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Sample records for ho dtpa-bis biotin

  1. Biotin

    PubMed Central

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

    2016-01-01

    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 homeostasis, and these roles are reviewed here. Possible effects of inadequate biotin intake, drug interactions, and inborn errors of metabolism are discussed, including putative effects on birth defects. PMID:19319844

  2. Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand

    NASA Astrophysics Data System (ADS)

    Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao

    2017-05-01

    A novel adenine (Ad) fluorescence probe (EuIII-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the EuIII-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the EuIII-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the EuIII-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using EuIII-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of EuIII-dtpa-bis(guanine) at 570 nm as a function of adenine (Ad) concentration in the range of 0.00-5.00 × 10- 5 mol L- 1 was observed. The detection limit is about 4.70 × 10- 7 mol L- 1.

  3. Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand.

    PubMed

    Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao

    2017-02-24

    A novel adenine (Ad) fluorescence probe (Eu(III)-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the Eu(III)-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the Eu(III)-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the Eu(III)-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using Eu(III)-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of Eu(III)-dtpa-bis(guanine) at 570nm as a function of adenine (Ad) concentration in the range of 0.00-5.00×10(-5)molL(-1) was observed. The detection limit is about 4.70×10(-7)molL(-1).

  4. Biotin

    MedlinePlus

    ... weight loss. It is also used orally for hair loss, brittle nails, skin rash in infants (seborrheic dermatitis), ... biotin deficiency.Skin rash in infants (seborrheic dermatitis).Hair loss. There is some preliminary evidence that hair loss ...

  5. Synthesis of specific SPECT-radiopharmaceutical for tumor imaging based on methionine: 99mTc-DTPA-bis(methionine).

    PubMed

    Hazari, Puja Panwar; Shukla, Gauri; Goel, Vijay; Chuttani, Krishna; Kumar, Nitin; Sharma, Rajnish; Mishra, Anil Kumar

    2010-02-17

    Methionine-diethylenetriaminepentaaceticacid-methionine [DTPA-bis(Met)] was synthesized by covalently conjugating two molecules of methionine (Met) to DTPA and was labeled with (99m)Tc in high radiochemical purity and specific activity (166-296 MBq/micromol). Kinetic analysis showed K(m) of 12.95 +/- 3.8 nM and a maximal transport rate velocity (V(max)) of 80.35 +/- 0.42 pmol microg protein(-1) min(-1) of (99m)Tc-DTPA-bis(Met) in U-87MG cells. DTPA-bis(Met) had dissociation constants (K(d)) of 0.067 and 0.077 nM in U-87MG and BMG, respectively. (35)S-methionine efflux was trans-stimulated by (99m)Tc-labeled DTPA conjugate demonstrating concentrative transport. The blood kinetic studies showed fast clearance with t(1/2) (F) = 36 +/- 0.5 min and t(1/2) (S) = 5 h 55 min +/- 0.85 min. U-87MG and BMG tumors saturated at approximately 2000 +/- 280 nmol/kg of (99m)Tc-DTPA-bis(Met). Initial rate of transport of (99m)Tc-DTPA-bis(Met) in U-87MG tumor was found to be 4.68 x 10(-4) micromol/kg/min. The tumor (BMG cell line, malignant glioma) grafted in athymic mice were readily identifiable in the gamma images. Semiquantitative analysis from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 +/- 3.99 and compared ROI with muscle (0.55 +/- 0.13). The tumor-to-contralateral muscle tissue ratio of (99m)Tc-DTPA-bis(Met) was found to be 23 +/- 3.3. Biodistribution revealed significant tumor uptake and good contrast in the U-87MG, BMG, and EAT tumor-bearing mice. In clinical trials, the sensitivity, specificity, and positive predictive values were found to be 87.8%, 92.8%, and 96.6%, respectively. (99m)Tc-DTPA-bis(Met) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging methionine-dependent human tumors and to quantify the ratio of MET(+)/HCY(-).

  6. Gd-Complexes of New Arylpiperazinyl Conjugates of DTPA-Bis(amides): Synthesis, Characterization and Magnetic Relaxation Properties.

    PubMed

    Ba-Salem, Abdullah O; Ullah, Nisar; Shaikh, M Nasiruzzaman; Faiz, Mohamed; Ul-Haq, Zaheer

    2015-04-29

    Two new DTPA-bis(amide) based ligands conjugated with the arylpiperazinyl moiety were synthesized and subsequently transformed into their corresponding Gd(III) complexes 1 and 2 of the type [Gd(L)H2O]·nH2O. The relaxivity (R1) of these complexes was measured, which turned out to be comparable with that of Omniscan®, a commercially available MRI contrast agent. The cytotoxicity studies of these complexes indicated that they are non-toxic, which reveals their potential and physiological suitability as MRI contrast agents. All the synthesized ligands and complexes were characterized with the aid of analytical and spectroscopic methods, including elemental analysis, 1H-NMR, FT-IR, XPS and fast atom bombardment (FAB) mass spectrometry.

  7. LAT-1 based primary breast cancer detection by [99m]Tc-labeled DTPA-bis-methionine scintimammography: first results using indigenously developed single vial kit preparation.

    PubMed

    Sharma, Sarika; Singh, Baljinder; Mishra, Anil K; Rathod, Deepti; Hazari, Puja Panwar; Chuttani, Krishna; Chopra, Shalini; Singh, Paramvir Mangat; Abrar, M L; Mittal, Bhagwant R; Singh, Gurpreet

    2014-09-01

    To evaluate the diagnostic utility of a single vial ready to label with [99m]Tc kit preparation of DTPA-bis-methionine (DTPA-bis-MET) for the detection of primary breast cancer. The conjugate (DTPA-bis-MET) was synthesized by covalently conjugating two molecules of methionine to DTPA and formulated as a single vial ready to label with [99m]Tc lyophilized kit preparations. Thirty female patients (mean age=47.5±11.8 years; range=21-69 years) with radiological/clinical evidence of having primary breast carcinoma were subjected to [99m]Tc-methionine scintigraphy. The whole body (anterior and posterior) imaging was performed on all the patients at 5 minutes, 10 minutes, 1 hour, 2 hours, and 4 hours following an intravenous administration of 555-740 MBq radioactivity of [99m]Tc-methionine. In addition, scintimammography (static images; 256×256 matrix) at 1, 2, and 4 hours was also performed on all the patients. The resultant radiolabel, that is, [99m]Tc-DTPA-bis-MET, yielded high radiolabeling efficiency (>97.0%), radiochemical purity (166-296 MBq/μmol), and shelf life (>3 months). The radiotracer primarily gets excreted through the kidneys and localizes in the breast cancer lesions with high target-to-nontarget ratios. The mean±SD ratios on the scan-positive lesions acquired at 1, 2, and 4 hours postinjection were 3.6±0.48, 3.10±0.24, and 2.5±0.4, respectively. [99m]Tc-methionine scintimammography demonstrated an excellent sensitivity and positive predictive value of 96.0% each for the detection of primary breast cancer. Ready to label single vial kit formulations of DTPA-bis-MET can be easily synthesized as in-house production and conveniently used for the scintigraphic detection of breast cancer and other methionine-dependent tumors expressing the L-type amino acid transporter-1 receptor. The imaging technique thus could be a potential substitute for the conventional single-photon emission computed tomography (SPECT)-based tumor imaging agents, especially

  8. LAT-1 Based Primary Breast Cancer Detection by [99m]Tc-Labeled DTPA-Bis-Methionine Scintimammography: First Results Using Indigenously Developed Single Vial Kit Preparation

    PubMed Central

    Sharma, Sarika; Mishra, Anil K.; Rathod, Deepti; Hazari, Puja Panwar; Chuttani, Krishna; Chopra, Shalini; Singh, Paramvir Mangat; Abrar, M.L.; Mittal, Bhagwant R.; Singh, Gurpreet

    2014-01-01

    Abstract Objective: To evaluate the diagnostic utility of a single vial ready to label with [99m]Tc kit preparation of DTPA-bis-methionine (DTPA-bis-MET) for the detection of primary breast cancer. Methods: The conjugate (DTPA-bis-MET) was synthesized by covalently conjugating two molecules of methionine to DTPA and formulated as a single vial ready to label with [99m]Tc lyophilized kit preparations. Thirty female patients (mean age=47.5±11.8 years; range=21–69 years) with radiological/clinical evidence of having primary breast carcinoma were subjected to [99m]Tc-methionine scintigraphy. The whole body (anterior and posterior) imaging was performed on all the patients at 5 minutes, 10 minutes, 1 hour, 2 hours, and 4 hours following an intravenous administration of 555–740 MBq radioactivity of [99m]Tc-methionine. In addition, scintimammography (static images; 256×256 matrix) at 1, 2, and 4 hours was also performed on all the patients. Results: The resultant radiolabel, that is, [99m]Tc-DTPA-bis-MET, yielded high radiolabeling efficiency (>97.0%), radiochemical purity (166–296 MBq/μmol), and shelf life (>3 months). The radiotracer primarily gets excreted through the kidneys and localizes in the breast cancer lesions with high target-to-nontarget ratios. The mean±SD ratios on the scan-positive lesions acquired at 1, 2, and 4 hours postinjection were 3.6±0.48, 3.10±0.24, and 2.5±0.4, respectively. [99m]Tc-methionine scintimammography demonstrated an excellent sensitivity and positive predictive value of 96.0% each for the detection of primary breast cancer. Conclusion: Ready to label single vial kit formulations of DTPA-bis-MET can be easily synthesized as in-house production and conveniently used for the scintigraphic detection of breast cancer and other methionine-dependent tumors expressing the L-type amino acid transporter-1 receptor. The imaging technique thus could be a potential substitute for the conventional single-photon emission computed

  9. Biological comparison of 149Pm-, 166Ho-, and 177Lu-DOTA-biotin pretargeted by CC49 scFv-streptavidin fusion protein in xenograft-bearing nude mice.

