15. 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)

    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/m2 (mean administered dose, 106.5-10.3 mCi/m2) 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/m2 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

  16. Avidin-biotin system radiolabelling with 153Sm and its experimental study in pre-targeting radioimmunoimaging with anti-CEA McAb

    International Nuclear Information System (INIS)

    Objective: To evaluate the usefulness of 153Sm labelled avidin-biotin system in pre-targeting radioimmunoimaging (RII)with anti-CEA McAb. Methods: Chelate DTPA-biotin (DB2) was first radiolabelled with 153Sm and then bound this 153Sm-DB2 to streptavidin (SA). The labelling of McAb with 153Sm was conducted using the bifunctional chelate cyclic DTPA anhydride (cDTPAa). Experimental studies were carried out in nude mice bearing human colon carcinoma. The authors established two- and three-step strategies for avidin-biotin system pre-targeting techniques. In three-step method A, tumor-bearing nude mice were first injected with McAb-biotin followed by cold avidin (Av) 2 days later and then 153Sm-DB2 1 day after. But in three-step method B, one group of mice was injected with McAb-SA followed by cold biotin 2 days later, and 4 h after biotin, by 153Sm-SA. While two-step protocols consisted of the injection of 153Sm-SA 48 h or 96 h after pre-targeting with McAb-bition (method A); and the injection of 153Sm-DB2 48 h after the use of McAb-SA (method B). SPECT imaging was performed and biodistribution was studied at 4, 24, 48 or 72 h after injection of 153Sm labelled compounds. Results: The authors' results showed that: 1) The three-step method A allowed faster blood clearance of the 153Sm-DB2 and yielded higher T/NT ratios (5.76 at 4 h and 12.94 at 24 h). 2) In the two-step method A, a significant accumulation of 153Sm-SA was observed in the tumor of nude mice which were injected 153Sm-SA 2 days after pre-targeting. The uptake in the tumor was also relatively high even in nude mice received 153Sm-SA 4 days after pre-targeting, demonstrating that the McAb-bition was still bound to the tumors and accessible to the 153Sm-SA even after long period of time. 3) Two- and three-step method A could increase the T/NT ratio in RII, shortening the imaging time (24 and 4 h, respectively). Conclusions: Aforementioned studies provide an experimental evidence for the improvement of pre

  17. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2015. Scientific opinion on biotin and contribution to normal energy-yielding metabolism: evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    deliver an opinion on the scientific substantiation of a health claim related to biotin and contribution to normal energy-yielding metabolism. The Panel considers that biotin, the food constituent that is the subject of the health claim, is sufficiently characterised. Contribution to normal energy......-yielding metabolism applies to all ages, including infants and young children (from birth to three years). The Panel concludes that a cause and effect relationship has been established between the dietary intake of biotin and contribution to normal energy-yielding metabolism. The following wording reflects the...... scientific evidence: ‘Biotin contributes to normal energy-yielding metabolism.’ The target population is infants and young children up to three years of age....

  18. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  19. Comparison of in situ DNA hybridization protocols using 35S-labeled and biotin-labeled probes in detection of human papillomavirus DNA sequences

    International Nuclear Information System (INIS)

    This paper describes a modified in situ DNA hybridization technique with 35S-labeled HPV DNA probes (Technique II) compared against the method (Technique I) routinely used by the authors, as well as against a new technique utilizing biotin-labeled probes (Technique III) the latter employed a 12-hour hybridization with biotin-labeled HPV DNA probes and reaction visualization with avidin, biotinylated alkaline phosphatase, and a substrate kit (Vector). The modifications in Technique II included omission of the 0.2 N HCI wash, increased proteinase-Κ concentration, elevated denaturation temperature (110-1200C), as well as replacement of poly-D-lysine as a slide-coating medium by Kodak Photo-Flo 200. In a series of cervical and penile human papillomavirus (HPV) lesions tested, identical HPV DNA types were discovered by Techniques I and II, whereas three specimens remained negative with Technique III

  20. 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)

    The biotin-avidin affinity system is exploited in pre-targeted imaging using avidin-conjugated antibodies. 18F-FDG is available at all PET centres. 18F-FDG forms oximes by reaction with oxyamine. Herein we describe the synthesis of oxyamine-funtionalised biotin, its 18F-labelling by conjugation with 18F-FDG and confirm its ability to interact with avidin.

  1. The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

    OpenAIRE

    Haines, D M; Clark, E.G.; Chelack, B J

    1989-01-01

    Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska sta...

  2. 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)

    External beam radiotherapy (EBRT) after conservative surgery for early breast cancer requires 5-7 weeks. For elderly patients and those distant from an RT center, attending for EBRT may be difficult or impossible. We investigated local toxicity, cosmetic outcomes, and quality of life in a new breast irradiation technique - intraoperative avidination for radionuclide therapy (IART) - in which avidin is administered to the tumor bed and 90Y-labelled biotin later administered intravenously to bind the avidin and provide irradiation. Reduced duration EBRT (40 Gy) is given subsequently. After surgery, 50 (ten patients), 100 (15 patients) or 150 mg (ten patients) of avidin was injected into the tumor bed. After 12-24 h, 3.7 GBq 90Y-biotin (beta source for therapeutic effect) plus 185 MBq 111In-biotin (gamma source for imaging and dosimetry) was infused slowly. Whole-body scintigraphy and SPECT/CT images were taken for up to 30 h. Shortened EBRT started 4 weeks later. Local toxicity was assessed by RTOG scale; quality of life was assessed by EORTC QOL-30. Of 35 patients recruited (mean age 63 years; range 42-74) 32 received IART plus EBRT. 100 mg avidin provided 19.5 ± 4.0 Gy to the tumor bed and was considered the optimum dose. No side-effects of avidin or 90Y-biotin occurred, with no hematological or local toxicity. Local G3 toxicity occurred in 3/32 patients during EBRT. IART plus EBRT was well accepted, with good cosmetic outcomes and maintained quality of life. IART plus reduced EBRT can accelerate irradiation after conservative breast surgery. (orig.)

  3. Intraoperative avidination for radionuclide treatment as a radiotherapy boost in breast cancer: results of a phase II study with {sup 90}Y-labeled biotin

    Energy Technology Data Exchange (ETDEWEB)

    Paganelli, Giovanni; De Cicco, Concetta; Carbone, Giuseppe; Pacifici, Monica [European Institute of Oncology, Division of Nuclear Medicine, Milan (Italy); Ferrari, Mahila E.; Cremonesi, Marta; Di Dia, Amalia [European Institute of Oncology, Division of Medical Physics, Milan (Italy); Pagani, Gianmatteo; Galimberti, Viviana; Luini, Alberto [European Institute of Oncology, Division of Senology, Milan (Italy); Leonardi, Maria Cristina; Ferrari, Annamaria; Orecchia, Roberto [European Institute of Oncology, Division of Radiotherapy, Milan (Italy); De Santis, Rita [Sigma-Tau SpA R and D, Rome (Italy); Zurrida, Stefano [European Institute of Oncology, Division of Senology, Milan (Italy); University of Milan School of Medicine, Milan (Italy); Veronesi, Umberto [European Institute of Oncology, Scientific Director, Milan (Italy)

    2010-02-15

    External beam radiotherapy (EBRT) after conservative surgery for early breast cancer requires 5-7 weeks. For elderly patients and those distant from an RT center, attending for EBRT may be difficult or impossible. We investigated local toxicity, cosmetic outcomes, and quality of life in a new breast irradiation technique - intraoperative avidination for radionuclide therapy (IART) - in which avidin is administered to the tumor bed and {sup 90}Y-labelled biotin later administered intravenously to bind the avidin and provide irradiation. Reduced duration EBRT (40 Gy) is given subsequently. After surgery, 50 (ten patients), 100 (15 patients) or 150 mg (ten patients) of avidin was injected into the tumor bed. After 12-24 h, 3.7 GBq {sup 90}Y-biotin (beta source for therapeutic effect) plus 185 MBq {sup 111}In-biotin (gamma source for imaging and dosimetry) was infused slowly. Whole-body scintigraphy and SPECT/CT images were taken for up to 30 h. Shortened EBRT started 4 weeks later. Local toxicity was assessed by RTOG scale; quality of life was assessed by EORTC QOL-30. Of 35 patients recruited (mean age 63 years; range 42-74) 32 received IART plus EBRT. 100 mg avidin provided 19.5 {+-} 4.0 Gy to the tumor bed and was considered the optimum dose. No side-effects of avidin or {sup 90}Y-biotin occurred, with no hematological or local toxicity. Local G3 toxicity occurred in 3/32 patients during EBRT. IART plus EBRT was well accepted, with good cosmetic outcomes and maintained quality of life. IART plus reduced EBRT can accelerate irradiation after conservative breast surgery. (orig.)

  4. 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)

    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)

  5. Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium.

    Science.gov (United States)

    Stevenson, M E; Blaschke, A P; Toze, S; Sidhu, J P S; Ahmed, W; van Driezum, I H; Sommer, R; Kirschner, A K T; Cervero-Aragó, S; Farnleitner, A H; Pang, L

    2015-07-01

    Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water. PMID:25888174

  6. Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

    Science.gov (United States)

    Rogez-Florent, Tiphaine; Goossens, Laurence; Drucbert, Anne-Sophie; Duban-Deweer, Sophie; Six, Perrine; Depreux, Patrick; Danzé, Pierre-Marie; Goossens, Jean-François; Foulon, Catherine

    2016-10-15

    This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives. PMID:27485269

  7. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    International Nuclear Information System (INIS)

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches

  8. Synthesis of a Cytokinin Linked by a Spacer to Dexamethasone and Biotin: Conjugates to Detect Cytokinin-Binding Proteins.

    Science.gov (United States)

    Wang, You; Letham, David S; John, Peter C L; Zhang, Ren

    2016-01-01

    Yeast cells expressing cDNA libraries have provided two new approaches to facilitate further identification of cytokinin-binding proteins and receptors. These are the yeast three hybrid (Y3H) system and fluorescence activated cell sorting (FACS). The Y3H system requires a synthetic hybrid ligand comprising an "anchor" moiety (e.g., dexamethasone) linked to a cytokinin via a spacer. In the yeast nucleus, this ligand by binding connects two fusion proteins leading to a reporter gene activation and detection and characterisation of cytokinin binding proteins. Herein is reported the first synthesis of dexamethasone-cytokinin ligands with a spacer linkage. This was attached to the purine ring of 6-benzylaminopurine (BAP) at positions 2, 8 or 9. To achieve this, dexamethasone was modified by periodate oxidation yielding a carboxylic group used for conjugation to the spacer by amide formation. Biotinyl derivatives of cytokinins for FACS included those synthesised by reaction of an activated ester of biotin with 8-(10-amino-decylamino) derivatives of BAP and BAP 9-riboside. Properties of the conjugates and some biological situations where they could be applicable are discussed briefly. PMID:27144549

  9. Introduction of new derivatives of biotin and DTPA for labeling of antibodies with 111 In ti detect malignant tumors

    International Nuclear Information System (INIS)

    Radiolabeled monoclonal antibodies, have created new innovations in diagnosis, research, and therapy of diseases in last 2 decades. One of the serious limitations of applications of radiolabeled antibodies in vivo is relatively low target to background activity. Various strategies have been proposed to solve this problem including pre-targeting methods that was suggested in 1989. Regarding importance of monoclonal antibodies and radioisotopes, based on pre-targeting strategy, we have introduced new derivative of biotin and DTPA to decrease background activity. DTPA-bio and new derivative (DTPA-bio-1 OX) were labeled with 111 In, labeled compounds and injected through tail veins into Balb/c mice, and percent of injected dose per gram of blood (% ID/g of blood ) was determined at 15, 30, 60, 120, 180 and 240 min after injection. Based on results, 111 In-DTPA-bio rapidly cleared from serum, indicating activity not bound to the target. While in the case of new derivative, by attaching 10 Adenine base (IOX) molecular weight of label is increased causing delayed clearance from serum. Therefore, there is enough time for label to accumulate in the target tissues. With advent of second generation of monoclonal antibodies and antibody engineering, pre targeting methods have changed greatly. It seems that derivatives we introduced will have and important role in new pre-targeting methods

  10. 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)

  11. Immobilization of oligonucleotide probes on silicon surfaces using biotin-streptavidin system examined with microscopic and spectroscopic techniques

    Science.gov (United States)

    Awsiuk, K.; Rysz, J.; Petrou, P.; Budkowski, A.; Bernasik, A.; Kakabakos, S.; Marzec, M. M.; Raptis, I.

    2014-01-01

    To immobilize effectively oligonucleotide probes on SiO2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin-biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin-b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin-b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m2) and second (1.31(±0.22) mg/m2) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m2) and fourth (0.41(±0.11) mg/m2) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  12. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport-NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885-were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  13. Transcriptome and Gene Ontology (GO Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Hong-Il Kim

    2016-03-01

    Full Text Available Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs, 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase, NCgl2516 (bioD, encoding dithiobiotin synthetase, NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885 were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

  14. Quantitative Measurement of Plasma 3-Hydroxyisovaleryl Carnitine by LC-MS/MS as a Novel Biomarker of Biotin Status in Humans

    OpenAIRE

    Horvath, Thomas D.; Stratton, Shawna L; Bogusiewicz, Anna; Pack, Lindsay; Moran, Jeffery; Mock, Donald M

    2010-01-01

    An increased plasma concentration of 3-hydroxyisovaleryl carnitine (3HIA-carnitine) results from impairment in the leucine catabolic pathway at the conversion of 3-methyl-crotonyl-CoA to 3-methylglutaconyl-CoA. The impairment is caused by reduced activity of the biotin-dependent enzyme 3-methylcrotonyl-CoA carboxylase. Here, we describe an LC-MS/MS method for the quantitation of 3HIA-carnitine in plasma and present preliminary evidence validating plasma 3HIA-carnitine as a novel biomarker of ...

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

    International Nuclear Information System (INIS)

    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 99mTc-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/99mTc(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 99mTc-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 99mTc(N)(Biot-Abu-CysOS)(PNP3) complex, whereas the 99mTc(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

  16. Pantothenic acid and biotin

    Science.gov (United States)

    ... the cabbage family Eggs Legumes and lentils Milk Mushrooms Organ meats Poultry White and sweet potatoes Whole- ... such as pregnancy). Women who are pregnant or breast-feeding need higher amounts. Ask your health care ...