    PubMed

    Lewis, Michael R; Zhang, Jiuli; Jia, Fang; Owen, Nellie K; Cutler, Cathy S; Embree, Mary F; Schultz, Jody; Theodore, Louis J; Ketring, Alan R; Jurisson, Silvia S; Axworthy, Donald B

    2004-02-01

    The radiolanthanides (149)Pm, (166)Ho, and (177)Lu possess a range of half-lives and alpha(-) beta(-) energies for targeted radiotherapy of cancer. (149)Pm-, (166)Ho-, and (177)Lu-DOTA-biotin were pretargeted to LS174T colorectal tumors in nude mice with CC49 scFvSA antibody-streptavidin fusion protein. Tumor uptakes of (149)Pm (22.9% ID/g), (166)Ho (30.2% ID/g), and (177)Lu (35.4% ID/g) peaked at 1-4 h. Rapid blood disappearance was accompanied by urinary excretion of 59-66% ID within 1 h. Biodistributions of these agents show promise for pretargeted radioimmunotherapy of cancer.

  10. Gd3+-DTPA-bis (N-methylamine) - anionic linear globular Dendrimer-G1; a more efficient MRI contrast media.

    PubMed

    Ghalandarlaki, N; Mohammadi, T D; Agha Babaei, R; Tabasi, M A; Keyhanvar, P; Mehravi, B; Yaghmaei, P; Cohan, R A; Ardestani, M S

    2014-02-01

    By advancing of molecular imaging techniques, magnetic resonance imaging (MRI) is becoming an increasingly important tool in early diagnosis. Researchers have found new ways to increase contrast of MRI images.Therefore some types of drug known as contrast media are produced. Contrast media improve the visibility of internal body structures in MRI images. Gadodiamide (Omniscan®) is one of these contrast media which is produced commercially and used clinically. In this study Gadodiamide was first synthesized and then qualitative and quantitative methods were carried out to ensure the proper synthesis of this drug then to increase the efficiency of this contrast medium use dendrimer that is one kind of nano particle. This dendrimer has a polyethylene glycol (PEG) core and citric acid branches. After dendrimer attached to Gadodiamide to ensure the proper efficient connection between them the stability studies were carried out and cytotoxicity of the drug was evaluated. Finally, after ensuring the non-toxicity of the drug, in vivo studies (injected into mice) MR imaging was performed to examine the impact of synthesis drug on the resolution of image.The result obtained from this study demonstrated that the attachment of Gadodiamide to dendrimer reduces its cytotoxicity and also improved resolution of image. Also the new contrast media (Gd3+-DTPA- bis [N-methylamine] - Dendrimer) - unlike Omniscan® - is biodegradable and able to enter the HEPG2 cell line. The results confirm the hypothesis that using dendrimer to synthesize this new nano contrast medium increases its effectiveness.

  11. Biotin and biotinidase deficiency

    PubMed Central

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

    2009-01-01

    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 in transcriptional repression of genes and genome stability. Biotin deficiency may be caused by insufficient dietary uptake of biotin, drug–vitamin interactions and, perhaps, by increased biotin catabolism during pregnancy and in smokers. Biotin deficiency can also be precipitated by decreased activities of the following proteins that play critical roles in biotin homeostasis: the vitamin transporters sodium-dependent multivitamin transporter and monocarboxylate transporter 1, which mediate biotin transport in the intestine, liver and peripheral tissues, and renal reabsorption; holocarboxylase synthetase, which mediates the binding of biotin to carboxylases and histones; and biotinidase, which plays a central role in the intestinal absorption of biotin, the transport of biotin in plasma and the regulation of histone biotinylation. Symptoms of biotin deficiency include seizures, hypotonia, ataxia, dermatitis, hair loss, mental retardation, ketolactic acidosis, organic aciduria and also fetal malformations. This review focuses on the deficiencies of both biotin and biotinidase, and the medical management of such cases. PMID:19727438

  12. Biotin and biotinidase deficiency.

    PubMed

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

    2008-11-01

    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 in transcriptional repression of genes and genome stability. Biotin deficiency may be caused by insufficient dietary uptake of biotin, drug-vitamin interactions and, perhaps, by increased biotin catabolism during pregnancy and in smokers. Biotin deficiency can also be precipitated by decreased activities of the following proteins that play critical roles in biotin homeostasis: the vitamin transporters sodium-dependent multivitamin transporter and monocarboxylate transporter 1, which mediate biotin transport in the intestine, liver and peripheral tissues, and renal reabsorption; holocarboxylase synthetase, which mediates the binding of biotin to carboxylases and histones; and biotinidase, which plays a central role in the intestinal absorption of biotin, the transport of biotin in plasma and the regulation of histone biotinylation. Symptoms of biotin deficiency include seizures, hypotonia, ataxia, dermatitis, hair loss, mental retardation, ketolactic acidosis, organic aciduria and also fetal malformations. This review focuses on the deficiencies of both biotin and biotinidase, and the medical management of such cases.

  13. Biotin in swine nutrition.

    PubMed

    Kornegay, E T

    1985-01-01

    For many years, it was believed that supplemental biotin was not needed in swine diets because of the wide distribution of biotin in feedstuffs used in the formulation of swine diets, and because of the known synthesis of biotin by the animal's intestinal microflora. However, interest in biotin nutrition for swine was rekindled in the mid-1970s when several field reports indicated a biotin deficiency in swine that was responsive to biotin supplementation in many cases. Results from university research are accumulating, especially from long-term sow studies (three to four parities), which suggest that supplemental biotin will improve litter size, conception rate, weaning-to-estrus interval, toe lesions, and haircoat condition. Milk biotin concentration and plasma biotin concentration of sows and piglets were also elevated when supplemental biotin was fed. Using presently available feedstuffs and under modern swine production conditions, a marginal biotin deficiency is possible. Swine producers experiencing poor reproductive performance in their sow herds, associated with excessive loss of hair and severe foot lesions, should evaluate the biotin content of their sow diet and consider supplemental biotin.

  14. Biotin dependency due to a defect in biotin transport

    PubMed Central

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

    2002-01-01

    We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-dependent carboxylases in PBMCs and cultured skin fibroblasts were normal, excluding biotin holocarboxylase synthetase deficiency. Despite extracellular biotin sufficiency, biotin withdrawal caused recurrent abnormal organic aciduria, indicating intracellular biotin deficiency. Biotin uptake rates into fresh PBMCs from the child and into his PBMCs transformed with Epstein Barr virus were about 10% of normal fresh and transformed control cells, respectively. For fresh and transformed PBMCs from his parents, biotin uptake rates were consistent with heterozygosity for an autosomal recessive genetic defect. Increased biotin breakdown was ruled out, as were artifacts of biotin supplementation and generalized defects in membrane permeability for biotin. These results provide evidence for a novel genetic defect in biotin transport. This child is the first known with this defect, which should now be included in the identified causes of biotin dependency. PMID:12070309

  15. [Importance of biotin metabolism].

    PubMed

    Rodríguez Meléndez, R

    2000-01-01

    Biotin is a water soluble enzyme cofactor that belongs to the vitamin B complex. In humans, biotin is involved in important metabolic pathways such as gluconeogenesis, fatty acid synthesis, and amino acid catabolism by acting a as prosthetic group for pyruvate carboxylase, propionyl-CoA carboxylase, beta-methylcrotinyl-CoA carboxylase, and acetyl-CoA carboxylase. Carboxylases are synthesized as apo-carboxylases without biotin and the active form is produced by their covalent binding of biotin to the epsilon-amino group of a lysine residue of the apocarboxylases. This reaction is catalyzed by the holo-carboxylase synthetase. The last step in the degradation of carboxylases, the cleavage of the biotinyl moiety from the epsilon-amino group lysine residues, is catalyzed by biotinidase and results in the release of free biotin, which can be recycled. Biotin regulates the catabolic enzyme propionyl-CoA carboxylase at the posttranscriptional level whereas the holo-carboxylase synthetase is regulated at the transcriptional level. Aside from its role in the regulation of gene expression of carboxylases, biotin has been implicated in the induction of the receptor for the asialoglycoprotein, glycolytic enzymes and of egg yolk biotin binding proteins. Biotin deficiency in humans is extremely rare and is generally associated with prolonged parenteral nutrition, the consumption of large quantities of avidin, usually in the form of raw eggs, severe malnutrition and, inherited metabolic disorders. In humans, there are autosomal recessive disorders of biotin metabolism that result from the disruption of the activity of biotinidase or holo-carboxylase synthetase.

  16. Biotin: From Nutrition to Therapeutics.

    PubMed

    Mock, Donald M

    2017-08-01

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

  17. Biotin-binding proteins and biotin transport to oocytes.

    PubMed

    White, H B

    1985-01-01

    The eggs of chickens and other birds contain two proteins that bind biotin. Both are homotetrameric proteins of similar size. In contrast to the well-characterized egg white avidin, egg yolk biotin-binding protein has a very acidic isoelectric point, binds biotin with lower affinity, and is usually saturated with biotin. Like other egg yolk proteins, biotin-binding protein appears to be synthesized in the liver, transported by the blood stream to the ovary and deposited in the developing oocyte. Since the yolk of a chicken egg contains over 90% of the biotin in an egg and all of the biotin is bound to biotin-binding protein, the function of biotin-binding protein is undoubtedly to transport biotin to the egg for future use by the developing embryo. Avidin is produced by the oviduct and in the egg it is presumed to deter microbial growth around the oocyte by sequestering biotin. Among the eggs examined, those from turkeys have the lowest amount of biotin-binding protein and the highest amount of avidin. Furthermore, the majority of the biotin in turkey eggs can be bound to avidin in the egg white, suggesting a nutritional role for avidin in turkeys. An assay has been developed to conveniently measure apo- and holobiotin-binding proteins.

  18. Concentrations of biotin metabolites in human milk.

    PubMed

    Mock, D M; Mock, N I; Stratton, S L

    1997-09-01

    Because estimates of the biotin requirement for infants currently are based on the biotin concentration in human milk, we sought to determine whether inactive biotin metabolites are present. Samples were collected for 7 weeks post partum from 15 healthy women. Biotin and the inactive metabolites bisnorbiotin and biotin sulfoxide were measured by means of a high-performance liquid chromatography avidin-binding assay. At 8 days post partum the proportion of biotin was 44%, bisnorbiotin 48%, and biotin sulfoxide 8%. Although biotin content increased post partum (p < 0.003), the metabolites remained an important portion of the total providing evidence that accurate measurement of biotin in human milk requires an assay that is specific for biotin.

  19. Biotin studies in pigs. 2. The biotin requirement of the growing pig.

    PubMed

    Kopinski, J S; Leibholz, J

    1989-11-01

    Twenty pigs weaned at 5 d were given pelleted diets based on maize flour and casein and supplemented with 0, 50, 100, 150 or 200 micrograms biotin/kg diet. The performance of the pigs was not influenced by the biotin content of the diets. Typical biotin deficiency symptoms (foot lesions and skin pustules) were observed in pigs given the unsupplemented diet and the diet supplemented with 50 micrograms biotin/kg diet. Tissue biotin concentration plateaued when 100 micrograms biotin/kg was added to the diet. Faecal biotin excretion was independent of dietary biotin intake, but increased with age. Urine biotin excretion at 102 d was significantly lower for the unsupplemented pigs than for the pigs given various levels of dietary biotin supplements. A dietary requirement of biotin for young pigs between 50 and 100 micrograms/kg diet is suggested from the results of the present experiment.

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

    PubMed

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

    2016-01-01

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

  1. Inborn errors of biotin metabolism.

    PubMed

    Nyhan, W L

    1987-12-01

    The important role of biotin in human physiology has been highlighted by the recognition of two newly discovered human inborn errors of the metabolism of biotin. The molecular defect in the neonatal-onset disease is in the enzyme holocarboxylase synthetase. The defect in the later infantile-onset disease is in the enzyme biotinidase. Both disorders present with impressive clinical manifestations involving the skin and hair. In the neonatal disease, alopecia totalis is associated with a bright red scaly total body eruption. In biotinidase deficiency, the alopecia is more patchy and the skin lesions resemble acrodermatitis enteropathica. Both disorders are complicated by recurrent episodes of life-threatening acidosis and massive ketosis.

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

    PubMed Central

    2010-01-01

    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 valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. Results In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. Conclusions The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography. PMID:20937134

  3. 21 CFR 182.8159 - Biotin.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Biotin. 182.8159 Section 182.8159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a) Product. Biotin. (b) Conditions of use. This substance...

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

    PubMed

    Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

    2014-01-01

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

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

    SciTech Connect

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

    1986-03-01

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

  6. Intestinal absorption of biotin in the rat

    SciTech Connect

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

    1986-07-01

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

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

    PubMed

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

    2015-12-01

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

  8. Biotin transport in the rat central nervous system.

    PubMed

    Lo, W; Kadlecek, T; Packman, S

    1991-12-01

    Previous studies in the biotin-deficient rat have shown that brain biotin concentrations and the activity of biotin-dependent carboxylases are relatively preserved in the face of biotin starvation and systemic biotin deficiency. These data suggested the existence of a concentration mechanism for biotin in brain, and the present studies were undertaken to further characterize brain biotin transport. We presently show that rat cerebrospinal fluid biotin concentrations are 2.5 times higher than serum concentrations, consistent with the existence of a concentrative mechanism for biotin. Further, we demonstrate uptake of 3H-biotin into rat brain from blood at physiologic biotin concentrations, using single pass clearance measurements of a brain uptake index. The calculated brain uptake indices for biotin, and the inhibition kinetics, are consistent with the possible existence of a low affinity mediated uptake mechanism. The results have implications for the pathophysiology of human biotin-responsive multiple carboxylase deficiency.

  9. Biotin biosynthesis, transport and utilization in rhizobia.

    PubMed

    Guillén-Navarro, Karina; Encarnación, Sergio; Dunn, Michael F

    2005-05-15

    Biotin, a B-group vitamin, performs an essential metabolic function in all organisms. Rhizobia are alpha-proteobacteria with the remarkable ability to form a nitrogen-fixing symbiosis in combination with a compatible legume host, a process in which the importance of biotin biosynthesis and/or transport has been demonstrated for some rhizobia-legume combinations. Rhizobia have also been used to delimit the biosynthesis, metabolic effects and, more recently, transport of biotin. Molecular genetic analysis shows that an orthodox biotin biosynthesis pathway occurs in some rhizobia while others appear to synthesize the vitamin using alternative pathways. In addition to its well established function as a prosthetic group for biotin-dependent carboxylases, we are beginning to delineate a role for biotin as a metabolic regulator in rhizobia.

  10. Pharmacological Effects of Biotin in Animals.

    PubMed

    Riveron-Negrete, Leticia; Fernandez-Mejia, Cristina

    2017-01-01

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

  11. 21 CFR 182.8159 - Biotin.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Biotin. 182.8159 Section 182.8159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a)...

  12. 21 CFR 182.8159 - Biotin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Biotin. 182.8159 Section 182.8159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a)...

  13. 21 CFR 182.8159 - Biotin.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Biotin. 182.8159 Section 182.8159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a)...

  14. 21 CFR 182.8159 - Biotin.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Biotin. 182.8159 Section 182.8159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a)...

  15. Biochemical Characterization of the Arabidopsis Biotin Synthase Reaction. The Importance of Mitochondria in Biotin Synthesis1

    PubMed Central

    Picciocchi, Antoine; Douce, Roland; Alban, Claude

    2001-01-01

    Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h−1 with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (Km = 30 μm) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K0.5 = 39 μm), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis. PMID:11706201

  16. Stimulation of Nitrobacter agilis by Biotin

    PubMed Central

    Krulwich, Terry A.; Funk, Helen B.

    1965-01-01

    Krulwich, Terry A. (Goucher College, Baltimore, Md.), and Helen B. Funk. Stimulation of Nitrobacter agilis by biotin. J. Bacteriol. 90:729–733. 1965.—Addition of biotin to nitrite-mineral medium greatly stimulated the autotrophic growth of four strains of Nitrobacter agilis. Comparisons of cultures of the organisms grown in parallel at 30 C in nitrite medium and in the medium supplemented with 150 mμg of biotin per ml showed that the vitamin promoted: (i) 2- to 4-fold greater rates of utilization of nitrite, and (ii) 100- to 1,000-fold greater populations of cells per milliliter. Avidin specifically inhibited the biotin stimulation of nitrite utilization at an avidin-biotin ratio of 133:1. Incubation of the four strains of N. agilis at 37 C imposed a requirement for biotin that could be met by daily addition of 150 mμg of the vitamin per ml of medium. The stimulatory effects of the vitamin at 30 C suggest that in N. agilis the synthesis of biotin is rate-limiting for growth. PMID:16562074

  17. Biotin absorption by distal rat intestine

    SciTech Connect

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

    1987-12-01

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

  18. Altered glucagon release in biotin deficiency.

    PubMed

    Klandorf, H; Clarke, B L; Brown, J

    1987-01-01

    Classical biotin deficiency in young chickens is characterized by a reduction in the activity of liver biotin-dependent enzymes which thus impairs gluconeogenesis during periods of food withdrawal. Because the normal bird maintains an elevated fasting blood glucose level, the ability of the classically biotin-deficient animal to resist changes induced by fasting has not been established. Whether the release of insulin and/or glucagon is affected by biotin deficiency has not been investigated and so determining the importance of these hormones in maintaining fasting glucose levels as well as their ability to respond to a challenge is the objective of the present study. Experimental animals (15-week-old cockerels) were fed a biotin-deficient diet for 9 weeks while control animals (n = 8) were pair-fed biotin-supplemented diets. Before fasting, concentrations of plasma glucose, insulin, and glucagon were not different between the two groups. After 72 h of fasting basal glucose levels remained the same in both groups and concentrations of plasma insulin were equally suppressed but concentrations of glucagon were significantly elevated in biotin-deficient animals (P less than 0.025). Ten minutes after an iv glucose challenge the magnitude of the increase in plasma insulin concentrations was equivalent for both groups of animals whereas the magnitude of the decline in plasma glucagon concentrations was greater for biotin-deficient birds. Twenty minutes after a protein challenge (purified casein diet) the levels of plasma glucagon in both groups were maximally increased although the concentrations remained elevated only in control animals. In conclusion the release of insulin and glucagon is not impaired in biotin-deficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in Candida albicans.