  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)

    A high effective specific activity (HESA) formulation of a biotin-containing 99mTc 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 99mTc-gluconate at room temperature, followed by elution of the HESA 99mTc-RP488 in saline (minimum specific activity ∼ 1000 TBq/mmol by amino acid analysis). Both HESA and LESA 99mTc-RP488 labeled at > 90% purity. In vitro, HESA 99mTc-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 99mTc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting 99mTc radiopharmaceuticals

  18. Maskless localized patterning of biomolecules on carbon nanotube microarray functionalized by ultrafine atmospheric pressure plasma jet using biotin-avidin system

    Science.gov (United States)

    Abuzairi, Tomy; Okada, Mitsuru; Purnamaningsih, Retno Wigajatri; Poespawati, Nji Raden; Iwata, Futoshi; Nagatsu, Masaaki

    2016-07-01

    Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.

  19. Dual Labeled Peptides as Tools to Study Receptors: Nanomolar Affinity Derivatives of TIPP (Tyr-Tic-Phe-Phe) Containing an Affinity Label and Biotin as Probes of δ Opioid Receptors

    OpenAIRE

    Aldrich, Jane V.; Kumar, Vivek; Murray, Thomas F.; Guang, Wei; Wang, Jia Bei

    2009-01-01

    A general strategy for the design of dual labeled peptides was developed and derivatives of the δ opioid receptor (DOR) selective antagonist TIPP (Tyr-Tic-Phe-PheOH) containing both an affinity label and biotin were prepared by solid phase synthesis. Tyr-Tic-Phe-Phe(p-X)-Asp-NH(CH2CH2O)2-CH2CH2NH-biotin (where X = N=C=S or NHCOCH2Br) exhibit nanomolar DOR affinity. The ability to detect receptors labeled with these peptides following solubilization and SDS-PAGE demonstrate the applicability o...

  20. Country report: Italy (Duatti). Development of a Kit Formulation for Labeling Biotin-Derived Ligands with the Re-188 Nitrido Core

    International Nuclear Information System (INIS)

    Within the scope of this CRP, we developed a series of small-molecule biotinylated Re-188 complexes containing the [188Re≡N]2+ core. These complexes have been prepared using different chemical strategies, but all of them incorporate a biologically active biotin group. The main interest for this effort was brought to us after the introduction by Paganelli et al. of a new approach for the treatment of residual malignant cells after surgical removal of a primary breast tumour, and dubbed IART (Intraoperative Avidination for Radionuclide Therapy) [1]. Since now, the IART approach has been applied using 90Y as therapeutic radionuclide and a DOTA-functionalized biotin ligand for coordination to the radiometal. A major drawback for using 90Y as a therapeutic radionuclide comes from the absence of any radioactive decay, associated with the main β-emission, that may allow imaging of the actual biodistribution of injected radioactivity in any individual patient. Other therapeutic radionuclides, such as 177Lu and 188Re, possess an additional γ emission that easily allows imaging of target’s uptake, and could be conveniently utilized to monitor the course of therapy. In particular, 188Re emits a β- particle having almost the same energy as that emitted by 90Y, but with an associated 155-keV γ-emission that can be efficiently employed for imaging the biodistribution of the injected radiocompound. Considering also that 188Re is produced through a 188W/188Re transportable generator system, and that deep similarities between the chemistry of congener rhenium and technetium usually allow 99mTc compounds to be used for a preliminary evaluation of the biodistribution of the analogous 188Re compounds, this makes 188Re as an interesting candidate for application to the IART approach

  1. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil and maintenance of normal hair pursuant to Article 13(5) of

    DEFF Research Database (Denmark)

    Tetens, Inge

    claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L.) and maintenance of normal hair. The Panel considers that the specified combination is sufficiently characterised. The claimed effects are “contributes to reduce......, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L.) and maintenance of normal hair....

  2. IART® (Intra-Operative Avidination for Radionuclide Therapy) for accelerated radiotherapy in breast cancer patients. Technical aspects and preliminary results of a phase II study with 90Y-labelled biotin

    OpenAIRE

    Paganelli, G.; De Cicco, C; M. E. Ferrari; McVie, G.; Pagani, G; Leonardi, M C; Cremonesi, M.; Ferrari, A.; Pacifici, M.; Di Dia, A; Botta, F; De Santis, R; Galimberti, V.; Luini, A.; Orecchia, R.

    2010-01-01

    Background: Breast conserving surgery (BCS) plus external beam radiotherapy (EBRT) is considered the standard treatment for early breast cancer. We have investigated the possibility of irradiating the residual gland, using an innovative nuclear medicine approach named IART® (Intra-operative Avidination for Radionuclide Therapy). Aim: The objective of this study was to determine the optimal dose of avidin with a fixed activity (3.7 GBq) of 90Y-biotin, in order to provide a boost of 20 Gy, foll...

  3. EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

    International Nuclear Information System (INIS)

    Introduction: As direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging. Methods: Human epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to 99mTc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice. Results: Whereas both 99mTc-Av-EGF and 99mTc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. 99mTc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). 99mTc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h. Conclusion: 99mTc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.

  4. Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

    Directory of Open Access Journals (Sweden)

    Ting-Yu Angela Liao

    Full Text Available Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

  5. Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

    Science.gov (United States)

    Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

    2015-01-01

    Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine. PMID:26716832

  6. A comparative evaluation of avidin-biotin ELISA and micro SNT for detection of antibodies to infectious bovine rhinotracheitis in cattle population of Odisha, India

    Directory of Open Access Journals (Sweden)

    Priyaranjan Das

    2014-08-01

    Full Text Available Aim: The present study was undertaken to serologically detect Infectious Bovine Rhinotracheitis (IBR in the cattle population of Odisha, India using micro-Serum neutralization test (micro SNT and Avidin-Biotin Enzyme linked immuno sorbent assay (AB ELISA and finding out their comparative efficacy to serve as a suitable diagnostic tool in field condition. Materials and Methods: The study was carried out using serum samples (n=180 collected randomly from cattle populations of nine districts of Odisha. Similarly vaginal swabs (n=26 from cattle having history of repeat breeding, abortion, vulvo-vaginitis and nasal swabs (n=8 from calves with respiratory symptoms and nasal discharge were collected aseptically, to ascertain the circulation of virus among the cattle population. Results: Virus isolation by cell culture and subsequent confirmation by polymerase chain reaction confirmed four isolates. Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively. The sensitivity and specificity of AB ELISA test was found to be 88.23% and 95.70% respectively taking micro SNT as gold standard and the kappa value between the two tests was 0.75. Conclusion: Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively, thus highlighting the circulation of virus among the livestock population of Odisha and that AB ELISA could be more efficiently applied for the sero-diagnosis of IBR virus infections at field conditions, with demand for more study on faster, efficient and large scale screening of the infected animals.

  7. Effect of pyruvate kinase gene deletion on the physiology of Corynebacterium glutamicum ATCC13032 under biotin-sufficient non-glutamate-producing conditions: Enhanced biomass production

    Directory of Open Access Journals (Sweden)

    Kazunori Sawada

    2015-12-01

    Full Text Available The effect of pyruvate kinase gene (pyk deletion on the physiology of Corynebacterium glutamicum ATCC13032 was investigated under biotin-sufficient, non-glutamate-producing conditions. In a complex medium containing 100 g/L glucose, a defined pyk deletion mutant, strain D1, exhibited 35% enhancement in glucose consumption rate, 37% increased growth and a 57% reduction in respiration rate compared to the wild-type parent. Significant upregulation of phosphoenolpyruvate (PEP carboxylase and downregulation of PEP carboxykinase activities were observed in the D1 mutant, which may have prevented over-accumulation of PEP caused by the pyk deletion. Moreover, we found a dramatic 63% reduction in the activity of malate:quinone oxidoreductase (MQO in the D1 mutant. MQO, a TCA cycle enzyme that converts malate to oxaloacetate (OAA, constitutes a major primary gate to the respiratory chain in C. glutamicum, thus explaining the reduced respiration rate in the mutant. Additionally, pyruvate carboxylase gene expression was downregulated in the mutant. These changes seemed to prevent OAA over-accumulation caused by the activity changes of PEP carboxylase/PEP carboxykinase. Intrinsically the same alterations were observed in the cultures conducted in a minimal medium containing 20 g/L glucose. Despite these responses in the mutant, metabolic distortion caused by pyk deletion under non-glutamate-producing conditions required amelioration by increased biomass production, as metabolome analysis revealed increased intracellular concentrations of several precursor metabolites for building block formation associated with pyk deletion. These fermentation profiles and metabolic alterations observed in the mutant reverted completely to the wild-type phenotypes in the pyk-complemented strain, suggesting the observed metabolic changes were caused by the pyk deletion. These results demonstrated multilateral strategies to overcome metabolic disturbance caused by pyk

  8. 生物素-亲和素技术锚定肝素于胰岛表面*☆%Heparin anchored to the surface of islet by avidin-biotin technique

    Institute of Scientific and Technical Information of China (English)

    田晓辉; 李杨; 丁小明; 宋焕瑾; 冯新顺; 薛武军

    2013-01-01

    BACKGROUND:Islet capil aries are damaged in the process of islet isolation, thereby affecting the nutrient supply of islets after transplantation. Heparin has a very important significance for the regeneration of blood vessels;meanwhile, heparin is commonly used in the clinical islet transplantation to inhibit thrombosis. But systemic heparin can increase the risk of bleeding. The avidin has two strong binding sites of biotin and heparin respectively. OBJECTIVE:To improve islet revascularization and decrease risk of bleeding resulting from heparin systemic application through anchoring the heparin on the surface of islet with avidin-biotin technique based on the characteristics of avidin. METHODS:Adult human pancreas were isolated and purified with Ricordi automation method, then the islets were incubated and cultured with 0, 0.5, 1, 1.5, 2 g/L biotin (including biotin-N-hydroxysuccinimide ester, N-hydroxy-succinimido-6-biotinyl amido hexanoate, biocytin hydrazine, biotin hydrazide and TFP-biotin), 1 g/L avidin, and 0.5, 1.0, 1.5 and 2.0 g/L heparin, the change of heparin was observed. RESULTS AND CONCLUSION:TFP-biotin had the best effect to mediate the islet surface heparinization, and there was no significant difference in the activity of islet before and after heparinization (P>0.05);the heparinized and unheparinized heparin islets had the similar insulin release reaction (P>0.05). Biotin-avidin technique is a safe and effective islet surface heparinization treatment method.%  背景:胰岛微血管在胰岛分离过程中被破坏,进而影响移植后胰岛的营养供应。肝素对于血管的再生具有非常重要的意义;同时,临床胰岛移植多应用肝素来抑制血栓形成,但全身肝素化增加了出血的风险。而亲和素同时具备生物素和肝素2个较强的结合位点。  目的:利用亲和素这一特性,应用生物素-亲和素技术,将肝素锚定于胰岛的表面,促进胰

  9. False positive reaction due to endogenous biotin activity in glandular epithelium of decidua Reação falso positiva em epitélio glandular da decídua devido a atividade endógena de biotina

    Directory of Open Access Journals (Sweden)

    Liliana Cruz Spano

    2005-06-01

    Full Text Available Biotin-labeled probe was used in an in situ hybridisation assay to localize virus infection in formalin-fixed, paraffin embedded tissues taken from eleven abortion cases. Probes for human cytomegalovirus (HCMV, human Parvovirus B19 (B19 and human adenovirus type 2 (HAd2, were labeled with biotin-11-dUTP by nick-translation reaction. Streptavidin-alkaline-phosphatase (SAP was used to detect biotin, followed by 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP solution. Positive reaction was observed in nucleus of glandular ephitelium cells of decidua either in positive or in negative control at first and second gestational trimester. The reaction was not inhibited with blocking solution for alkaline phosphatase endogenous activity and it persisted even with probes omission. The use of adequate negative control permitted to reveal the presence of nuclear biotin in glandular epithelium of decidua, responsible for false positivity in detection systems involving streptavidin biotin system (StrepABC. The stained cells resembled to cytophatic effect due to herpesvirus, which could induce further misinterpretation. The results obtained in this study strongly recommend that DNA detection by in situ hybridisation reaction in gestational endometrium should be done without using StrepABC system.Sondas marcadas com biotina foram utilizadas neste trabalho para detecção de infecção viral por hibridização in situ em tecidos fixados com formalina e embebidos em parafina de 11 casos obtidos de abortamento. Sondas para citomegalovírus humano (HCMV, parvovírus B19 humano (B19 e adenovírus humano tipo 2 (HAd2, foram marcadas com biotina-11-dUTP através da reação de nick-translation. Estreptavidina conjugada com fosfatase alcalina (SAP seguida por solução de 4-nitro-azul de tetrazolio/5-bromo-4-cloro-3-indolil fosfato (NBT/BCIP foram utilizadas para detecção da biotina após a reação de hibridização. Reação positiva foi

  10. 微生物法测定多种维生素片中的生物素含量%Microorganism determination of biotin content in multi-vitamins tablets

    Institute of Scientific and Technical Information of China (English)

    黄进丽; 杨祖伟

    2015-01-01

    目的:利用微生物法测定多种维生素片中生物素的含量。方法在GB 5413.19-2010分析方法的基础上,通过将乳酸杆菌培养基改成MRS肉汤培养基来制备菌悬液,样品经过65℃~70℃水浴超声提取,在630 nm 波长下用酶标仪测定培养液吸光度,对生物素含量进行定量检测。与国标法进行标准曲线、精密度、准确度对比试验,以及对新建立的方法进行加标回收试验。结果生物素浓度在0.01~0.1 ng/mL范围内与吸光度呈现良好的二次曲线关系,相关系数r2>0.999,精密度和准确度良好。在80%、100%、120%添加水平下,生物素的回收率分别为99.8%、96.1%、97.2%。结论方法操作简单、灵敏度高、处理量大,适合生产企业的多种维生素片的大批量质量控制和检测。%Objective To determine the content of biotin in multi-vitamins tablets by microbial assay. Methods On the basis of GB 5413.19 2010 analysis method, bacteria suspension were prepared by lactic acid bacteria culture medium to (MRS) broth culture medium, the sample was extracted by water bath ultrasonic after 65℃~70℃;then culture medium absorbance was measured by absorbance enzyme standard instrument under the 630 nm, and to quantitatively detect the content of biotin. The standard curve, precision and accuracy of contrast test were compared with the national standard method, and the new method had carried on the standard addition recovery test. Results Biotin concentration within the scope of 0.01 to 0.1 ng/mL and absorbance had a good conical relationship, the correlation coefficient r2>0.999, and had a good precision and accuracy. The recovery rates of biotin were 99.8%, 96.1%and 97.2%under adding level 80%, 100%, and 120%, respectively. Conclusion The method is simple, high sensitivity and large quantity, and it is suitable for the mass production enterprises of various vitamin pills quality control and testing.