    PubMed

    Ahmad Hussin, Nur; Pathirana, Ruvini U; Hasim, Sahar; Tati, Swetha; Scheib-Owens, Jessica A; Nickerson, Kenneth W

    2016-09-21

    Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee's medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee's medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1-4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO₂. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting

  20. Zinc and biotin deficiencies after pancreaticoduodenectomy.

    PubMed

    Yazbeck, N; Muwakkit, S; Abboud, M; Saab, R

    2010-01-01

    We report zinc and biotin deficiencies after pancreaticoduodenectomy in a 16 year old female presenting clinically with marked alopecia, total body hair loss, dry skin with scales, and maculopathy with significant vision loss. These micronutrient deficiencies likely occurred due to resection of the duodenum and proximal jejunum, sites of primary absorption of several micronutrients and their protein carriers, including zinc and biotin. Early diagnosis is essential to prevent irreversible sequelae. Adequate supplementation of zinc and biotin as well as dietary advice is needed for clinical improvement.

  1. A Review of the Use of Biotin for Hair Loss.

    PubMed

    Patel, Deepa P; Swink, Shane M; Castelo-Soccio, Leslie

    2017-08-01

    Biotin has gained commercial popularity for its claimed benefits on healthy hair and nail growth. Despite its reputation, there is limited research to support the utility of biotin in healthy individuals. To systematically review the literature on biotin efficacy in hair and nail growth. We conducted a PubMed search of all case reports and randomized clinical trials (RCTs) using the following terms: (biotin and hair); (biotin and supplementation and hair); (biotin supplementation); (biotin and alopecia); (biotin and nails); (biotin and dermatology), and (biotin recommendations). We found 18 reported cases of biotin use for hair and nail changes. In all cases, patients receiving biotin supplementation had an underlying pathology for poor hair or nail growth. All cases showed evidence of clinical improvement after receiving biotin. Though its use as a hair and nail growth supplement is prevalent, research demonstrating the efficacy of biotin is limited. In cases of acquired and inherited causes of biotin deficiency as well as pathologies, such as brittle nail syndrome or uncombable hair, biotin supplementation may be of benefit. However, we propose these cases are uncommon and that there is lack of sufficient evidence for supplementation in healthy individuals.

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

    PubMed Central

    Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

    2014-01-01

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

  3. Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency.

    PubMed

    Dupuis, L; Leon-Del-Rio, A; Leclerc, D; Campeau, E; Sweetman, L; Saudubray, J M; Herman, G; Gibson, K M; Gravel, R A

    1996-07-01

    Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency (MCD). We have identified six different point mutations in the HCS gene in nine patients with MCD. Two of the mutations are frequent among the MCD patients analyzed. Four of the mutations cluster in the putative biotin-binding domain as deduced from the corresponding Escherichia coli enzyme and consistent with an explanation for biotin-responsiveness based on altered affinity for biotin. The two others may define an additional domain involved in biotin-binding or biotin-mediated stabilization of the protein.

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

    PubMed

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

    2012-01-13

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

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

    PubMed Central

    2012-01-01

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

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

    SciTech Connect

    Sano, Takeshi; Cantor, C.R.

    1995-04-11

    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 streptavidin, indicating that the mutation of tryptophan 120 to phenylalanine has no significant effect on the basic properties of streptavidin. However, its biotin-binding affinity was reduced substantially, to approximately 10{sup 8} M{sup {minus}1}, indicating that the contact made by tryptophan 120 to biotin has a considerable contribution o the extremely tight biotin binding by streptavidin. The mutant retained bound biotin over a wide pH range or with the addition of urea up to 6 M at neutral pH. However, bound biotin was efficiently released by the addition of excess free biotin due, presumably, to exchange reactions. Electrophoretic analysis revealed that the intersubunit contact made by tryptophan 120 to biotin through the dimer-dimer interface is the major interaction responsible for the biotin-induced, tighter subunit association of streptavidin. In addition, the mutant has weaker subunit association than natural streptavidin even in the absence of biotin, indicating that tryptophan 120 also contributes to the subunit association of tetramers in the absence of biotin. 37 refs., 6 figs.

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

    PubMed

    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.

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

    PubMed

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

    2017-08-15

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

  9. Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from Rhizobium etli.

    PubMed

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

    2011-11-15

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

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

    PubMed Central

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

    2011-01-01

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

  11. Availability of avidin-bound biotin to the chicken embryo.

    PubMed

    White, H B; Orth, W H; Schreiber, R W; Whitehead, C C

    1992-10-01

    Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.

  12. Biosynthesis of Biotin in Microorganisms V. Control of Vitamer Production

    PubMed Central

    Birnbaum, Jerome; Pai, Chik H.; Lichstein, Herman C.

    1967-01-01

    Use of a yeast-lactobacillus differential microbiological assay permitted investigation into the synthesis of biotin vitamers by a variety of bacteria. A major portion of the biotin activity was found extracellularly. The level of total biotin (assayable with yeast) greatly exceeded the level of true biotin (assayed with lactobacillus). Values for intracellular biotin generally showed good agreement between the assays, suggesting the presence of only true biotin within the cells. Bioautographic analysis of the medium after growth of each organism revealed the presence of large amounts of a vitamer which corresponded to dl-desthiobiotin on the basis of Rf value and biological activity. Biotin, when detected at all, was at very low concentrations. Also, an avidin-uncombinable vitamer was synthesized by a majority of the bacteria. Addition of d-biotin to the growth medium prevented completely the synthesis of both vitamers of biotin. d-Biotin-d-sulfoxide had no effect on the synthesis of desthiobiotin or the avidin-uncombinable vitamer. Addition of dl-desthiobiotin did not prevent its own synthesis nor that of the other vitamer. Control of vitamer synthesis is therefore highly specific for d-biotin. The avidin-uncombinable vitamer was produced only at repressed levels in the presence of high concentrations of both d-biotin and dl-desthiobiotin, which suggested that it is not a degradation product of these substances. A possible mechanism for the overproduction of the biosynthetic precursors of biotin is presented. PMID:4965364

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

    PubMed

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

    2017-05-01

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

  14. Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives.

    PubMed

    Ebner, Andreas; Marek, Markus; Kaiser, Karl; Kada, Gerald; Hahn, Christoph D; Lackner, Bernd; Gruber, Hermann J

    2008-01-01

    Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 microM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 microM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 microg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.

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

    PubMed

    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.

  16. Serum Biotin Levels in Women Complaining of Hair Loss

    PubMed Central

    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

  17. Biotin selective polymer nano-films

    PubMed Central

    2014-01-01

    Background The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements. Results Biotin-selective thin (3–5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N,N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts. Conclusions Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems. PMID:24655809

  18. 21 CFR 582.5159 - Biotin.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Biotin. 582.5159 Section 582.5159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. 21 CFR 582.5159 - Biotin.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Biotin. 582.5159 Section 582.5159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  20. 21 CFR 582.5159 - Biotin.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Biotin. 582.5159 Section 582.5159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  1. 21 CFR 582.5159 - Biotin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Biotin. 582.5159 Section 582.5159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  2. 21 CFR 582.5159 - Biotin.

    Code of Federal Regulations, 2010 CFR

    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, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  3. Biotin and biotin analogues specifically modify the fluorescence decay of avidin.

    PubMed

    Mei, G; Pugliese, L; Rosato, N; Toma, L; Bolognesi, M; Finazzi-Agrò, A

    1994-09-30

    Avidin, a basic tetrameric glycoprotein, isolated from hen egg-white, binds up to four molecules of biotin with exceptionally high affinity. The presence of tryptophanyl residues in the active site pointed out the opportunity of correlating the protein fluorescence with biotin binding. We have performed both steady state and dynamic fluorescence experiments using biotin or biotin-derived molecules (biotinamine, diaminobiotin and iminobiotin) as ligands. The fluorescence decay data can only be fitted by two continuous distributions of lifetimes which may reflect the presence of static or dynamic microheterogeneity in the environment of the tryptophan residues. We observed that the binding of biotin, biotinamine and iminobiotin reduces the widths of both distributions to discrete lifetimes thus indicating a more homogenous environment for the emitting tryptophan residues. Instead, the binding of diaminobiotin, which lacks the imidazolone ring, affects one lifetime distribution only. The binding of biotin also affects the rotational correlation time of avidin, which becomes shorter, suggesting a more compact structure of the ligated protein. The utility of analyzing the fluorescence in terms of distributions appears to be further warranted.