  11. 罗氏沼虾18S rRNA基因生物素标记探针的制备及应用%Preparation and application of the biotin-labeled probe of 18S rRNA gene in Macrobrachium rosenbergii

    Institute of Scientific and Technical Information of China (English)

    高风英; 叶星; 白俊杰; 吴锐全; 劳海华; 简清; 罗建仁

    2005-01-01

    Probes are essential for study of gene expression and regulation. In this study, a method was established to prepare the biotin-labeled probe for 18S rRNA gene of freshwater prawn, Macrobrachium rosenbergii. And the labeled method was used to produce a lysozyme gene probe, then applied in analysis of lysozyme gene expression. Primers were designed according to the nucleotide sequences of 18S rRNA of Decalxxta in order to isolate the 18S rRNA gene sequences of M. rosenbergii. Total genomic DNA was isolated from hepatopancreas of the freshwater prawn. A specific DNA fragment with desired size was amplified by PCR using the total DNA as templates. The DNA fragment was inserted into pGEM-T Easy vector and sequenced. The result of BLAST and alignment analysis confirmed that the DNA fragment isolated was the 18S rRNA gene of M. rosenbergii, which was 418 nt in length.Biotin-labeled probe of the 18S rRNA was then produced by PCR using the recombinant plasmid as templates. The biotin-21-dTTP and the non-labeled dNTP were added to the PCR reaction system. Ratio of the biotin-21-dTTP and the non-labeled dTFP was 3 to 1.The yield of the labeled probe is 300 ng·μL-1. The detection limit of the probe is 60 pg. A biotin-labeled probe of lysozyme gene was prepared by the same label method, and the yield of the lysozyme gene probe is 500 ng·μL-1. These biotin-labeled probes were applied in Northern dot blotting analysis of tissue distribution of lysoyzme mRNA of M. rosenbergii. Signals were scanned and quantified by Analysis System of Biology Image. The signal intensity ratio of the lysozyme to 18S rRNA represents the relative expression level of lysozyme mRNA. The results showed that the lysozyme mRNA existed in all the tissues checked, including eye,muscle, gill, hepatopancreas, haemocytes and intestine. But lysoyzme mRNA levels varied among different tissues. The highest level was found in the intestine, and the second was in the hepatopancreas and the lowest was in the

  12. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil and maintenance of normal hair pursuant to Article 13(5) of Regulation (EC) No 1924/2006

    OpenAIRE

    Tetens, Inge

    2012-01-01

    Following two applications from Nutrilinks Sarl, submitted pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L.) and maintenance of normal hair. The Panel considers that t...

  13. Scientific Opinion on the substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil and maintenance of normal hair pursuant to Article 13(5) of Regulation (EC) No 1924/2006

    OpenAIRE

    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA)

    2012-01-01

    Following two applications from Nutrilinks Sarl, submitted pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L.)...

  14. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    Science.gov (United States)

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  15. Scientific Opinion on the substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil and maintenance of normal hair pursuant to Article 13(5 of Regulation (EC No 1924/2006

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Dietetic Products, Nutrition and Allergies

    2012-07-01

    Full Text Available

    Following two applications from Nutrilinks Sarl, submitted pursuant to Article 13(5 of Regulation (EC No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies (NDA was asked to deliver an opinion on the scientific substantiation of a health claim related to a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L. and maintenance of normal hair. The Panel considers that the specified combination is sufficiently characterised. The claimed effects are “contributes to reduce hair loss” and “increases the number of hair”. The target population proposed by the applicant is healthy adults in the general population. The Panel considers that maintenance of normal hair is a beneficial physiological effect. The applicant identified one publication as being pertinent to the health claim. This study did not use the food which is the subject of the claim. No conclusions can be drawn from this study for the scientific substantiation of the claim. The Panel concludes that a cause and effect relationship has not been established between the consumption of a combination of thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, D-biotin and pumpkin seed oil (Cucurbita pepo L. and maintenance of normal hair.

  16. Synthesis of 6-PEtN-α-D-GalpNAc-(1–>6-β-D-Galp-(1–>4-β-D-GlcpNAc-(1–>3-β-D-Galp-(1–>4-β-D-Glcp, a Haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof

    Directory of Open Access Journals (Sweden)

    Andreas Sundgren

    2010-07-01

    Full Text Available Background: In bacteria with truncated lipopolysaccharide structures, i.e., lacking the O-antigen polysaccharide part, core structures are exposed to the immune system upon infection and thus their use as carbohydrate surface antigens in glycoconjugate vaccines can be considered and investigated. One such suggested structure from Haemophilus influenzae LPS is the phosphorylated pentasaccharide 6-PEtN-α-D-GalpNAc-(1→6-β-D-Galp-(1→4-β-D-GlcpNAc-(1→3-β-D-Galp-(1→4-β-D-Glcp.Results: Starting from a spacer-containing lactose derivative a suitably protected lacto-N-neotetraose tetrasaccharide structure was constructed through subsequential couplings with two thioglycoside donors, a glucosamine residue followed by a galactose derivative, using NIS/AgOTf as promoter. Removal of a silyl protecting group at the primary position of the non-reducing end residue afforded an acceptor to which the terminal α-galactosamine moiety was introduced using a 2-azido bromo sugar and halide assisted coupling conditions. Global deprotection afforded the non-phosphorylated target pentasaccharide, whereas removal of a silyl group from the primary position of the non-reducing end residue produced a free hydroxy group which was phosphorylated using H-phosphonate chemistry to yield the phosphoethanolamine-containing protected pentasaccharide. Partial deprotection afforded the phosphorylated target pentasaccharide with a free spacer amino group but with a protected phosphoethanolamino group. Conjugation of the spacer amino group to biotin or dimethyl squarate followed by deprotection of the phosphoethanolamino group and, in the case of the squarate derivative, further reaction with a protein then afforded the title conjugates.Conclusion: An effective synthesis of a biologically interesting pentasaccharide structure has been accomplished. The target pentasaccharide, an α-GalNAc substituted lacto-N-neotetraose structure, comprises a phosphoethanolamine motif and

  17. Structural DNA Nanotechnology:Stabilization of DNA Nanostructures via Streptavidin-Biotin Interactions%结构DNA纳米技术:利用链霉亲和素-生物素相互作用增强DNA纳米结构的稳定性

    Institute of Scientific and Technical Information of China (English)

    张磊; 徐子晨

    2013-01-01

    Rational design and assembly of nanometer-size objects have been a major goal of nanotechnology and precision engineering.The predictability of duplex interactions and helix geometry of DNA make it an excellent building block for the construction of nanometer-scale structures.DNA nanostructures are prone to dissociation as a result of the change of solution condition.A streptavidinbiotin complexation unit was introduced into the DNA nanostructure to improve its stability.Both the gel test and the melting temperature test prove that the stability of DNA nanostructures can be improved after streptavidin-biotin complexation.This approach is broadly applicable to solving similar problems in structural DNA nanotechnology.%实现纳米尺寸物体的合理设计与组装是纳米技术与精密工程的主要目标之一.DNA因其双链相互作用和螺旋几何构型的可预测性而使其成为构建纳米尺度结构的优秀建筑基元.DNA纳米结构在溶液状况改变时易分解.为提高DNA纳米结构的稳定性,一个链霉亲和素-生物素复合单元被引入到该纳米结构中.凝胶测试与熔点测试均证实链霉亲和素-生物素复合有助于提高DNA纳米结构的稳定性.该方法可广泛用于解决结构DNA纳米技术中的类似问题.

  18. Padronização da técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina para a identificação de sangue ingerido por Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 Enzyme-linked Immunosorbent Assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia longipalpis bloodmeals (Lutz & Neiva, 1912

    Directory of Open Access Journals (Sweden)

    Ana Maria Marassá

    2004-12-01

    Full Text Available A identificação de sangue ingerido pelos insetos é um importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dos métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os imunológicos. O estudo teve como objetivo, padronizar a técnica imunoenzimática de captura e titular amostras de sangue ingerido em fêmeas de flebotomíneos ingurgitadas de Lutzomyia longipalpis criadas em laboratório e alimentadas experimentalmente em rato. Em vista da alta sensibilidade, favorecida pelo sistema avidina-biotina, foi possível a realização de pelo menos noventa testes, de cada uma das amostras em duplicata, e constatar a presença de sangue para todas as amostras com períodos de 12 e 24 horas pós-ingestão, observando-se diferença significativa entre os respectivos títulos.Bloodmeals taken by insects constitute an important parameter for clarifying aspects of the transmission of zoonoses, including leishmaniases. Immunological assays can be used to investigate the attraction of vectors to animals, which may be hosts of these parasitoses. The objective of this study was to standardize a sandwich enzyme-linked immunosorbent assay and titer samples with different time periods of digestion, in laboratory-bred Lutzomyia longipalpis fed on rats. In the light of the high sensitivity that the biotin-avidin method permits, the technique provided at least ninety repeat tests for each sample and identified recent bloodmeals taken by these insects. Bloodmeals were detectable up to 12 and 24h after blood ingestion, and a significant difference between these titers was observed.

  19. Radioimmunotargeting with modified streptavidin-biotin. Final report

    International Nuclear Information System (INIS)

    For the past three years, the author has designed and produced several useful streptavidin variants, characterized their properties both in vitro and in vivo, and developed methods and strategies needed for characterization of each molecule and the entire radioimmunotargeting approaches using these molecules. In molecular design of these streptavidin mutants, an enhanced molecular modeling method, including empirical free energy calculations was used which is of great assistance in designing streptavidin mutants effectively and intelligently. When the original proposal was submitted in 1992, the reviewers were rather skeptical about the proposal to design successfully a various streptavidin mutants, particularly a dimeric streptavidin and multi-flavor streptavidins. These were clearly very challenging tasks, but now the author has the molecules in hand. This demonstrates the power of the molecular modeling method he is using and suggests the feasibility of further enhancements of these and other streptavidin variants by the molecular modeling capability. The author has also developed signal amplification methods which could introduce large amounts of radioisotopes specifically at target tumor sites in the radioimmunotargeting approaches

  20. Studies on chemical modification of papain by 5-chlorosulfonyl-2-oxobenzimidazole as biotin model compound

    OpenAIRE

    石橋, 文秀; 森藤, 昌樹; 根来, 千晴; 園田, 章; 片山, あずさ; 武部, 靖

    2009-01-01

     5-chlorosulfonyl-2-oxobenzimidazole(1) was synthesized.  On adding 1 to the suspension of papain in acetonitrile containing formamide, 1 was introduced into the papain in a yield of 7%, suggesting that 1 modified papain chemically to give 2-oxobenzimidazolesulfonyl papain(OBI- papain).  Also, it was found in-terestingly that papain activity of OBI-papain was maintained and that SH group in the active center in the large cleft of papain was free.  Accordingly, It expects that OBI-papain might...

  1. Dermatose responsiva à biotina em cão Dermatosis responsive to Biotin in a dog

    OpenAIRE

    Sandra Prudente Nogueira; Márcio Antonio Brunetto; Juliana Toloi Jeremias; Márcia de Oliveira Sampaio Gomes; Eliana Teshima; Aulus Cavalieri Carciofi

    2010-01-01

    Os transtornos da pele e dos pelos são parte importante na prática clínica de pequenos animais. Numerosos fatores nutricionais afetam a homeostase, a qualidade e o aspecto da pelagem. As vitaminas do complexo B incluem compostos hidrossolúveis necessários como coenzimas em diversas funções celulares envolvidas no metabolismo energético e na síntese tecidual. A biotina, em especial, é necessária nas reações de carboxilação, participando da síntese de ácidos graxos, aminoácidos e purinas pelo t...

  2. Biotin-avidin-conjugated metal sulfide nanoclusters for simultaneous electrochemical immunoassay of tetracycline and chloramphenicol

    International Nuclear Information System (INIS)

    We report on a protocol for a simultaneous competitive immunoassay for tetracycline (TC) and chloramphenicol (CAP) on the same sensing interface. Conjugates of TC and of CAP with bovine serum albumin were first co-immobilized on a glassy carbon electrode modified with gold nanoparticles. In parallel, monoclonal anti-TC and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters, respectively. In a typical assay, the immobilized haptens and the added target analytes competed for binding to the corresponding antibodies on the nanoclusters. Subsequently, Cd(II) and Pb(II) ions are released from the surface of the corresponding nanoclusters by treatment with acid and then were detected by square wave anodic stripping voltammetry. The currents at the peak potentials for Cd(II) and Pb(II) were used as the sensor signal for TC and CAP, respectively. This multiplex immunoassay enables the simultaneous determination of TC and CAP in a single run with dynamic ranges from 0.01 to 50 ng mL−1 for both analytes. The detection limits for TC and for CAP are 7.5 pg mL−1 and 5.4 pg mL−1, respectively. No obvious nonspecific adsorption and cross-reactivity was observed in a series of analyses. Intra-assay and inter-assay coefficients of variation were less than 10 %. The method was evaluated by analyzing TC and CAP in spiked samples of milk and honey. The recoveries range from 88 % to 107 % for TC, and from 91 % to 119 % for CAP. (author)

  3. Bioactivation of water-soluble peptidic quantum dot through biotin-streptavidin binding

    Science.gov (United States)

    Dif, A.; Touchet, S.; Nagarajan, S.; Baudy-Floc'h, M.; Dahan, M.; Piehler, J.; Marchi-Artzner, V.

    2008-02-01

    This paper describes the preparation of bioactive water-soluble fluorescent CdSe/ZnS semi-conductor quantum dots with a small hydrodynamic diameter of 10 nm. These quantum dots are functionalized with a biotinylated peptide that can be introduced at different ratios onto the surface of the quantum dots. Their ability to bind to streptavidin in solution is tested by using gel electrophoresis and fluorescence resonance energy transfer with a fluorescent labeled-streptavidin. The binding of these quantum dots to Agarose micrometric beads coated with streptavidin is also analyzed by fluorescent optical microscopy. A synthetic pegylated peptide is successfully used to prevent the non specific adsorption of streptavidin onto the quantum dots. A specific binding to the streptavidin results in the formation of a very stable streptavidin-quantum dot complex without any significant aggregation. The average number of streptavidin per quantum dot is found to be to 4 at the most. Such bioactivate quantum dots can be further conjugated to any biotinylated biomolecule and used in biological medium.