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

    PubMed

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

    2016-09-01

    Given that nutritional biotin deficiency in Japanese infants has been reported, a straightforward method for estimating biotin level is needed. The biotin content in infant formula, breast milk, and the sera of infants fed with various types of formula were measured using avidin-binding assay. A commercially available ELISA kit was used for the measurement of biotin in 54 types of formula, including hydrolysate formulas for milk allergy, as well as in breast milk and in the sera of 27 infants fed with these formulas. The biotin content reached the recommended value in only five formulas. All of the hydrolysate formulas and more than half of the special formulas contained biotin <0.1 μg/dL. Serum biotin was low in infants fed only with the hydrolysate formulas, and one of them had alopecia related to biotin deficiency. While many were asymptomatic, infants fed with formulas lacking biotin are at risk of developing symptomatic disease. The addition of biotin to breast milk substitutes was finally approved in the middle of 2014, however pediatricians in Japan should still be vigilant with regard to nutritional biotin deficiency in infants for the time being. © 2016 Japan Pediatric Society.

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

    SciTech Connect

    Ogita, Takeshi ); Knowles, J.R. )

    1988-10-18

    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 {sup 18}O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5{prime}-(({alpha},{beta}-{sup 18}O,{beta},{beta}-{sup 18}O{sub 2})triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, they showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an {sup 18}O label directly from ({sup 18}O)bicarbonate into the product P{sub i}. This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.

  6. Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin.

    PubMed

    Pazy, Yael; Kulik, Tikva; Bayer, Edward A; Wilchek, Meir; Livnah, Oded

    2002-08-23

    We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs

  7. Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

    PubMed

    Hiller, Y; Bayer, E A; Wilchek, M

    1991-09-01

    The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

  8. Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

    PubMed Central

    Hiller, Y; Bayer, E A; Wilchek, M

    1991-01-01

    The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 6. Fig. 7. Fig. 9. PMID:1898347

  9. Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system.

    PubMed

    Ahmed, Raju; Spikings, Emma; Zhou, Shaobo; Thompsett, Andrew; Zhang, Tiantian

    2014-04-01

    Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin-avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin-streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin-streptavidin detection system.

  10. Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein.

    PubMed Central

    White, H B; Whitehead, C C

    1987-01-01

    In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in

  11. Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein.

    PubMed

    White, H B; Whitehead, C C

    1987-02-01

    In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in

  12. Avidin traps biotin diffusing out of chicken egg yolk.

    PubMed

    Bush, L; White, H B

    1989-01-01

    1. The unequal distribution of biotin and biotin-binding proteins between the yolk and albumen of freshly laid chicken eggs provides the potential for time-dependent redistribution of biotin that could affect egg quality, biotin availability, and hatchability. 2. Avidin-bound biotin was measured in albumen next to the shell and next to the yolk in eggs stored up to 23 days. 3. Biotin bound to biotin-binding proteins (BBP-I and BBP-II) was measured at the center and periphery of yolk from the same eggs. 4. After 11 days of storage, significant amounts of biotin from the yolk began to accumulate in the albumen adjacent to the yolk. 5. This transfer is attributed to a change in the vitelline membrane that permits diffusion of biotin, not BBP-I or BBP-II, out of the yolk. 6. The dynamics of this phenomenon suggest that in addition to its antimicrobial role, avidin may be involved in the utilization of biotin by the chick embryo.

  13. Biotinidase deficiency: factors responsible for the increased biotin requirement.

    PubMed

    Baumgartner, E R; Suormala, T; Wick, H; Bausch, J; Bonjour, J P

    1985-01-01

    Inability to recycle biotin from endogenous biocytin in congenital biotinidase deficiency is associated with increased requirement of exogenous free biotin. We have observed that severe biotin depletion with clinical and biochemical consequences occurs within 12 days after birth in a newborn patient and within 15-20 days after withdrawal of biotin supplementation in four other patients. Our studies have shown that: Urinary loss of biotin and biocytin are major causes for this rapid biotin depletion. Intestinal absorption of biotin seems to be normal at least at the loading dose of 1.5 micrograms/kg. At normal or subnormal plasma biotin concentrations biocytin is found in low concentrations (below 1 nmol l-1) in plasma of patients but at much higher concentrations in urine (100-600 nmol l-1). An oral load of biocytin results in patients in unchanged biotin levels but in a marked rise of biocytin in plasma followed by rapid renal excretion of biocytin whereas in controls biotin levels in plasma increase rapidly and biocytin remains below detection levels.

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

    PubMed

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

    2015-04-01

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

  15. Regulation of immunological and inflammatory functions by biotin.

    PubMed

    Kuroishi, Toshinobu

    2015-12-01

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

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

    SciTech Connect

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

    1986-03-01

    The authors have extended their previous studies of biotin absorption in rat proximal jejunum (PJ) to examine biotin absorptive capacity of rat ileum (I) and proximal colon (PC) using in vivo intestinal loop technique. Intestinal loops (2.5 cm) were filled with 0.3 ml of solution containing (/sup 3/H)-biotin and (/sup 14/C)-inulin in phosphate buffer, pH 6.5. Biotin absorption was determined on the basis of luminal biotin disappearance after correction for inulin recovery and averaged (pmol/loop-10 min; X +/- SEM). In related experiments, 5-cm loops of PJ, distal I (DI), or PC were filled with 0.5 ml of solution of similar composition (1.0 ..mu..M 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.

  17. Final report on the safety assessment of biotin.

    PubMed

    Fiume, M Z

    2001-01-01

    Biotin is a water-soluble vitamin used as a hair-conditioning agent and a skin-conditioning agent in many cosmetic products at concentrations ranging from 0.0001% to 0.6%. Although Biotin does absorb some ultraviolet (UV) radiation, the absorption shows no peaks in the UVA or UVB region. Biotin is rapidly metabolized and excreted in urine. Little acute oral toxicity is seen in animal tests. Short-term and subchronic toxicity studies likewise found no evidence of toxicity. Although intradermal injection of a small quantity of Biotin (0.1 ml) into guinea pig skin did not produce skin irritation, Biotin (0.1% at pH 7.3) did produce slight, transient ocular irritation in rabbit eyes. Biotin was not mutagenic in bacterial tests, but positive results were found in a Tradescantia micronucleus test. There was evidence of an increase in the number of resorptions in rats receiving Biotin by subcutaneous injection, with concomitant decreases in fetal, uterine, and placental weights. Another study of mice receiving Biotin orally or by subcutaneous injection found no differences between control and treatment groups. Although there is one case study reporting an urticarial reaction in the literature, there are a very large number of individuals exposed to Biotin on a daily basis, and there is not a parallel appearance of irritation, sensitization, or other adverse reactions. Based on these available data, it was concluded that Biotin is safe as used in cosmetic formulations.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    2004-01-01

    Abstract 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 cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn. PMID:15188952

  20. Biotin nutritional status of vegans, lactoovovegetarians, and nonvegetarians.

    PubMed

    Lombard, K A; Mock, D M

    1989-09-01

    Urinary excretion of biotin (total avidin-binding substances) was measured in adults and children who were adhering to one of the following self-selected diets: strict vegetarian (vegan), lactoovovegetarian, or mixed (containing meat and dairy products as well as plant-derived foods). In a subset of subjects, plasma biotin concentrations were also measured. In adults the biotin excretion rate was significantly greater in the vegan group than in either the lactoovovegetarian or the mixed-diet groups; the latter were not significantly different from one another. In children the biotin excretion rates in both the vegan group and the lactoovovegetarin group were significantly greater than in the mixed-diet group. A similar trend (vegan greater than lactoovovegetarian greater than mixed) was detected in the plasma concentrations of biotin of adults and children but differences were not generally statistically significant. These observations provide evidence that the biotin nutritional status of vegans is not impaired.

  1. Functional loop dynamics of the streptavidin-biotin complex.

    PubMed

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

    2015-01-20

    Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop(3-4) 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 loop(3-4) 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 loop(3-4) and biotin. (3) The closure of loop(3-4) is concerted to the stable binding of biotin to streptavidin. When the loop(3-4) is in its open-state, biotin moves out of the binding pocket, indicating that the interactions between the loop(3-4) and biotin are essential in trapping biotin in the binding pocket. (4) In the tetrameric streptavidin system, the conformational change of the loop(3-4) 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.

  2. Functional Loop Dynamics of the Streptavidin-Biotin Complex

    NASA Astrophysics Data System (ADS)

    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.

  3. Interaction of immobilized avidin with an aequorin-biotin conjugate: an aequorin-linked assay for biotin.

    PubMed

    Feltus, A; Ramanathan, S; Daunert, S

    1997-12-01

    Biotinylated recombinant aequorin was used in the development of a heterogeneous bioluminescence binding assay for biotin. This assay is based on a competition between a biotinylated aequorin conjugate and biotin for the binding sites of avidin immobilized on solid particles. Dose-response curves were obtained that relate solid-phase aequorin activity to the concentration of biotin. Under certain experimental conditions these curves were biphasic; i.e., as the biotin concentration increased, the solid-phase aequorin activity first increased reaching a maximum and then decreased at higher biotin concentrations. This "hook" effect was observed with four different types of immobilization supports. The effect was more pronounced when low concentrations of aequorin-biotin conjugate were used, and diminished at a high conjugate concentration. This behavior indicates a possible positive cooperativity in the interaction between the immobilized avidin and biotin. Scatchard plot analysis was also consistent with a positive cooperativity mechanism. By using the ascending portion of the dose-response curve, the detection limit of the assay for biotin was 1 x 10(-15) M (100 zmol of biotin in the sample). Copyright 1997 Academic Press.

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

    PubMed Central

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

    2016-01-01

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

  5. Minfong Ho: Politics in Prose

    ERIC Educational Resources Information Center

    Wiggins, Joy L.

    2006-01-01

    In this article, the author interviews Minfong Ho, an award-winning Thai writer of children's and young adult novels. Ho was born in Burma to Chinese parents in 1951, raised in Singapore and Thailand, educated in Bangkok, Taiwan, and at Cornell University in New York. Ho's first novel, "Sing to the Dawn," won first prize from the Council of…

  6. Minfong Ho: Politics in Prose

    ERIC Educational Resources Information Center

    Wiggins, Joy L.

    2006-01-01

    In this article, the author interviews Minfong Ho, an award-winning Thai writer of children's and young adult novels. Ho was born in Burma to Chinese parents in 1951, raised in Singapore and Thailand, educated in Bangkok, Taiwan, and at Cornell University in New York. Ho's first novel, "Sing to the Dawn," won first prize from the Council of…

  7. Biotin ameliorates muscle cramps of hemodialysis patients: a prospective trial.

    PubMed

    Oguma, Shiro; Ando, Itiro; Hirose, Takuo; Totsune, Kazuhito; Sekino, Hiroshi; Sato, Hiroshi; Imai, Yutaka; Fujiwara, Masako

    2012-01-01

    Patients with renal failure undergoing hemodialysis often have muscle cramps during and after the dialysis therapy. Muscle cramps are defined as the sudden onset of a prolonged involuntary muscle contraction accompanied with severe pain, resulting in early termination of a HD session and inadequate dialysis. The etiology of the cramps is unknown and effective anti-cramp medicine is not available. We have hypothesized that water-soluble vitamins are deficient in HD patients. Accordingly, we administrated biotin to 14 patients who had frequent muscle cramps during HD sessions. Oral administration of 1 mg/day biotin promptly reduced the onset and the severity of cramps in 12 patients both during and after HD. Then, the plasma biotin levels were measured by an enzyme-linked immunosorbent assay method (ELISA) in HD patients, including 14 patients with cramps and 13 patients without cramps, and 11 healthy volunteers. Plasma biotin levels were elevated in 27 HD patients at baseline compared with healthy volunteers [451 (377 - 649) vs. 224 (148 - 308) ng/l, median (lower-upper quartiles); p < 0.0001]. Unexpectedly, among the 14 cramp patients, the biotin levels were significantly higher in biotin-ineffective 7 patients than biotin-effective 7 patients [1,064 (710 - 1,187) vs. 445 (359 - 476) ng/l; p < 0.001]. Thus, the biotins measured by ELISA may consist of not only intact biotin but also its metabolites that do not function as a vitamin. In conclusion, biotin administration is one choice to relieve HD patients from muscle cramps regardless of their elevated plasma biotin levels.

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

    Agrawal, Sudhanshu; Said, Hamid M.

    2016-01-01

    The water-soluble biotin (vitamin B7) is indispensable for normal human health. The vitamin acts as a cofactor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte-derived DCs. Culture of DCs in a biotin-deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of the proinflammatory cytokines TNF-α, IL-12p40, IL-23, and IL-1β compared with LPS-activated DCs cultured in biotin-sufficient (control) and biotin-oversupplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN-γ and IL-17 indicating Th1/Th17 bias in T cells compared with cells maintained in biotin control or biotin-oversupplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency. PMID:27413170

  10. A Biotin Biosynthesis Gene Restricted to Helicobacter

    PubMed Central

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

    2016-01-01

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

  11. A Biotin Biosynthesis Gene Restricted to Helicobacter.

    PubMed

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

    2016-02-12

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

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

    PubMed Central

    Du, C; 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 proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin. PMID:8702286

  13. Biotin Production and Utilization in a Sewage Treatment Lagoon 1

    PubMed Central

    Fillipi, Gordon M.; Vennes, John W.

    1971-01-01

    Biotin, in a sewage oxidation lagoon also receiving potato processing wastes, was observed to increase two logs during the summer period of waste stabilization and then to decline to near earlier concentrations. Three organisms, Aerobacter aerogenes, Chlorella vulgaris, and Thiocapsa floridana, were at least partially responsible for these fluctuations; the latter two organisms were associated with biotin utilization and the former with biotin production. Since copious quantities of biotin are produced in these waste treatment facilities, the vitamin may act as a useful indicator of microbial action on certain organic molecules, especially in domestic and industrial wastes such as those from municipalities and potato and sugar beet processing plants. Furthermore, the presence of biotin in rivers and streams may be indicative of the discharge of incompletely stabilized wastes from these sources. PMID:4939123

  14. Structural origins of high-affinity biotin binding to streptavidin.

    PubMed

    Weber, P C; Ohlendorf, D H; Wendoloski, J J; Salemme, F R

    1989-01-06

    The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

  15. Tropospheric HO determination by FAGE

    NASA Technical Reports Server (NTRS)

    Hard, T. M.; Obrien, R. J.; Chan, C. Y.; Mehrabzadeh, A. A.

    1986-01-01

    In the detection of tropospheric HO by laser excited fluorescence, and alternative air-sampling method, named FAGE (Fluorescence Assay with Gas Expansion) was introduced. Here the air is expanded through a nozzle prior to excitation, in order to improve the ratio of the HO signal to the scattered, fluorescent, and photolytic backgrounds. The improvement comes from the differing pressure dependence of the intensities of these four terms, as well as the distinguishability of their temporal waveforms at low pressures when excited by a pulsed laser. HO has been excited by a YAG/dye laser. Other lasers and pumping paths may perform as well or better in this method. With FAGE, chemical modulation of the HO signal was achieved by hydrocarbon addition to the nozzle flow, converting photolytic HO from an interference to a background. Chemical calibration of the instrumental response to external HO was also achieved, by hydrocarbon decay, at HO concentrations within the ambient range.

  16. The Biotin/Avidin complex adhesion force

    NASA Astrophysics Data System (ADS)

    Balsera, Manel A.; Izrailev, Sergei; Stepaniants, Sergey; Oono, Yoshitsugu; Schulten, Klaus

    1997-03-01

    The vitamin Biotin and the protein avidin form one of the strongest non-covalent bonds between biological molecules. We have performed molecular and stochastic dynamic modeling of the unbinding of this complex(Izrailev et al., Biophysical Journal, In press). These simulations provide insight into the effect of particular residues and water on the tight binding of the system. With the aid of simple phenomenological models we have related qualitatively our results to Atomic Force Microscopy adhesion force measurements (E.-L. Florin, V. T. Moy and H. E. Gaub Science) 264:415-417 and kinetic dissociation experiments( A. Chilcotti and P. S. Stayton, J. Am. Chem. Soc.) 117:10622-10628. We will discuss the difficulties preventing a more quantitative understanding of the unbinding force and kinetics.

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

    PubMed Central

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

    2006-01-01

    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 the rate of fetal malformations in ICR mice. Biotin status was controlled by feeding diets with varying egg white concentration. In dams and fetuses, biotin status was assessed by hepatic biotin content and hepatic activity of the biotin-dependent enzyme propionyl-CoA carboxylase; in dams, status was also assessed by urinary excretion of biotin and 3-hydroxyisovaleric acid. Malformations were assessed morphologically. Biotin was measured by HPLC/avidin-binding assay. Propionyl-CoA carboxylase (PCC) activity was determined by H14CO3 incorporation. 3-Hydroxyisovaleric acid concentration was determined by GC/MS. Although no overt signs of deficiency appeared, metabolic disturbances caused by biotin deficiency were detectable in dams and fetuses. These disturbances increased with increasing egg white. Fetal biotin status correlated significantly with maternal biotin status (fetal vs. dam hepatic biotin, r = 0.671; fetal vs. dam PCC activity, r = 0.70). The incidences of malformations were strikingly dependent on egg white concentration. We conclude that in ICR mice, marginal maternal biotin deficiency causes fetal biotin deficiency. We speculate that the fetal malformations are primarily the consequence of fetal biotin deficiency. Because murine malformations appeared at degrees of biotin deficiency that are similar to those in human gestation, we speculate that some human fetal malformations may be caused by biotin deficiency. PMID:12888630

  18. Early Mechanistic Events in Biotin Dissociation from Streptavidin

    SciTech Connect

    Hyre, D. E.