  4. (Strept)avidin-biotin interactions at amalgam electrodes covered by thiol monolayer

    Czech Academy of Sciences Publication Activity Database

    Josypčuk, Bohdan; Mareček, Vladimír; Yosypchuk, O.

    Ústí nad Labem: BEST servis, 2012 - (Navrátil, T.; Fojta, M.), s. 155-158 ISBN 978-80-905221-0-7. [Moderní elektrochemické metody /32./. Jetřichovice (CZ), 21.05.2012-25.05.2012] R&D Projects: GA ČR GAP206/11/1638; GA ČR GAP208/12/1645; GA AV ČR IAA400400806 Institutional support: RVO:61388955 Keywords : voltammetry * amalgam electrodes * monolayer Subject RIV: CG - Electrochemistry

  5. Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology.

    Science.gov (United States)

    Mallin, Hendrik; Hestericová, Martina; Reuter, Raphael; Ward, Thomas R

    2016-05-01

    Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d. PMID:27031496

  6. A SNARE-like protein and biotin are implicated in soybean cyst nematode virulence

    Science.gov (United States)

    Some phytoparasitic nematodes have the ability to infect and reproduce on plants that are normally considered resistant to nematode infection. Such nematodes are referred to as virulent and the mechanisms they use to evade or suppress host plant defenses are not well understood. Here, we report the ...

  7. The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

    Czech Academy of Sciences Publication Activity Database

    Malý, J.; Lampová, H.; Semerádtová, A.; Štofik, M.; Kováčik, Lubomír

    2009-01-01

    Roč. 20, č. 38 (2009), s. 385101-385101. ISSN 0957-4484 Grant ostatní: GA AV ČR(CZ) KAN200520702; GA ČR(CZ) GP203/07/P412; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50110509 Keywords : electrochemical stripping detection * DNA hybridization * sulfide nanoparticle Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.137, year: 2009

  8. Optimized biotin-hydrazide enrichment and mass spectrometry analysis of peptide carbonyls

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Wojdyla, K; Jensen, O. N.; Møller, Ian Max; Rogowska-Wrzesinska, A.

    Irreversible cell damage through protein carbonylation is the result of reaction with reactive oxygen species (ROS) and has been coupled to many diseases. The precise molecular consequences of protein carbonylation, however, are still not clear. The localization of the carbonylated amino acid is ...

  9. Electrochemical detection of osmium-modified peptides and streptavidin-biotin complex

    Czech Academy of Sciences Publication Activity Database

    Billová, Sabina; Brázdová, Marie; Havran, Luděk; Kizek, René; Masařík, Michal; Paleček, Emil

    Brno : Masarykova univerzita, 2002. s. 62. ISBN 80-210-2777-0. [Pracovní setkání biochemiků a molekulárních biologů /6./. 07.02.2002, Brno] Institutional research plan: CEZ:AV0Z5004920 Keywords : OSO4 * 2,2-bipyridine * electrochemical detection Subject RIV: BO - Biophysics

  10. C-C bond forming reactions catalyzed by artificial metalloenzymes based on the biotin-streptavidin technology

    OpenAIRE

    Chatterjee, Anamitra

    2015-01-01

    The research in Ward group is merging organometallic chemistry with Biotechnology. With the aim of exploiting most attractive and complementary features of catalysts, it is proposed to merge homogeneous and enzymatic catalysis to yield artificial metalloenzymes. In the past, the incorporation of an active metal moiety within a protein environment afforded hybrid catalysts with very promising properties, including high activities and selectivity, reminiscent of both homogeneous and enzymatic c...

  11. Incorporation of bioactive ligand to methacrylate hydrogel surface through avidin-biotin complex for targeted cell cultivation

    Czech Academy of Sciences Publication Activity Database

    Hobzová, Radka; Kostina, Nina Yu.; Mareková, Dana; Širc, Jakub; Michálek, Jiří

    Kathmandu : Nepal Polymer Institute, 2011. s. 194. ISBN 9937-2-3292-9. [POLYCHAR 19 - World Forum on Advanced Materials. 20.03.2011-24.03.2011, Kathmandu] R&D Projects: GA ČR GA304/07/1129; GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50390703 Keywords : hydrogels * surface * modification Subject RIV: FJ - Surgery incl. Transplants

  12. DFT study on the isomerization and tautomerism in vitamins B3 (niacin), B5 (pantothenic acid) and B7 (biotin)

    Science.gov (United States)

    Valadbeigi, Younes; Farrokhpour, Hossein; Tabrizchi, Mahmoud

    2014-05-01

    Isomerization and tautomerism of the three water soluble vitamins including B3, B5 and B7 were studied applying density functional theory using B3LYP method in gas and aqueous phases. Activation energies (Ea), Gibbs free energies of activation (ΔG#), and imaginary frequencies of the transition state structures were calculated for all the isomerization and tautomerism reactions. Activation energies of the neutral → zwitterion (amine-enamine) tautomerism in vitamin B3 were 310-360 kJ/mol where these values for the keto-enol tautomerism were 100-130 kJ/mol. It was found that water molecule catalyzes the tautomerism and decreases the activation energies about 90-160 kJ/mol.

  13. Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

    Science.gov (United States)

    Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

    2015-12-01

    The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications. PMID:26434388

  14. Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System

    OpenAIRE

    Ting-Yu Angela Liao; Alice Lau; Sunil Joseph; Vesa Hytönen; Zakaria Hmama

    2015-01-01

    Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we deve...

  15. Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1

    OpenAIRE

    Zhu, Min; Gong, Xue; Hu, YongHong; Ou, Weijun; Wan, Yakun

    2014-01-01

    Background Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, hi...

  16. APPLICATION OF AVIDIN-BIOTIN TECHNOLOGY AND ADSORPTIVE TRANSFER STRIPPING SQUARE-WAVE VOLTAMMETRY FOR DETECTION OF DNA HYBRIDIZATION AND AVIDIN IN TRANSGENIC AVIDIN MAIZE

    Science.gov (United States)

    The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgeni...

  17. Analysis of pirlimycin residues in beef muscle, milk and honey by a biotin-streptavidin-amplified enzyme-linked immunosorbent assay

    Science.gov (United States)

    Food contamination caused by veterinary drug residues is a world-wide public health concern and requires continuous monitoring. In this paper, we describe a biotin–streptavidin-amplified ELISA (BA-ELISA) for detecting pirlimycin residues in beef, milk, and honey. The IC50 value of the BA-ELISA was...

  18. Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize

    Czech Academy of Sciences Publication Activity Database

    Masařík, Michal; Kizek, René; Kramer, K. J.; Billová, Sabina; Brázdová, Marie; Jelen, František; Howard, J. A.

    2003-01-01

    Roč. 75, č. 11 (2003), s. 2663-2669. ISSN 0003-2700 R&D Projects: GA ČR GA203/02/0422; GA AV ČR IAA1163201; GA AV ČR IBS5004107 Institutional research plan: CEZ:AV0Z5004920 Keywords : carbon electrodes * mercury electrodes * streptavidin Subject RIV: BO - Biophysics Impact factor: 5.250, year: 2003

  19. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  20. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    OpenAIRE

    Hong-Il Kim; Jong-Hyeon Kim; Young-Jin Park

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Bl...

  1. Cluster (Cyanobacteria): 1158:3915 [

    Lifescience Database Archive (English)

    Full Text Available 1158:3915 Bifunctional protein birA (Includes: Biotin ... operon repressor; Biotin --(acetyl-CoA-carb ... oxylase) synthetase (Biotin --protein ligase)) 1150:7038 482564:922|272129:3709 ...

  2. Protein (Cyanobacteria): 392180 [

    Lifescience Database Archive (English)

    Full Text Available ZP_07113914.1 1117:24513 1150:7038 1158:3915 272129:3709 Bifunctional protein birA (Includes: Biotin ... otin operon repressor; Biotin --(acetyl-CoA-carboxylase) synthetase (Biotin --prot ...

  3. SwissProt search result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 (P50747) Biotin --protein ligase (EC 6.3.4.-) (Biotin ... apo-protein ligase) [In ... cludes: Biotin --[methylmalonyl-CoA-carboxytransferase] ligase (EC ... 6.3.4.9); Biotin --[propionyl-CoA-carboxylase [ATP-hydrolyzing

  4. NCBI nr-aa BLAST: CBRC-XTRO-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0052 ref|NP_795108.1| biotin ... carboxylase/biotin ... carboxyl carrier protein [Pseudomon ... as syringae pv. tomato str. DC3000] gb|AAO58803.1| biotin ... carboxylase/biotin ... carboxyl carrier protein [Pseud ...

  5. SwissProt search result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 (Q920N2) Biotin --protein ligase (EC 6.3.4.-) (Biotin ... apo-protein ligase) [In ... cludes: Biotin --[methylmalonyl-CoA-carboxytransferase] ligase (EC ... 6.3.4.9); Biotin --[propionyl-CoA-carboxylase [ATP-hydrolyzing

  6. NCBI nr-aa BLAST: CBRC-XTRO-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0052 ref|YP_272793.1| biotin ... carboxylase/biotin ... carboxyl carrier protein [Pseudomon ... as syringae pv. phaseolicola 1448A] gb|AAZ37384.1| biotin ... carboxylase/biotin ... carboxyl carrier protein [Pseud ...

  7. SwissProt search result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 (P48445) Biotin --protein ligase (EC 6.3.4.-) (Biotin ... Apo-protein ligase) [In ... cludes: Biotin --[methylmalonyl-CoA-carboxytransferase] ligase (EC ... 6.3.4.9); Biotin --[propionyl-CoA-carboxylase [ATP-hydrolyzing

  8. A streptavidin mutant with altered ligand-binding specificity

    OpenAIRE

    Reznik, Gabriel O.; Vajda, Sandor; Sano, Takeshi; Cantor, Charles R.

    1998-01-01

    The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between b...

  9. Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene–streptavidin–magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

    Directory of Open Access Journals (Sweden)

    Piyasak Chaumpluk et al

    2007-01-01

    Full Text Available Combinations of PCR-based amplification platform using 5' thiolated and biotinylated specific primers, S1 nuclease–PCR products treatment, ferrocene–streptavidin (Fc–Stv–magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5' terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene–streptavidin–magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.

  10. Detection of Filarial Larvae in Mosquitoes with Wuchereria Bancrofti Specific DNA Probe Labeled with Biotin/PCR System%生物素DNA探针/PCR系统用于班氏丝虫蚊媒监测的研究

    Institute of Scientific and Technical Information of China (English)

    黄炳成; 常江; 韩广东; 张玉林; 刘凤梅; 王士华; 陈锡欣; 王凯; 甄天民; 李连云; 贾凤菊; 刘玉俊; 程义亮

    2000-01-01

    目的:评价班氏丝虫特异生物素DNA探针/PCR系统用于丝虫病防治后期蚊媒监测的效果.方法:应用长臂光敏生物素标记的班氏丝虫种特异的DNA片段(490bp)为探针,结合聚合酶链反应技术,对实验室感染蚊虫和现场捕获蚊虫进行检测.结果:该探针只与班氏丝虫感染蚊提取的DNA杂交,不与马来丝虫感染蚊提取的DNA、其他动物丝虫DNA以及正常蚊DNA杂交.同批人工感染班氏微丝蚴蚊虫,人工解剖镜检阳性率为21.28%,探针检测阳性率为30.67%,差异无显著性(P>0.05).在平均感染度为1.6条幼虫/蚊情况下,50只蚊虫中有1只感染蚊即可被探针检出.在河南省班氏丝虫病流行区自病家和病村其他户分别捕获蚊虫,应用探针每20只为一组进行检测,蚊虫最低阳性率分别为0.75%和0.19%,检测非病村正常蚊均为阴性.结论:该DNA探针/PCR系统的敏感性和特异性均达到实用水平,为丝虫病防治后期的监测增添了一新的有用工具.

  11. Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin: a potential mechanism of long term toxicity by organophosphorus agents

    OpenAIRE

    Grigoryan, Hasmik; Schopfer, Lawrence M.; Thompson, Charles M.; Alvin V Terry; Masson, Patrick; Lockridge, Oksana

    2008-01-01

    Chronic low dose exposure to organophosphorus poisons (OP) results in cognitive impairment. Studies in rats have shown that OP interfere with microtubule polymerization. Since microtubules are required for transport of nutrients from the nerve cell body to the nerve synapse, it has been suggested that disruption of microtubule function could explain the learning and memory deficits associated with OP exposure. Tubulin is a major constituent of microtubules. We tested the hypothesis that OP bi...

  12. Automated microbiological assay for d-biotin in multi-vitamin tablets%应用自动浊度微生物分析仪测定维生素片剂中的生物素

    Institute of Scientific and Technical Information of China (English)

    黎旭茹; 张沁; 刘春丽

    2006-01-01

    目的 建立应用自动浊度微生物分析仪定量测定多种维生素营养片剂中生物素含量的方法.方法 应用自动浊度微生物分析仪对样品进行自动稀释,自动检测标准溶液和样品溶液中的菌体生长浊度,通过测定细菌生长和繁殖强度间接地测定出样品中的生物素含量.结果 方法的线性范围0.05~0.9 ng/ml,曲线相关系数r为0.9972,精密度为3.75%,回收率平均值为102.37%.结论 方法操作简单、灵敏度高、处理量大,适合生产企业的批量质量控制和检测.

  13. Prepare Macromolecules and Quantum Dots Multilayer Films by Bioconjugating Biotin and Streptavidin%生物素与链霉亲合素偶联法制备量子点-透明质酸复合膜

    Institute of Scientific and Technical Information of China (English)

    李国有; 王云起; 张文豪; 蔡继业

    2007-01-01

    首先利用一种阳离子型聚电解质(多聚赖氨酸)修饰玻璃基底,并与阴离子型聚电解质(生物素化透明质酸)在静电引力下自组装,然后以生物素化透明质酸和链霉亲合素化量子点为材料,通过生物素和链霉亲合素之间的特异性结合力,层层自组装法制备了量子点一、二、三层膜.用紫外可见吸收光谱和荧光光谱对其光学性质进行表征;原子力显微镜成像表明三种膜表面均一、平整,每层的量子点都分散均匀,没有严重的聚集情况.