    2002-01-01

    The streptavidin-biotin system has provided a unique opportunity to investigate the molecular details of ligand dissociation pathways. An underlying mechanistic question is whether ligand dissociation proceeds with a relatively ordered process of bond breaking and ligand escape. Here we report a joint computational and crystallographic study of the earliest events in biotin dissociation. In molecular dynamics potential of mean force simulations, a water molecule from a defined access channel intercalated into the hydrogen bond between Asp 128 and biotin, bridging them and stabilizing an intermediate state. In forced biotin dissociation simulations, this event led to subsequent bond breaking steps and ligand escape. In equilibrium simulations, the water molecule was sometimes observed to move back to the access channel with re-formation of the biotin hydrogen bond. Analysis of streptavidin crystal structures revealed a close overlap of crystallographically defined and simulated waters in the water access channel. These results suggest that biotin dissociation is initiated by stochastic coupling of water entry with lengthening of a specific biotin hydrogen-bonding interaction.

  19. Biotin deficiency in an infant fed with amino acid formula.

    PubMed

    Fujimoto, Wataru; Inaoki, Makoto; Fukui, Toru; Inoue, Yoshito; Kuhara, Tomiko

    2005-04-01

    Biotin deficiency is rarely encountered in an infant on weaning from breast and formula feeding. It is characterized by alopecia and scaly, erythematous dermatitis distributed around the body orifices. We report a 5-month-old Japanese infant with typical skin lesions who had been diagnosed as a neonate with dyspepsia and fed only an amino acid formula. Serum and urine levels of biotin were below the normal range, but zinc and biotinidase were within normal range. Urinary excretion of 3-methylcrotonylglycine, 3-hydroxyisovaleric acid, and methylcitric acid was significantly elevated. Daily oral supplementation with 1 mg of biotin resulted in dramatic improvement of the periorificial dermatitis and hair growth together with a complete disappearance of the organic aciduria. Our case shows that the characteristic skin manifestations are the most important clue to the diagnosis of biotin deficiency and demonstrated that urinary excretion of biotin and organic aciduria, rather than the serum concentration of biotin, are the sensitive indicators for evaluating the patient's status of biotin deficiency.

  20. Tropospheric HO2 determination by FAGE

    NASA Technical Reports Server (NTRS)

    Hard, Thomas M.; George, L. A.; Obrien, Robert J.

    1994-01-01

    In the present work, HO2 is determined by FAGE, a method which employs low-pressure laser-excited fluorescence to observe the hydroxyl radical HO, using chemical modulation to distinguish the desired signal from the background. In the HO2 determination mode, NO is added to the low-pressure flowing sample, converting HO2 to HO.

  1. Low biotin content of infant formulas made in Japan.

    PubMed

    Watanabe, T; Fukui, T

    1998-07-01

    Since minor vitamins such as biotin are not yet registered as food additives in Japan, these cannot be used to supplement infant formulas or foods, despite their nutritional value. Therefore, the biotin contents of infant formulas made in Japan were determined and compared with those from the United States (US). The Japanese products were obtained from five companies, while the US products were limited to those from one company. The average content of total biotin in 11 Japanese products for nursing of normal infants (standard formulas) was 1.04 micrograms/100 kcal (0.69 microgram/100 ml), which was significantly lower than that in two US products (2.56 micrograms/100 kcal, 1.71 micrograms/100 ml). The total biotin content in 26 Japanese infant formulas specially prepared for medical treatment and prevention of disease (special formulas) was 0.45 microgram/100 kcal (0.30 microgram/100 ml) on average. This was less than a fifth of the level in the three US products (1.82 micrograms/100 kcal, 1.22 micrograms/100 ml). There was no difference in the proportion of free biotin (the percent of active biotin to total biotin) between the Japanese and US standard formulas (67.7% and 77.2%, respectively). However, in special formulas the mean proportion of the free biotin in the Japanese products (29.4%) was lower than that in the US products (71.2%). The nutrient intake of infants is limited to the maternal milk and/or infant formulas. These findings suggest that Japanese infant formulas should be appropriately improved to maintain growth and good health. Biotin should be registered as a food additive in Japan so that it can be used to fortify infant formulas.

  2. Biotin-dependent Enzymes: the Work of Feodor Lynen

    PubMed Central

    Kresge, Nicole; Simoni, Robert D.; Hill, Robert L.

    2009-01-01

    The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (+)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of the Reaction (Lane, M. D., Young, D. L., and Lynen, F. (1964)J. Biol. Chem.239, 2858–2864)

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

    PubMed

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

    2015-12-16

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

  4. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    PubMed

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  5. Multiple carboxylase deficiency: inherited and acquired disorders of biotin metabolism.

    PubMed

    Baumgartner, E R; Suormala, T

    1997-01-01

    Acquired biotin deficiency and the two known congenital disorders of biotin metabolism, biotinidase and holocarboxylase synthetase (HCS) deficiency, all lead to deficiency of the 4 biotin-dependent carboxylases, i.e. to multiple carboxylase deficiency (MCD). The underlying mechanism in HCS-deficiency, discovered in 1981, is decreased affinity of HCS for biotin impairing the formation of holocarboxylases at physiological biotin levels. In biotinidase deficiency, discovered in 1983, MCD results from progressive development of biotin-deficiency due to inability to liberate and recycle biotin which is lost in urine as biocytin. MCD leads to typical organic aciduria and severe life-threatening illness. Main symptoms and signs are feeding difficulties, neurologic abnormalities (hypotonia, impaired consciousness, seizures, ataxia) and cutaneous changes (rash, alopecia). However, the clinical presentation and age of onset are extremely variable, and organic aciduria may initially be absent in biotinidase deficiency. Therefore, the definitive diagnosis requires enzyme studies. MCD can be detected in lymphocytes obtained before treatment and biotinidase deficiency is confirmed or excluded by a colorimetric enzyme assay in plasma. Newborn screening for biotinidase deficiency has resulted in the detection of patients with partial deficiency (10-30% of mean normal activity) in addition to patients with profound deficiency (0-10%). Severe illness has been observed mainly in patients with O-activity or a Km-mutation, detection of which requires detailed investigation. HCS-deficiency has to be confirmed by enzyme assay in cultured cells. Both congenital disorders respond clinically and biochemically to oral biotin therapy. Whereas 10 mg/day or less is sufficient to treat profound biotinidase deficiency, the optimal biotin dose for patients with HCS-deficiency must be assessed individually. The prognosis of both disorders is good if biotin therapy is introduced early and continued

  6. Kinetic spectrophotometric determination of biotin in pharmaceutical preparations.

    PubMed

    Walash, M I; Rizk, M; Sheribah, Z A; Salim, M M

    2008-09-01

    A simple accurate kinetic spectrophotometric method was developed for the determination of biotin in pure form and pharmaceutical preparations. The proposed method is based on a catalytic acceleration of biotin on the reaction between sodium azide and tri-iodide in an aqueous solution. Concentration range of 4-16 μg/mL for biotin was determined by measuring the decrease in the absorbance of tri-iodide at 348 nm by a fixed time method. The decrease in absorbance after 14 min from the initiation of the reaction was markedly correlated to the concentration with correlation coefficient of 0.9999. The detection limit (LOD) of biotin was 0.18 μg/mL while quantitation limit (LOQ) was 0.54 μg/mL. The percentage recovery of the spiked samples was 100.08 ± 0.761. The proposed procedure was successfully applied for the determination of biotin in its pharmaceutical preparations with mean recoveries of 101.17 ± 2.05 and 97.87 ± 1.50 for biotin ampoules and capsules, respectively. The results obtained were in good agreement with those obtained using official method.

  7. Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

    PubMed Central

    Napier, Brooke A.; Meyer, Lena; Bina, James E.; Miller, Mark A.; Sjöstedt, Anders; Weiss, David S.

    2012-01-01

    Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15–60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens. PMID:23071317

  8. Kinetic Spectrophotometric Determination of Biotin in Pharmaceutical Preparations

    PubMed Central

    Walash, M. I.; Rizk, M.; Sheribah, Z. A.; Salim, M. M.

    2008-01-01

    A simple accurate kinetic spectrophotometric method was developed for the determination of biotin in pure form and pharmaceutical preparations. The proposed method is based on a catalytic acceleration of biotin on the reaction between sodium azide and tri-iodide in an aqueous solution. Concentration range of 4-16 μg/mL for biotin was determined by measuring the decrease in the absorbance of tri-iodide at 348 nm by a fixed time method. The decrease in absorbance after 14 min from the initiation of the reaction was markedly correlated to the concentration with correlation coefficient of 0.9999. The detection limit (LOD) of biotin was 0.18 μg/mL while quantitation limit (LOQ) was 0.54 μg/mL. The percentage recovery of the spiked samples was 100.08 ± 0.761. The proposed procedure was successfully applied for the determination of biotin in its pharmaceutical preparations with mean recoveries of 101.17 ± 2.05 and 97.87 ± 1.50 for biotin ampoules and capsules, respectively. The results obtained were in good agreement with those obtained using official method. PMID:23675096

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

    PubMed

    Wang, Jingheng; Beckett, Dorothy

    2017-08-01

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

  10. Supplemental biotin for swine. I. Influence on feedlot performance, plasma biotin and toe lesions in developing gilts.