  14. Matrix Interference in Serum Total Thyroxin (T4) Time-resolved Fluorescence Immunoassay (TRFIA) and Its Elimination With the Use of Streptavidin-biotin Separation Technique

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In development of serum total thyroxin TRFIA using the surface second-antibody (S-Ab) as separation agent, a significant bias of measurement caused by matrix interference when the surface S-Ab shows a relatively low binding capacity for primary anti-T4 monoclonal antibody (McAb) is studied. The bias ranges from 10% to 78%, depending on the matrix of individual samples and the binding capacity of the surface S-Ab prepared. So, a new separation system based on the use of a highly active surface streptavidin and biotinylated anti-T4 McAb is employed. The results indicate

  15. GenBank blastx search result: AK061906 [KOME

    Lifescience Database Archive (English)

    Full Text Available moderately similar to ACETYL-/PROPIONYL-COENZYME A CARBOXYLASE ALPHA CHAIN [CONTAINS: BIOTIN CARBOXYLASE (EC 6.3.4.14); BIOTIN CARBOXYL CARRIER PROTEIN (BCCP)].|PRI PRI 1e-102 +1 ...

  16. Genetics Home Reference: holocarboxylase synthetase deficiency

    Science.gov (United States)

    ... the body is unable to use the vitamin biotin effectively. This disorder is classified as a multiple ... impaired activity of certain enzymes that depend on biotin. The signs and symptoms of holocarboxylase synthetase deficiency ...

  17. GenBank blastx search result: AK061906 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061906 001-041-H10 AY185124.1 Campylobacter jejuni putative biotin ... decarboxylase (accC) gene, ... partial cds; and putative biotin ... decarboxylase (accB), Cj1292, putative UDP GlcNAc ...

  18. GenBank blastx search result: AK061906 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061906 001-041-H10 AF042099.1 Sulfolobus metallicus putative carboxyl transferase (pccB), biotin ... in carboxylase (accC), and biotin ... carboxyl carrier protein (accB) genes, complete cd ...

  19. Functionalization of embedded thiol-ene waveguides for evanescent wave-induced fluorescence detection in a microfluidic device

    DEFF Research Database (Denmark)

    Feidenhans, Nikolaj A.; Jensen, Thomas Glasdam; Lafleur, Josiane P.; Kutter, Jörg P.

    functionalized with biotin using photografting. The biotin was used for immobilization of fluorescently labelled streptavidin, and experiments revealed a linear correlation between streptavidin concentration and fluorescent intensity. To further demonstrate the attractiveness of using thiol-ene for optofluidic...

  20. GenBank blastx search result: AK060716 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060716 001-031-D11 AF042099.1 Sulfolobus metallicus putative carboxyl transferase (pccB), biotin ... in carboxylase (accC), and biotin ... carboxyl carrier protein (accB) genes, complete cd ...

  1. Genetics Home Reference: biotinidase deficiency

    Science.gov (United States)

    ... the body is unable to recycle the vitamin biotin. If this condition is not recognized and treated, ... making an enzyme called biotinidase. This enzyme recycles biotin, a B vitamin found in foods such as ...

  2. Sodium-Dependent Multivitamin Transporter Gene Is Regulated at the Chromatin Level by Histone Biotinylation in Human Jurkat Lymphoblastoma Cells1–3

    OpenAIRE

    Zempleni, Janos; Gralla, Michael; Camporeale, Gabriela; Hassan, Yousef I.

    2009-01-01

    The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, holocarboxylase synthetase (HCS) mediates covalent binding of biotin to histones; biotinylation of lysine-12 in histone H4 (K12BioH4) causes gene repression. Here we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesize that nuclear translocation of HCS increases in response to biotin supplementation; ...

  3. MOLECULARLY IMPRINTED POLYMERS

    OpenAIRE

    Hall, Andrew J.

    2014-01-01

    The present invention refers to new classes of polymerisable monomers targeting biotin, a biotin derivative, a biotin analogue or a biotinylated molecule and related structures, as well as molecularly imprinted polymers obtainable by polymerisation of at least one of these monomers and at least one cross-linking monomer in the presence of a suitable template molecule. The obtained polymers may be used for separation of biotin and related small molecules, together with larger biotinylated mole...

  4. Noncovalent Immobilization of Streptavidin on In Vitro- and In Vivo-Biotinylated Bacterial Magnetic Particles▿

    OpenAIRE

    Maeda, Yoshiaki; Yoshino, Tomoko; Takahashi, Masaaki; Ginya, Harumi; Asahina, Junko; Tajima, Hideji; Matsunaga, Tadashi

    2008-01-01

    Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In ...

  5. Disease: H01231 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H01231 Biotin-responsive basal ganglia disease (BBGD) Biotin-responsive basal ganglia disease (B ... olic disease hsa04977(80704) Vitamin digestion and absorption ... SLC19A3 [HSA:80704] [KO:K14610] Biotin [DR:D00029] ...

  6. SwissProt search result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 (P46363) BirA bifunctional protein [Includes: Biotin ... operon repressor; Biotin ... [acetyl-CoA-carboxylase] synthetase (EC 6.3.4.15) (Biotin --protein ligase)] BIRA_HAEIN 1e-16 ...

  7. SwissProt search result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 (P42975) BirA bifunctional protein [Includes: Biotin ... operon repressor; Biotin ... [acetyl-CoA-carboxylase] synthetase (EC 6.3.4.15) (Biotin --protein ligase)] BIRA_BACSU 9e-11 ...

  8. Locus: 5368 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available Hs.371350 H. sapiens - S: gi|29824592|ref|NC_000021.3|NC_000021 NC_000021 Homo sapiens mRNA for ... HCS, complete cds. biotin -[acetyl-CoA-carboxylase] ligase activity | biotin - ... methylcrotonoyl-CoA-carboxylase] ligase activity | biotin -[methylmalonyl-CoA-carboxytransferase] ligase acti ...

  9. Drug: D07586 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D07586 Drug Biotin ... sodium salt; Medebiotin ... (TN) C10H15N2O3S. Na 266.0701 266.2925 D07586.gif Vit ... ONS A11HA Other plain vitamin preparations A11HA05 Biotin ... D07586 Biotin ... sodium salt PubChem: 51091898 Ligand ...

  10. Gene : CBRC-XTRO-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0052 Novel UN D UNKNOWN BCCA_MYCLE 1e-141 55% ref|YP_296657.1| Biotin /lipoyl attach ... amoyl-phosphate synthetase large chain, N-terminal:Biotin ... carboxylase, C-terminal [Ralstonia eutropha JMP134 ... ] gb|AAZ61813.1| Biotin /lipoyl attachment:Carbamoyl-phosphate synthase L c ...

  11. GenBank blastx search result: AK061906 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061906 001-041-H10 AF317651.1 Methanosarcina barkeri pyruvate carboxylase biotin -containing su ... ATP-binding subunit PYCA (pycA), and bifunctional biotin ... ligase/biotin ... operon repressor PYCB (birA) genes, ...

  12. NCBI nr-aa BLAST: CBRC-XTRO-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0052 ref|YP_296657.1| Biotin /lipoyl attachment:Carbamoyl-phosphate synthase L chain ... amoyl-phosphate synthetase large chain, N-terminal:Biotin ... carboxylase, C-terminal [Ralstonia eutropha JMP134 ... ] gb|AAZ61813.1| Biotin /lipoyl attachment:Carbamoyl-phosphate synthase L c ...

  13. NCBI nr-aa BLAST: CBRC-XTRO-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0052 ref|YP_233608.1| Biotin /lipoyl attachment:Carbamoyl-phosphate synthase L chain ... amoyl-phosphate synthetase large chain, N-terminal:Biotin ... carboxylase, C-terminal [Pseudomonas syringae pv. ... syringae B728a] gb|AAY35570.1| Biotin /lipoyl attachment:Carbamoyl-phosphate synthase L c ...

  14. Studie über die gesunde Haut von Masthühnern und ihre Veränderungen bei einem experimentell erzeugten Biotinmangel

    OpenAIRE

    Waese, Kerstin

    2010-01-01

    Chicken in biotin-deficiency develop typical skin lesions, especially in the weight bearing metatarsal pad: In biotin-deficiency the thickness of epidermis increase. Shape and size of the dermal papillae are changed.In biotin-deficient epidermis the intercellular spaces are dilated and the synthesis and exocytosis of intercellular cementing substance is disturbed.

  15. Locus: 4550 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available Hs.449863 H. sapiens - S: gi|29824588|ref|NC_000017.5|NC_000017 NC_000017 Homo sapiens acetyl-Co ... ds ATP binding | acetyl-CoA carboxylase activity | biotin ... binding | biotin ... carboxylase activity | biotin ... car ...

  16. Streptavidin sensor and its sensing mechanism based on water-soluble fluorescence conjugated polymer

    Science.gov (United States)

    Chen, Yanguo; Hong, Peng; Xu, Baoming; He, Zhike; Zhou, Baohan

    2014-03-01

    Fluorescence quenching effect of water-soluble anionic conjugated polymer (CP) (poly[5-methoxy-2-(3-sulfopoxy)-1,4-phenylenevinylene] (MPS-PPV)) by [Re(N-N)(CO)3(py-CH2-NH-biotin)](PF6) [N-N=2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline; py-CH2-NH-biotin=N-[(4-pyridyl) methyl] biotinamide] (Re-Biotin) and fluorescence recovery in the presence of streptavidin (or avidin) were investigated using Re-Biotin as quencher tether ligand (QTL) probe. Meanwhile, the mechanisms of fluorescence quenching and recovery were discussed to provide new thoughts to design biosensor based on water-soluble CPs. The results indicate that the sensing mechanisms of streptavidin sensor or avidin sensor, using Re-Biotin as QTL probe, are the same and stable, whether in non-buffer system (aqueous solution) or different buffer systems [0.01 mol·L-1 phosphate buffered solution (pH = 7.4), 0.1 mol·L-1 ammonium carbonate buffered solution (pH = 8.9)]. There exists specific interactions between streptavidin (or avidin) and biotin of Re-Biotin. Fluorescence quenching and recovery processes of MPS-PPV are reversible. Mechanisms of Re-Biotin quenching MPS-PPV fluorescence can be interpreted as strong electrostatic interactions and charge transferences between Re-Biotin and MPS-PPV. Fluorescence recovery mechanisms of Re-Biotin-MPS-PPV system can be interpreted as specific interactions between streptavidin (or avidin) and biotin of Re-Biotin making Re-Biotin far away from MPS-PPV. Avidin or strptavidin as re-Biotin probe can not only be quantitatively determinated, but also be identified.

  17. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

    Science.gov (United States)

    Jiao, Chensong; Summerlin, Matthew; Bruzik, Karol S; Hanakahi, Leslyn

    2015-10-20

    Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems. PMID:26397942

  18. Expression of streptavidin gene in bacteria and plants

    International Nuclear Information System (INIS)

    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

  19. Structural investigation of the interactions of biotinylruthenocene with avidin.

    Science.gov (United States)

    Strzelczyk, Paweł; Bujacz, Anna; Plażuk, Damian; Zakrzewski, Janusz; Bujacz, Grzegorz

    2013-06-25

    The crystal structure of avidin, a protein from hen egg white, was determined in the form of a complex with biotinylruthenocene. This biotin-derived organometallic ligand is a potential anticancer agent for targeted therapy based upon avidin-biotin technology. Isothermal titration calorimetry experiments, involving avidin complexes with biotin (vitamin H or B7) derivatives, show differences in their affinity to the protein in comparison to its avidin-biotin complex, the strongest known biochemical interaction in Nature. The crystal structure of the first complex of avidin with biotinylruthenocene, determined at 2.5Å resolution (PDB: 4I60), shows unique interactions of the ruthenocene moiety with avidin. Biotin derivatives exhibit weaker affinity to avidin then biotin, which allows their wider use in biotechnology. The specific properties of biotinylruthenocene and the knowledge of its interactions with avidin may be useful in biochemical, medical, and nanotechnological applications. PMID:23603015

  20. Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics

    OpenAIRE

    Senevirathne, Chamara; Pflum, Mary Kay H.

    2013-01-01

    Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase-catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag ...

  1. Functional Characterization of Sodium-dependent Multivitamin Transporter (SMVT) in MDCK-MDR1 cells and its Utilization as a Target for Drug Delivery

    Science.gov (United States)

    Luo, Shuanghui; Kansara, Viral S.; Zhu, Xiaodong; Pal, Dhananjay; Mitra, Ashim. K.

    2008-01-01

    The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [3H] biotin uptake and transport was delineated. Transepithelial permeability of the biotin conjugated prodrug i.e. biotin-saquinavir was also studied. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to confirm the existence of SMVT in MDCK-MDR1 cells. Biotin uptake was Na+, pH, and temperature dependent, but energyindependent. Transepithelial transport studies of biotin-saquinavir in MDCK-MDR1, wild type MDCK, and Caco-2 cells revealed that permeability of biotin-saquinavir was similar in all three cell lines. A band of SMVT mRNA at 862 bp was identified by RT-PCR. A sodium-dependent multivitamin transporter, SMVT, responsible for biotin uptake and transport, was identified and functionally characterized in MDCK-MDR1 cells. Therefore, MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of biotin conjugated prodrugs such as HIV protease inhibitors. PMID:16749865

  2. Biocompatible post-polymerization functionalization of a water soluble poly(p-phenylene ethynylene)

    OpenAIRE

    Swager, Timothy Manning; Vanveller, Brett Steven

    2010-01-01

    A biocompatible post-polymerization functionalization reaction takes advantage of a polymer's structural motif for the controllable attachment of biotin as a model biosensor that responds to streptavidin.

  3. Drug: D00029 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available utic category of drugs in Japan [BR:br08301] 3 Agents affecting metabolism 31 Vitamins 319 Miscellaneous vitamins... 3190 Miscellaneous vitamins D00029 Biotin (JP16/USP/INN) Anatomical Therapeutic Chemi...AIN VITAMIN PREPARATIONS A11HA Other plain vitamin preparations A11HA05 Biotin D00029 Biotin (JP16/USP/INN) ...D00029 Drug Biotin (JP16/USP/INN); Bioepiderm (TN) C10H16N2O3S 244.0882 244.3106 D00029.gif Vitamin Sam...e as: C00120 Therapeutic category: 3190 ATC code: A11HA05 Vitamin B7 (Vitamin H) Therape

  4. Padronização da técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina para a identificação de sangue ingerido por Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) Enzyme-linked Immunosorbent Assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia) longipalpis bloodmeals (Lutz & Neiva, 1912)

    OpenAIRE

    Ana Maria Marassá; Cleide Aschenbrenner Consales; Eunice Aparecida Bianchi Galati

    2004-01-01

    A identificação de sangue ingerido pelos insetos é um importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dos métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os imunológicos. O estudo teve como objetivo, padronizar a técnica imunoenzimática de captura e titular amostras de sangue ingerido em fêmeas de flebotomíneos ingurgitadas de Lutzomyia longipal...

  5. Sequence Classification: 244729 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|62389589|ref|YP_224991.1| BIOTIN CARB...OXYLASE AND BIOTIN CARBOXYL CARRIER PROTEIN || http://www.ncbi.nlm.nih.gov/protein/62389589 ...

  6. NCBI nr-aa BLAST: CBRC-OPRI-01-0495 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0495 ref|YP_454585.1| biotin synthesis ... protein BioC [Sodalis glossinidius str. 'mor ... sitans'] dbj|BAE74180.1| biotin synthesis ... protein BioC [Sodalis glossinidius str. 'morsitans ...

  7. M-aminophenyltrialkylstannane

    Science.gov (United States)

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

    1990-12-11

    m-Radiohalo-aniline is a stable intermediate for preparing biotin-m-radiohalo-anilide to be used as an imaging agent or therapeutic agent. The invention also contemplates m-aminophenyltrialkylstannane which can be radiohalogenated and linked to biotin. No Drawings

  8. NMSBA: Sandia Biotech 2016 Report

    Energy Technology Data Exchange (ETDEWEB)

    Ruffing, Anne [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2016-06-01

    The objective of this project is to modify the FluorAbody plasmid previously developed by Sandia Biotech to include a binding site for biotin by introducing the biotin carboxyl carrier protein (BCCP)and a gold binding protein (GBP) into a loop of the red fluorescent protein (mRFP).

  9. Arabidopsis CDS blastp result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 At1g37150.4 68414.m04644 holocarboxylase synthetase 2 (HCS2.d) identical to ... eptor splice sites; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family; contains TIGRfa ... m profile TIGR00121: biotin --acetyl-CoA-carboxylase ligase; 6e-97 ...

  10. Arabidopsis CDS blastp result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 At1g37150.1 68414.m04641 holocarboxylase synthetase 2 (HCS2.d) identical to ... eptor splice sites; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family; contains TIGRfa ... m profile TIGR00121: biotin --acetyl-CoA-carboxylase ligase; 1e-72 ...

  11. Arabidopsis CDS blastp result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 At1g37150.3 68414.m04643 holocarboxylase synthetase 2 (HCS2.d) identical to ... eptor splice sites; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family; contains TIGRfa ... m profile TIGR00121: biotin --acetyl-CoA-carboxylase ligase; 4e-82 ...

  12. Arabidopsis CDS blastp result: AK111447 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111447 002-183-B06 At1g04640.1 biotin /lipoate A/B protein ligase family protein similar to lip ... liana] GI:15887052; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family 3e-86 ...

  13. Arabidopsis CDS blastp result: AK110051 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110051 002-160-C11 At1g04640.1 biotin /lipoate A/B protein ligase family protein similar to lip ... liana] GI:15887052; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family 7e-23 ...

  14. Arabidopsis CDS blastp result: AK243419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243419 J100066K03 At1g37150.2 68414.m04642 holocarboxylase synthetase 2 (HCS2.d) identical to ... eptor splice sites; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family; contains TIGRfa ... m profile TIGR00121: biotin --acetyl-CoA-carboxylase ligase; 1e-100 ...

  15. Arabidopsis CDS blastp result: AK061075 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061075 006-206-E01 At1g04640.1 biotin /lipoate A/B protein ligase family protein similar to lip ... liana] GI:15887052; contains Pfam profile PF03099: Biotin /lipoate A/B protein ligase family 3e-86 ...

  16. Disease: H00180 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00180 Holocarboxylase synthetase deficiency Holocarboxylase synthetase (HLCS) deficiency is an ... autosomal recessive disorder of biotin ... metabolism that results from holocarboxylase synth ... ases. In humans, four carboxylases are known to be biotin ylated by HLCS. They are pyruvate carboxylase, prop ...

  17. Biotinylation of Immunoglobulins

    International Nuclear Information System (INIS)

    The biotinylation of monoclonal antibodies (MAbs) represents a useful tool to prepare in house MAbs containing a number of biotin molecules, which do not compromise the MAb immunoreactivity, and can be used as a first step in the pretargeting approach, which foresees as a final step the intravenous injection of radiolabelled biotin

  18. Biotinidase Deficiency Accompanying Hair Changes and Periorificial Lesions: A Case Report

    Directory of Open Access Journals (Sweden)

    Sema Aytekin

    2011-11-01

    Full Text Available Biotinidase deficiency is impairment of biotin metabolism characterized by various dermatological, ophthalmic and neurological symptoms. Autosomal recessive trait is a disorder. Skin findings such as alopecia, periorificial dermatitis and seborrhoeic dermatitis lesions are seen. Clinical signs improved dramatically with biotine treatment. We presented a 6-year-old male patient with periorificial lesions, alopecia and microscopic hair shaft defects.

  19. Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences.

    Science.gov (United States)

    Marttila, Ari T; Hytönen, Vesa P; Laitinen, Olli H; Bayer, Edward A; Wilchek, Meir; Kulomaa, Markku S

    2003-01-01

    The strong interaction between avidin and biotin is so tight (dissociation constant 10(-15) M) that conditions usually sufficient for protein denaturing fail to dislodge biotin from the avidin-biotin complex. This kind of irreversible binding hinders the use of avidin in applications such as affinity purification or protein immobilization. To address this concern, we have constructed a series of mutants of the strategically positioned Tyr-33 in order to study the role of this residue in biotin binding, and to create avidin variants with more reversible ligand-binding properties. Unexpectedly, an avidin mutant in which Tyr-33 was replaced with phenylalanine (Avm-Y33F) displayed similar biotin-binding characteristics to the native avidin, indicating that the hydrogen bond formed between the hydroxy group of Tyr-33 and the carbonyl oxygen of biotin is not as important for the tight binding of biotin as previously suggested. In terms of the reversibility of biotin binding, Avm-Y33H was the most successful substitution constructed in this study. Interestingly, the binding of this mutant exhibited clear pH-dependence, since at neutral pH it bound to the biotin surface in an irreversible fashion, whereas, at pH 9, 50% of the bound protein could be released with free biotin. Furthermore, although Tyr-33 is located relatively distant from the monomer-monomer interfaces, the mutagenesis of this residue also weakened the quaternary structure of avidin, indicating that the high ligand binding and the high stability of avidin have evolved together and it is difficult to modify one without affecting the other. PMID:12358604

  20. DERIVATIZACIÓN Y CARACTERIZACIÓN ESPECTROSCÓPICA DE UN BIOPOLÍMERO A BASE DE L -LISINA CON ANÁLOGOS DE LA D-BIOTINA: CO -POLI(L-LISINA-GRAFT -(ε-N-[X-D -BIOTINIL]-L-LISINA

    Directory of Open Access Journals (Sweden)

    Flavio Dolores Martínez-Mancera

    2016-01-01

    Full Text Available We report the single-step derivatization reaction of a biopolymer based onL -lysine with D -biotin analogs:Co -poly(L -lysine-graft-(ε-N -[X-D-biotinyl]-L -lysine (PLL-X-Biotin. The valeric acid carboxylate of D -biotin is activated to an NHS ester for direct modification of amine groups in proteins and other macromolecules. NHS esters react by nucleophilic attack of an amine in the carbonyl group, releasing the NHS group, and forming a stable amide linkage. NHS-X-Biotin is the simplest biotinylation reagent commercially available. In contrast withD -biotin, it has a longer spacer arm off the valeric acid side chain allowing better binding potential for avidin or streptavidin probes. Derivatization of poly(L -lysine (PLL with NHS-X-Biotin led to a copolymer PLL-X-Biotin. UV-Visible, IR-FT and 1H NMR characteristics derived from synthesis are briefly discussed.

  1. Detection and cellular localisation of the synthetic soluble macromolecular drug carrier pHPMA

    International Nuclear Information System (INIS)

    Synthetic macromolecules such as copolymers of N-(2-hydroxypropyl)methacrylamide (pHPMA) are potential carriers for the delivery of drugs owing to their ability to passively accumulate in solid tumours [enhanced permeation and retention (EPR) effect]. To gain further knowledge about the biodistribution and the cellular localisation, poly(HPMA) was prepared for labelling by introducing biotin molecules. Biotinylated pHPMA (5 mol%) was intravenously injected into tumour-bearing rats and the accumulation of biotin-pHPMA was visualised using a streptavidin-alkaline phosphatase technique at day 7 post injection. In spite of the high solubility of pHPMA copolymers and the lack of attachment to cell structures, the biotinylated polymer could be easily detected in tissues fixed in 10% paraformaldehyde-phosphate buffer at 4 C for 48 h. While biotin-pHPMA could be detected intracytoplasmically in liver and spleen, a predominantly interstitial localisation was observed within the anaplastic prostate carcinoma (Dunning R3327-AT1). How biotin as a label influences the biodistribution of poly(HPMA) was assessed by scintigraphy, autoradiography and histology comparing homopolymer poly(HPMA) with biotin-pHPMA. The organ distribution patterns of the two polymers correlated well, except with respect to kidney. It is assumed that the accumulation of biotin-pHPMA in the distal tubuli is due to a biotin transporter in the brush border membrane. The technique presented is useful for a more comprehensive understanding of the biodistribution of soluble macromolecules. (orig.)

  2. Simple test system for single molecule recognition force microscopy

    International Nuclear Information System (INIS)

    We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG800 diamine was glutarylated, the mono-adduct NH2-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy

  3. Functional Characterization of Sodium-dependent Multivitamin Transporter (SMVT) in MDCK-MDR1 cells and its Utilization as a Target for Drug Delivery

    OpenAIRE

    Luo, Shuanghui; Kansara, Viral S.; Zhu, Xiaodong; Pal, Dhananjay; Mitra, Ashim K.

    2006-01-01

    The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [3H] biotin uptake and transport was delineated. Transepithelial permeability of the biotin conjugated prodrug i.e....

  4. Identificação do sangue ingerido por Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 e Lutzomyia (Lutzomyia almerioi (Galati & Nunes, 1999 pela técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina Blood meals identification of Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 e Lutzomyia (Lutzomyia almerioi (Galati & Nunes, 1999 by enzime-linked immunossorbent assay biotin-avidin

    Directory of Open Access Journals (Sweden)

    Ana Maria Marassá

    2006-04-01

    Full Text Available Lutzomyia longipalpis e Lutzomyia almerioi, espécies integrantes da fauna flebotomínea da Serra da Bodoquena, no Estado de Mato Grosso do Sul, têm sido objeto de estudo devido às suas elevadas abundâncias no Assentamento Guaicurus, foco de leishmaniose tegumentar humana e visceral canina. Em pesquisas que vem sendo realizadas neste acampamento para a identificação de vetores destas parasitoses, foram capturados no período de 2002 a 2004, com armadilhas automáticas luminosas, instaladas em ambiente peridoméstico (galinheiro, 83 exemplares ingurgitados de Lutzomyia longipalpis e Lutzomyia almerioi. O presente estudo teve como objetivo a investigação do hábito alimentar para ave das fêmeas de ambas as espécies de flebotomíneos, mediante o emprego da técnica imunoenzimática de captura,comparando-se a reatividade durante os anos de 2002 a 2004. Dentre 57 amostras de Lutzomyia longipalpis e 26 de Lutzomyia almerioi, foram encontradas 72% reagentes para ave em Lutzomyia longipalpis e 96% em Lutzomyia almerioi, o que justifica o estudo do hábito alimentar na região, como medida de prevenção e instituição de vigilância epidemiológica.Lutzomyia longipalpis and Lutzomyia almerioi, phlebotomine species from the fauna of Serra da Bodoquena, in the State of Mato Grosso do Sul, Brazil, have been studied, particularly due to the fact of their abundance and occurrence, the Guaicurus settlement, focus of human tegumentary and canine visceral leishmaniasis. In researches that are being carried out in this settlement for identifying the vectors of these parasitosis, 83 engorged females belonging to the species Lutzomyia longipalpis and Lutzomyia almerioi were captured with automatic light traps from 2002 up to 2004 in the peridomiciliary environment of the Guaicurus settlement (hennery.The aim of this study was the investigation on bird feeding habit of females of both the phlebotomine species by the enzyme-linked immunosorbent assay technique, comparing the reactivity during the period from 2002 up to 2004. Of the 57 samples of Lutzomyia longipalpis and 26 of Lutzomyia almerioi that have been tested, 72% from Lutzomyia longipalpis and 96% from Lutzomyia almerioi were reactive, which justifies the feeding habit study in the region as a prevention measure and the institution of an epidemiological survey.

  5. Disease: H01182 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H01182 Biotinidase deficiency; BTD deficiency; Late-onset multiple carboxylase deficiency Biotin ... eSH: D028921 OMIM: 253260 PMID:21696988 Wolf B The neurology ... of biotinidase deficiency. Mol Genet Metab 104:27- ...

  6. Sequence Classification: 891264 [

    Lifescience Database Archive (English)

    Full Text Available rane domain containing major facilitator subfamily member; mRNA levels negatively regulated by iron deprivation and biotin; Vht1p || http://www.ncbi.nlm.nih.gov/protein/6321502 ...

  7. “Clickable” polymer nanoparticles: a modular scaffold for surface functionalization

    OpenAIRE

    Krovi, Sai Archana; Smith, DeeDee; Nguyen, SonBinh T.

    2010-01-01

    The versatility of copper-catalyzed alkyne-azide coupling (CuAAC) in functionalizing drug-loaded polymer nanoparticles is demonstrated via the modification of surfaces of acetylene-functionalized PNPs with folate, biotin, and gold nanoparticles.

  8. Organophosphorus Compound DEPBT as a Coupling Reagent for Oligopeptides and Peptoids Synthesis: Studies on Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Some oligopeptidcs and peptoids were synthesized by applying the organophosphorus compound DEPBT as a coupling rcagent. D-Biotin-OOBt was obtained unexpcctcdly. A proposed reaction mechanism for DEPBT-mediated coupling was proved.

  9. Organophosphorus Compound DEPBT as a Coupling Reagent for Oligopeptides and Peptoids Synthesis:Studies on Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    HuiLIU; YunHuaYE; 等

    2002-01-01

    Some oligopeptides and peptoids were synthesized by applying the organophosphorus compound DEPBT as a coupling reagent. D-Biotin-OOBt was obtained unexpectedly. A proposed reaction mechanism for DEPBT-mediated coupling was proved.

  10. Towards the development of a direct electrochemical biodetector of avidin based on the poly(chloro amino β-styryl terthiophene)-coated glassy carbon electrode

    KAUST Repository

    Mehenni, Hakim

    2012-03-30

    In this study, a simple and direct biodetector was proposed, which was based on biotin immobilized onto a conducting polymer-coated electrode, for the detection of avidin, a highly stable glycoprotein found in egg-whites. Biotin was immobilized onto the electrode by covalent coupling to the primary amine group on the poly 3′-(3-chloro-4-amino-β-styryl)-(2,2′: 5′,2″-terthiophene) (PCAST), and the biotinavidin interaction was monitored by cyclic voltammetry. Incubation of the PCAST/biotin-modified-coated electrode with avidin in a phosphate buffered saline solution caused a significant change to its cyclic voltammogram, which was explained by the binding of avidin by biotin, and resulted in restricted ion transfer to and from the conducting polymer. This change was then utilized to detect avidin at 4 × 10 -6molL -1. © 2012 CSIRO.

  11. Synthesis and in Vitro evaluation of ''1''8''8Re-biotinyl-hydrazino-etda

    International Nuclear Information System (INIS)

    Pretargeting strategies have overcome many drawbacks associated with the use of directly labelled MoAbs in the diagnosis / treatment of various solid tumors. In particular the avidin-biotin system has received much attention due to extremely high affinity between avidin and biotin. An EDTA derivative of biotin has been synthesized (yield-35%). In order or label biotin derivative (biotinyl-hydrazino-EDTA) , stannous ion was used to reduce ''1''8''8ReO4 (VII) to lower oxidation state and weak chelating agent glucoheptonate as stabilizer and trans chelating agent. Thin layer chromatography and high performance liquid chromatography techniques were employed to monitor the radiolabeling efficiency. The radiolabeling yield of ''1''8''8Re-EDTAB1 was >95%. The radiolabeled product was found to bind to avidin (70-80%), thereby demonstrating retention of its biological integrity

  12. Study and application of imaging agents for infection and inflammation

    International Nuclear Information System (INIS)

    Situation of current study and clinic application of main imaging agents for infection and inflammation is summarized. These agents include radiolabelled small molecular compounds, leucocytes, large molecular proteins, liposomes, antibiotics, biotins and etc

  13. Fortify Your Knowledge about Vitamins

    Medline Plus

    Full Text Available ... B vitamins (thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B-6, vitamin B-12 and folate). AAFP cites two ... redness of the skin, upset stomach. B-6 (pyridoxine, pyridoxal, and pyridoxamine): Nerve damage to the limbs, ...

  14. B Vitamins Test

    Science.gov (United States)

    ... name: Pyridoxal phosphate (PLP) Other ways to measure: Vitamin B6 functional test, Urine 4-pyridoxic acid, urine xanthurenic acid B7, Biotin Also known as: Vitamin H, Vitamin B-w Role: B7 is a ...

  15. Vitamin B12

    Science.gov (United States)

    ... with other B vitamins, such as niacin, riboflavin, vitamin B6, and magnesium. A prescription form of vitamin B12 ... Nutrition Board. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. ...

  16. Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

    Science.gov (United States)

    Kumaraswamy, Sriram; Bergstedt, Troy; Shi, Xiaobo; Rininsland, Frauke; Kushon, Stuart; Xia, Wensheng; Ley, Kevin; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-05-01

    Sensor formats have been developed for detecting the activity of proteolytic enzymes based on fluorescent conjugated polymer superquenching. These sensors employ a reactive peptide sequence within a tether linking a quencher to a biotin. The peptide binds to sensors containing colocated biotin-binding protein and fluorescent polymer by means of biotin-biotin binding protein interactions, resulting in a strong quenching of polymer fluorescence. Enzyme-mediated cleavage of the peptide results in a reversal of the fluorescence quenching. These assays for protease activity are simple, sensitive, fast, and have the specificity required for screening chemical libraries for novel protease inhibitors in a high-throughput screening assay environment. These assays have been demonstrated for enterokinase, caspase-3/7, and -secretase.

  17. A radioimmunoassay for chicken avidin

    International Nuclear Information System (INIS)

    A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I 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 [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin. (author)

  18. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  19. Measuring Single-Bond Rupture Forces Using High Electric Fields in Microfluidic Channels and DNA Oligomers as Force Tags

    OpenAIRE

    Breisch, Stefanie; Gonska, Julian; Deissler, Helmut; Stelzle, Martin

    2005-01-01

    The disruption force of specific biotin-streptavidin bonds was determined using DNA oligomers as force tags. Forces were generated by an electric field acting on a biotinylated fluorescently labeled DNA oligomer. DNA oligomers were immobilized via biotin-streptavidin bonds on the walls of microfluidic channels. Channel layout and fluid-based deposition process were designed to enable well-defined localized deposition of the oligomers in a narrow gap of the microchannel. Electric fields of up ...

  20. Microfluidic system to detect select DNA fragments using agglutination process

    OpenAIRE

    Chhina, Sumanpreet Kaur

    2011-01-01

    This thesis investigates the design, fabrication, and testing of an easy-to-use, disposable and portable microfluidic system for DNA amplification detection; this is suitable for point-of-care testing (POCT) applications. The microfluidic system utilizes biotin-labelled DNA to agglutinate streptavidin-coated microspheres. The microfluidic system is designed to retain aggregates of cross-linked microspheres as opposed to single microspheres, indicating the detection of biotin-labelled DNA. The...

  1. The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts

    OpenAIRE

    Kill, IR; Bridger, JM; Campbell, KHS; Maldonado-Codina, G; Hutchison, CJ

    1991-01-01

    The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence ...

  2. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Šácha, Pavel; Bařinka, Cyril; Knedlík, Tomáš; Starková, Jana; Lubkowski, J.; Konvalinka, Jan

    2012-01-01

    Roč. 82, č. 1 (2012), s. 106-115. ISSN 1046-5928 R&D Projects: GA MŠk 1M0508; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : affinity purification * biotin acceptor peptide * recombinant protein expression * biotin-protein ligase (BirA) * co-localization * PSMA Subject RIV: CE - Biochemistry Impact factor: 1.429, year: 2012

  3. Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

    OpenAIRE

    Forster, A C; McInnes, J L; Skingle, D C; Symons, R H

    1985-01-01

    A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-buta...

  4. A synthetic time-delay circuit in mammalian cells and mice.

    Science.gov (United States)

    Weber, Wilfried; Stelling, Jörg; Rimann, Markus; Keller, Bettina; Daoud-El Baba, Marie; Weber, Cornelia C; Aubel, Dominique; Fussenegger, Martin

    2007-02-20

    Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-kappaB activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 microM, and are adjustably shut off at biotin levels exceeding 10 microM (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing. PMID:17296937

  5. Kinase-Catalyzed Biotinylation

    OpenAIRE

    Senevirathne, Chamara; Green, Keith D.; Pflum, Mary Kay H.

    2012-01-01

    Kinase-catalyzed protein phosphorylation plays an essential role in a variety of biological processes. Methods to detect phosphoproteins and phosphopeptides in cellular mixtures will aid in cell biological and signaling research. Our laboratory recently discovered the utility of γ-modified ATP analogues as tools for studying phosphorylation. Specifically, ATP-biotin can be used for labeling and visualizing phosphoproteins from cell lysates. Because the biotin tag is suitable for protein detec...

  6. What determines the strength of noncovalent association of ligands to proteins in aqueous solution?

    OpenAIRE

    Miyamoto, S; Kollman, P A

    1993-01-01

    Free energy perturbation methods using molecular dynamics have been used to calculate the absolute free energy of association of two ligand-protein complexes. The calculations reproduce the significantly more negative free energy of association of biotin to streptavidin, compared to N-L-acetyltryptophanamide/alpha-chymotrypsin. This difference in free energy of association is due to van der Waals/dispersion effects in the nearly ideally performed cavity that streptavidin presents to biotin, w...

  7. AcEST: BP912817 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000023_C02 513 Adiantum capillus-veneris mRNA. clone: YMU001_000023_C02. BP912817 - Show ... t sp_hit_id Q06862 Definition sp|Q06862|ACCC_ANASP Biotin ... carboxylase OS=Anabaena sp. (strain PCC 7120) Alig ... cant alignments: (bits) Value sp|Q06862|ACCC_ANASP Biotin ... carboxylase OS=Anabaena sp. (strain ... 245 8e-65 ...

  8. Screening Effect of PEG on Avidin Binding to Liposome Surface Receptors

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, Ole G.; Jørgensen, Kent

    This study investigates the screening effect of poly(ethylene glycol)-phospholipids (PE-PEG) on the interaction of avidin with PEGylated liposomes containing surface-bound biotin ligands. The influence of grafting density and lipopolymer chain length is examined. A simple fluorescence assay....... Furthermore. it is found that none of the lipopolymers completely prevents avidin from reaching the surface-bound biotin ligands....

  9. Dicty_cDB: CHB590 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHB590 (Link to dictyBase) - - - Contig-U13863-1 CHB590P (Link to Original ... chromosome 2... 123 7e-27 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 122 1e-26 AF414940_1( ... haliana holocarboxyla... 121 3e-26 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 119 ...

  10. Dicty_cDB: AFI685 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AF (Link to library) AFI685 (Link to dictyBase) - - - Contig-U13863-1 AFI685P (Link to Original ... thaliana chromosome 2... 75 2e-22 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 75 ... rinus CCE9901... 76 7e-20 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 76 2e-19 BC153857_1( ...

  11. Dicty_cDB: SFJ133 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SF (Link to library) SFJ133 (Link to dictyBase) - - - Contig-U13863-1 SFJ133P (Link to Original ... thaliana chromosome 2... 131 3e-29 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 129 ... inus CCE9901... 124 4e-27 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 122 2e-26 AK168925_1( ...

  12. AcEST: BP913171 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000027_C08 408 Adiantum capillus-veneris mRNA. clone: YMU001_000027_C08. BP913171 CL860Co ... t sp_hit_id Q06881 Definition sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-CoA carboxylase ... cant alignments: (bits) Value sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-C... 37 0.030 s ...

  13. Dicty_cDB: SFD294 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SF (Link to library) SFD294 (Link to dictyBase) - - - Contig-U13863-1 SFD294E (Link to Original ... s lucimarinus CCE9901... 167 9e-40 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 166 ... 1e-39 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 159 2e-37 AP005804_18 ...

  14. AcEST: BP912679 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000021_F06 573 Adiantum capillus-veneris mRNA. clone: YMU001_000021_F06. BP912679 CL860Co ... t sp_hit_id P49786 Definition sp|P49786|BCCP_BACSU Biotin ... carboxyl carrier protein of acetyl-CoA carboxylase ... cant alignments: (bits) Value sp|P49786|BCCP_BACSU Biotin ... carboxyl carrier protein of acetyl-C... 35 2e-04 s ...

  15. Dicty_cDB: SFJ562 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SF (Link to library) SFJ562 (Link to dictyBase) - - - Contig-U13863-1 SFJ562P (Link to Original ... thaliana chromosome 2... 75 4e-25 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 75 ... a Group genom... 64 9e-22 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 76 6e-21 (Q920N2) Rec ...

  16. Dicty_cDB: SFF405 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SF (Link to library) SFF405 (Link to dictyBase) - - - Contig-U13863-1 SFF405Z (Link to Original ... thaliana chromosome 2... 87 4e-24 G84651( G84651 )biotin ... holocarboxylase synthetase [imported] - Ara... 85 ... rinus CCE9901... 85 3e-23 ( P50747 ) RecName: Full=Biotin --protein ligase; EC=6.3.4... 68 4e-22 (Q920N2) Rec ...

  17. AcEST: BP915012 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000065_E02 510 Adiantum capillus-veneris mRNA. clone: YMU001_000065_E02. BP915012 CL860Co ... t sp_hit_id Q06881 Definition sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-CoA carboxylase ... cant alignments: (bits) Value sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-C... 37 6e-05 s ...

  18. AcEST: BP915507 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000072_D11 512 Adiantum capillus-veneris mRNA. clone: YMU001_000072_D11. BP915507 CL860Co ... t sp_hit_id Q06881 Definition sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-CoA carboxylase ... cant alignments: (bits) Value sp|Q06881|BCCP_ANASP Biotin ... carboxyl carrier protein of acetyl-C... 37 6e-05 s ...

  19. Storable Arylpalladium(II) Reagents for Alkene Labeling in Aqueous Media

    OpenAIRE

    Simmons, Rebecca L.; Yu, Robert T.; Myers, Andrew G.

    2011-01-01

    We show that arylpalladium(II) reagents linked to biotin and indocyanine dye residues can be prepared by decarboxylative palladation of appropriately substituted electron-rich benzoic acid derivatives. When prepared under the conditions described, these organometallic intermediates are tolerant of air and water, can be stored for several months in solution in dimethylsulfoxide, and permit biotin- and indocyanine dye-labeling of functionally complex olefinic substrates in water by Heck-type co...

  20. Sequence Classification: 893227 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|6324384|ref|NP_014454.1| Putative transmembran...largonic acid; BIO5 is in a cluster of 3 genes (BIO3, BIO4, and BIO5) that mediate biotin synthesis; Bio5p || http://www.ncbi.nlm.nih.gov/protein/6324384 ... ...e protein involved in the biotin biosynthesis pathway; responsible for uptake of 7-keto 8-aminope

  1. Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes

    Science.gov (United States)

    Hodenius, Michael; Hieronymus, Thomas; Zenke, Martin; Becker, Christiane; Elling, Lothar; Bornemann, Jörg; Wong, John E.; Richtering, Walter; Himmelreich, Uwe; De Cuyper, Marcel

    2012-09-01

    This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC14:0PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g-1 Fe3O4 and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r2 relaxivities and moderately high r1 values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.

  2. Surface Functional Poly(lactic Acid Electrospun Nanofibers for Biosensor Applications

    Directory of Open Access Journals (Sweden)

    Edurne González

    2016-01-01

    Full Text Available In this work, biotin surface functionalized hydrophilic non-water-soluble biocompatible poly(lactic acid (PLA nanofibers are created for their potential use as biosensors. Varying concentrations of biotin (up to 18 weight total percent (wt % were incorporated into PLA fibers together with poly(lactic acid-block-poly(ethylene glycol (PLA-b-PEG block polymers. While biotin provided surface functionalization, PLA-b-PEG provided hydrophilicity to the final fibers. Morphology and surface-available biotin of the final fibers were studied by Field Emission Scanning Electron Microscopy (FESEM and competitive colorimetric assays. The incorporation of PLA-b-PEG block copolymers not only decreased fiber diameters but also dramatically increased the amount of biotin available at the fiber surface able to bind avidin. Finally, fiber water stability tests revealed that both biotin and PLA-b-PEG, migrated to the aqueous phase after relatively extended periods of water exposure. The functional hydrophilic nanofiber created in this work shows a potential application as a biosensor for point-of-care diagnostics.

  3. Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes

    International Nuclear Information System (INIS)

    This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)–biotin surface groups (SPIO–PEG–biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid–PEG–biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC14:0PE) to activated α-biotinylamido-ω –N-hydroxy-succinimidcarbonyl–PEG (NHS–PEG–biotin). Physical characterization of the oleate and PEG–biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of − 39 mV (oleate coated particles) and − 14 mV (PEG–biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62–73 emu g−1 Fe3O4 and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r2 relaxivities and moderately high r1 values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP. (paper)

  4. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer

  5. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Science.gov (United States)

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+)) and MDA-MB-453 cells (CD44-), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+) is a commonly used cancer-stem-cell biomarker, our

  6. Peptide nanofibers modified with a protein by using designed anchor molecules bearing hydrophobic and functional moieties.

    Science.gov (United States)

    Miyachi, Ayaka; Takahashi, Tsuyoshi; Matsumura, Sachiko; Mihara, Hisakazu

    2010-06-11

    Self-assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a beta-sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H-PKFKIIEFEP-OH) self-assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H-ALEAKFAAFEAKLA-NH(2)). The surface-exposed biotin moiety on the fibers could capture an anti-biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe-Phe or Ile-Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti-biotin antibody through the chelation of Ni(2+) ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials. PMID:20419712

  7. Monitoring ligand-receptor interactions by photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jeney, Sylvia [M E Mueller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056 (Switzerland); Mor, Flavio; Forro, Laszlo [Laboratory of Complex Matter Physics (LPMC), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland); Koszali, Roland [Institute for Information and Communication Technologies (IICT), University of Applied Sciences of Western Switzerland (HEIG-VD), Rue Galilee 15, CH 1401 Yverdon-les-bains (Switzerland); Moy, Vincent T, E-mail: sylvia.jeney@unibas.ch, E-mail: vmoy@miami.edu [Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136 (United States)

    2010-06-25

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  8. Use of ionizing radiation in the regulation of amino acid synthesis of micro organisms. Part of a coordinated programme on radiation microbiology

    International Nuclear Information System (INIS)

    The effects of ionizing radiations on the production of glutamic acid (from glucose) by Corynebacterium glutamicum was investigated. Experiments were carried out with resting cell systems and with growing cultures of C. glutamicum. The growing cultures produced optimum yields of glutamic acid (25-30% of theoretical) in culture medium containing 1,0μg/l of biotin. The yield was virtually zero when 25μg/l of biotin was supplied. Resting cells from a medium containing growth-limiting concentrations of biotin (1μg/l) gave good yield of glutamic acid (approximately 27%), while cells harvested from a biotin-rich medium produced only traces of glutamate. Pre-irradiated cells of C. glutamicum produced less glutamic acid than unirradiated cells, and continuously irradiated (3,03 and 4,76 rad/h resting cells accumulated less glutamic acid than the corresponding unirradiated controls. Considerable increase in the glutamate produced by C. glutamicum during growth in the presence of 25μg/l of biotin was induced by continuously irradiating the cultures from the time of inoculation. The increases in the actual concentration of glutamate and in the precentage yield vary from approximately 2-fold to 4-fold. A dose rate of 4.0 krad/h was the most effective of the ones tested

  9. Identifying paths of allosteric communication in the protein BirA through simulations

    Science.gov (United States)

    Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

    Biotin ligase/repressor (BirA) is a bifunctional enzyme which adenylates biotin and transfers the product, biotinyl-5'-AMP (bio-5'-AMP) to biotin carboxyl carrier protein (BCCP). In the absence of BCCP, bio-5'-AMP promotes the dimerization of BirA. In dimer form, the BirA.bio-5'-AMP complex is able to bind to the biotin operator and prevents further synthesis of biotin. The bio-5'-AMP binds away from the dimer interface, so it is acting as an allosteric activator. We perform all-atom molecular dynamics simulations with BirA to look at fluctuations within the protein at equilibrium. We simulate apoBirA, liganded BirA, as well as two mutants, M211A and V219A. In agreement with experimental observations, several loops of the protein become stabilized for the liganded BirA when compared to the apo protein. In addition, changes in the dimer interface are observed for the M211A and V219A mutations, which are located in the ligand binding region. Using inter-residue correlation coefficients and pair energies a communication network through the protein is constructed. With this network we have identified paths which have the potential to be important in allosteric activation of BirA. These paths and the methods we use to identify them will be presented.

  10. Development of an avidin sensor based on the poly(methoxy amino-β-styryl terthiophene)-coated glassy carbon electrode

    KAUST Repository

    Mehenni, Hakim

    2012-03-01

    In this study, a simple and direct biosensor was proposed, which was based on biotin immobilized onto a conducting polymer-coated electrode, for the determination of avidin, a highly stable glycoprotein found in egg whites. Biotin was immobilized onto the electrode by covalent coupling to the primary amine group on poly-3′-(2-methoxy-5-amino-β-styryl)-(2,2′: 5′,2″-terthiophene) (PMAST), and the biotin-avidin interaction was monitored by square-wave voltammetry. Incubation of the PMAST/biotin-modified coated electrode with avidin in a phosphate-buffered saline solution caused a significant change to its square-wave voltammogram, which was explained by the binding of avidin by biotin, and resulted in restricted ion transfer to and from the conducting polymer. This change was then utilized to determine avidin. Importantly, we found a linear relationship for the avidin sensor in the range of 4 × 10 -14 to 3 × 10 -4 mol/L, and the detection limit was determined to be approximately 10 -14 mol/L. © 2012 Published by NRC Research Press.

  11. Poly(glycidyl methacrylate) grafted CdSe quantum dots by surface-initiated atom transfer radical polymerization: Novel synthesis, characterization, properties, and cytotoxicity studies

    Energy Technology Data Exchange (ETDEWEB)

    Bach, Long Giang; Islam, Md. Rafiqul [Department of Imaging System Engineering, Pukyong National University, Busan 608-737 (Korea, Republic of); Lee, Doh Chang [Department of Chemical and Biomolecular Engineering, KAIST Institute for the Nanocentury (KINC), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of); Lim, Kwon Taek, E-mail: ktlim@pknu.ac.kr [Department of Imaging System Engineering, Pukyong National University, Busan 608-737 (Korea, Republic of)

    2013-10-15

    A novel approach for the synthesis of poly(glycidyl methacrylate) grafted CdSe quantum dot (QDs) (PGMA-g-CdSe) was developed. The PGMA-g-CdSe nanohybrids were synthesized by the surface-initiated atom transfer radical polymerization of glycidyl methacrylate from the surface of the strategic initiator, CdSe-BrIB QDs prepared by the interaction of 2-bromoisobutyryl bromide (BrIB) and CdSe-OH QDs. The structure, morphology, and optical property of the PGMA-g-CdSe nanohybrids were analyzed by FT-IR, XPS, TGA, XRD, TEM, and PL. The as-synthesized PGMA-g-CdSe nanohybrids having multi-epoxide groups were employed for the direct coupling of biotin via ring-opening reaction of the epoxide groups to afford the Biotin-f-PGMA-g-CdSe nanobioconjugate. The covalent immobilization of biotin onto PGMA-g-CdSe was confirmed by FT-IR, XPS, and EDX. Biocompatibility and imaging properties of the Biotin-f-PGMA-g-CdSe were investigated by MTT bioassay and PL analysis, respectively. The cell viability study suggested that the biocompatibility was significantly enhanced by the functionalization of CdSe QDs by biotin and PGMA.

  12. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

    Science.gov (United States)

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L; Fenouillet, Emmanuel; Jones, Ian M; Barclay, A Neil

    2016-01-01

    In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents. PMID:26986548

  13. A variable gene delivery carrier-biotinylated chitosan/polyethyleneimine

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yi-Chen; Young, Tai-Horng [Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, Taipei 106, Taiwan (China); Chang, Fu-Hsiung [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Wei, Ming-Feng, E-mail: thyoung@ntu.edu.t [Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan (China)

    2010-12-15

    A variable gene delivery system has been developed based on conjugating chitosan to biotin through a functionalized poly(ethylene glycol) (PEG) spacer, which can be used to further bind different molecules on the outer layer of a polymer/DNA complex by streptavidin (SA)-biotin linkage. In this study, TAT-conjugated SA was used as the model molecule to prove the conjugation function of the prepared complex. In addition, low-molecular-weight poly(ethyleneimine) (PEI) was added into the polymer/DNA complex to increase the transfection efficiency. The results of the luciferase assay show that the transfection efficiency of the prepared complex was significantly correlated with the amount of PEI and was further enhanced when TAT was conjugated to the complex by SA-biotin linkage. Considered to have negligible cytotoxic effects, the variable gene delivery complex prepared in this study would be of considerable potential as carriers for in vitro applications.

  14. Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring

    Directory of Open Access Journals (Sweden)

    Miroslav Fojta

    2008-01-01

    Full Text Available Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE, followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

  15. Specific detection of proteins using Nanomechanical resonators

    DEFF Research Database (Denmark)

    Fischer, Lee MacKenzie; Wright, V.A.; Guthy, C.; Yang, N.; McDermott, M.T.; Buriak, J.M.; Evoy, S.

    2008-01-01

    of probes onto their surfaces in order to enable the specificity of the detection. Such nanoresonator-based specific detection of proteins is here reported using streptavidin as target system, and immobilized biotin as probe. Nanomechanical resonators resistant to stiction were first realized from...... silicon carbonitride using a novel fabrication method. Vapor-phase deposition of mercaptopropyl trimethoxysilane was performed, and an added mass of 2.22 +/- 0.07 fg/mu m(2) was measured. This linker molecule was used to attach biotin onto the devices, enabling the specific detection of streptavidin. A...... mass of 3.6 fg/mu m(2) was attributed to the added streptavidin, corresponding to one molecule per 27 nm(2). The specificity of this recognition was confirmed by exposing the devices to a solution of streptavidin that was already saturated with biotin. An additional negative control was also performed...

  16. On the lipid head group hydration of floating surface monolayers bound to self-assembled molecular protein layers

    DEFF Research Database (Denmark)

    Lösche, M.; Erdelen, C.; Rump, E.; Ringsdorf, H.; Kjær, K.; Vaknin, D.

    The structure of monomolecular layers of the protein streptavidin, specifically bound to biotin-functionalized lipid monolayers at aqueous surfaces, has been characterized. Neutron and X-ray reflectivity measurements allowed an assessment of the organization of these self-assembled systems with...... molecular resolution. Emphasis here is placed on the hydration of the lipid head groups in the bound state. For three functionalized lipids with spacers of different lengths between the biotin and their chains it was observed that the head groups were dehydrated in monolayers of the pure lipids, which were...... kept at low surface pressure before protein adsorption. The introduction of dipole moments at the interface by the admixture of phospholipids or the application of lateral pressure on the lipid monolayer before protein adsorption were found to impose an extension of the spacer moieties. The biotin...

  17. Surveying colloid sedimentation by coplanar waveguides.

    Science.gov (United States)

    Duţu, C A; Vlad, A; Roda-Neve, C; Avram, I; Sandu, G; Raskin, J-P; Melinte, S

    2016-06-01

    By using coplanar waveguides, direct access to the dielectric properties of aqueous solutions of polystyrene beads with different diameters from 330 nm to 10 μm is provided. The relative variation of the transmission parameter with respect to water is monitored, ranging from [Formula: see text] obtained for a 9.5% solution with 330 nm diameter beads to ∼22% for 10 μm diameter particles at the same concentration. To highlight its applicability in biosensing, the technique was further employed to survey the clustering between biotin and streptavidin-coated beads. The transmission parameter displays a ∼50% increase for mixtures containing nine volumes of biotin and one volume of streptavidin-modified beads (4.5 ng μl(-1) of streptavidin) and reaches ∼400% higher values when equal volumes of biotin and streptavidin-coated beads (22.5 ng μl(-1) of streptavidin) were mixed. PMID:27114467

  18. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus; Nygaard, Frank; Sen, Jette Wagtberg

    2013-01-01

    prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of...... biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein...... complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor...

  19. Surveying colloid sedimentation by coplanar waveguides

    Science.gov (United States)

    Duţu, C. A.; Vlad, A.; Roda-Neve, C.; Avram, I.; Sandu, G.; Raskin, J.-P.; Melinte, S.

    2016-06-01

    By using coplanar waveguides, direct access to the dielectric properties of aqueous solutions of polystyrene beads with different diameters from 330 nm to 10 μm is provided. The relative variation of the transmission parameter with respect to water is monitored, ranging from ∼ {3}% obtained for a 9.5% solution with 330 nm diameter beads to ∼22% for 10 μm diameter particles at the same concentration. To highlight its applicability in biosensing, the technique was further employed to survey the clustering between biotin and streptavidin-coated beads. The transmission parameter displays a ∼50% increase for mixtures containing nine volumes of biotin and one volume of streptavidin-modified beads (4.5 ng μl–1 of streptavidin) and reaches ∼400% higher values when equal volumes of biotin and streptavidin-coated beads (22.5 ng μl–1 of streptavidin) were mixed.

  20. A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles.

    Science.gov (United States)

    Wang, Qi; Huang, Xi; Fu, Xuan; Deng, Huan; Ma, Meihu; Cai, Zhaoxia

    2016-06-01

    Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160ng/mL. The lower limit of detection was 0.59ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay. PMID:26978788