    PubMed

    Bryant, K L; Kornegay, E T; Knight, J W; Webb, K E; Notter, D R

    1985-01-01

    Three trials, utilizing a total of 240 crossbred gilts, were conducted to study the influence of 0 (NB) or 220 (SB) microgram supplemental biotin/kg of diet on feedlot performance, plasma biotin and development of toe lesions in developing gilts. Corn-soybean meal diets were fed from weaning to 92 kg body weight. Gilts were housed on expanded-metal floors to 50 kg body weight and on partially slatted concrete floors until completion of the trials. Feedlot performance, hair and structural soundness scores were not different (P greater than .10) between NB and SB gilts. Plasma biotin (PB) levels were elevated (P less than .01) when supplemental biotin was included in the diet. Gilts consuming SB diets had fewer (P less than .01) toe lesions/gilt and lower (P less than .01) toe lesion severity scores/gilt compared with gilts fed NB diets. Heel-horn erosion and heel cracks were the most frequent toe lesions observed. Fewer gilts (P less than .01) developed side-wall toe cracks when fed SB diets. Supplemental biotin reduced the frequency of individual toes containing heel-horn erosion (P less than .10), heel cracks (P less than .05) and side-wall toe cracks (P less than .05) with the severity of these lesions not affected (P greater than .10) by supplemental biotin. These results suggest that biotin levels in corn-soybean meal diets are adequate for feedlot performance, and that supplemental biotin can improve hoof integrity, but will not prevent toe lesions.

  11. In vivo biotinylation of bacterial magnetic particles by a truncated form of Escherichia coli biotin ligase and biotin acceptor peptide.

    PubMed

    Maeda, Yoshiaki; Yoshino, Tomoko; Matsunaga, Tadashi

    2010-09-01

    Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.

  12. Biotin and Lipoic Acid: Synthesis, Attachment and Regulation

    PubMed Central

    Cronan, John E.

    2014-01-01

    Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses

  13. Biotin analogues with antibacterial activity are potent inhibitors of biotin protein ligase.

    PubMed

    Soares da Costa, Tatiana P; Tieu, William; Yap, Min Y; Zvarec, Ondrej; Bell, Jan M; Turnidge, John D; Wallace, John C; Booker, Grant W; Wilce, Matthew C J; Abell, Andrew D; Polyak, Steven W

    2012-06-14

    There is a desperate need to develop new antibiotic agents to combat the rise of drug-resistant bacteria, such as clinically important Staphylococcus aureus. The essential multifunctional enzyme, biotin protein ligase (BPL), is one potential drug target for new antibiotics. We report the synthesis and characterization of a series of biotin analogues with activity against BPLs from S. aureus, Escherichia coli, and Homo sapiens. Two potent inhibitors with K i < 100 nM were identified with antibacterial activity against a panel of clinical isolates of S. aureus (MIC 2-16 μg/mL). Compounds with high ligand efficiency and >20-fold selectivity between the isozymes were identified and characterized. The antibacterial mode of action was shown to be via inhibition of BPL. The bimolecular interactions between the BPL and the inhibitors were defined by surface plasmon resonance studies and X-ray crystallography. These findings pave the way for second-generation inhibitors and antibiotics with greater potency and selectivity.

  14. Effects of cecal oxytetracycline infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts.

    PubMed

    Hamilton, C R; Veum, T L

    2012-11-01

    The objective of this 49-d experiment was to test effects of cecal oxytetracycline (OTC) infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts. Twenty-eight crossbred gilts with an initial age of 160 d and BW of 120 kg were surgically fitted with a T-cannula in the terminal ileum, a cecal fistula, and an indwelling catheter in the anterior vena cava, and allotted to 7 dietary treatments. Treatments with the basal semipurified (SP) diet fed at 1.86 kg/d were: SP-1, negative control; SP-2, positive control with 270 μg of biotin/kg; SP-3, with spray-dried egg albumen (EA, 100 g/d) and OTC (2.56 g/d by cecal infusion); and SP-4, with EA, OTC, and 700 μg of biotin/kg. Treatments with the basal corn-soybean meal (CS) diet fed at 1.80 kg/d were: CS-1, negative control; CS-2, with EA and OTC; and CS-3, with EA, OTC, and 700 μg of biotin/kg. Response criteria were: fecal bacteria counts; plasma concentrations of biotin, glucose, and urea N (PUN); liver pyruvate carboxylase (PC) activity; kidney and epithelial tissue histology; ileal and fecal biotin concentrations; ileal and total tract N and energy utilization; daily gilt observation; and BW gain. Blood samples were collected every 7 d with serial samples collected on d 49. Total urine collections and fecal grab samples were made twice daily from d 44 to 49. Gilts were killed on d 50 and liver, kidney, and skin samples were collected. No gilts had symptoms of biotin deficiency. There were no treatment differences in BW gain, plasma glucose concentrations, liver PC activity, kidney and epithelial tissue histology, or fecal bacteria counts. Ileal and total tract N and energy digestibilities (%) did not differ among treatments within the same protein source, with greater (P ≤ 0.05) values for gilts on the SP treatments than the CS treatments. However, N retained/N absorbed and N retained/N intake (%) were less (P ≤ 0.05) and PUN concentrations were greater (P ≤ 0.05) for SP

  15. HO:LULF and HO:LULF Laser Materials

    NASA Technical Reports Server (NTRS)

    Barnes, Norman P. (Inventor); Morrison, Clyde A. (Inventor); Filer, Elizabeth D. (Inventor); Jani, Mahendra G. (Inventor); Murray, Keith E. (Inventor); Lockard, George E. (Inventor)

    1998-01-01

    A laser host material LULF (LuLiF4) is doped with holmium (Ho) and thulium (Tm) to produce a new laser material that is capable of laser light production in the vicinity of 2 microns. The material provides an advantage in efficiency over conventional Ho lasers because the LULF host material allows for decreased threshold and upconversion over such hosts as YAG and YLF. The addition of Tm allows for pumping by commonly available GaAlAs laser diodes. For use with flashlamp pumping, erbium (Er) may be added as an additional dopant. For further upconversion reduction, the Tm can be eliminated and the Ho can be directly pumped.

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

    PubMed

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

    2015-01-01

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

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

    PubMed

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

    2015-07-15

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

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

    PubMed Central

    Ghosal, Abhisek; Jellbauer, Stefan; Kapadia, Rubina; Raffatellu, Manuela

    2015-01-01

    Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium (S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines. PMID:25999427

  19. Effect of biotin supplementation of a barley-wheat diet on restoration of healthy feet, legs and skin of biotin deficient sows.

    PubMed

    Misir, R; Blair, R

    1986-03-01

    Sixteen sows with induced biotin deficiency were divided into groups of four and fed an egg-white-free barley-wheat based diet without added biotin (control) or supplemented with one of three levels of synthetic d-biotin (100, 200 or 400 micrograms kg-1) for two parities in order to determine the optimal level required to reverse symptoms of the deficiency and to evaluate the relationship among dietary biotin levels, stage of recovery and serum biotin concentrations. Biotin supplementation significantly reduced (P less than 0.05) the incidence of lesions (on the foot pads, above the dew claws and on the skin) and also dry skin, but had no effect (P greater than 0.05) on hoof cracks, hair score or hair density. Time after supplementation had significant effects on reversal of most biotin deficiency symptoms. Interactions between biotin supplementation and time after supplementation suggested that these symptoms were reversed more quickly when higher levels of biotin were fed. Serum biotin concentrations of biotin-supplemented sows were increased with time and exceeded 600 ng litre-1 after 0.5, 1.0 and 1.5 months following biotin supplementation of the basal diet with 400, 200 and 100 micrograms kg-1. In contrast, serum biotin concentrations of the control sows remained low and peaked at 411 ng litre-1 after 2.5 months. The data suggested that supplementation of a barley-wheat diet to provide 147 to 217 micrograms available biotin kg-1 might be adequate to restore healthy feet, legs and skin and concurrently maintain high serum biotin concentrations.

  20. {sup 163}Ho based experiments

    SciTech Connect

    Gastaldo, Loredana

    2015-07-15

    The analysis of the endpoint region of the calorimetrically measured {sup 163}Ho electron capture spectrum is a very promising way to determine the mass of the electron neutrino. The achievable sensitivity of {sup 163}Ho-based experiments and the experimental challenges will be presented. Three large collaborations aim at developing large scale experiments able to reach sub-eV sensitivity. Presently pilot experiments are performed to demonstrate the possibility to calorimetrically measure high precision and high statistics {sup 163}Ho spectra. The different approaches as well as the state of the art of the experimental efforts for the three collaborations will be discussed.

  1. Recent development of biotin conjugation in biological imaging, sensing, and target delivery.

    PubMed

    Ren, Wen Xiu; Han, Jiyou; Uhm, Soojin; Jang, Yu Jin; Kang, Chulhun; Kim, Jong-Hoon; Kim, Jong Seung

    2015-07-04

    Despite encouraging results from preliminary studies of anticancer therapies, the lack of tumor specificity remains an important issue in the modern pharmaceutical industry. New findings indicate that biotin or biotin-conjugates could be favorably assimilated by tumor cells that over-express biotin-selective transporters. Furthermore, biotin can form stable complexes with avidin and its bacterial counterpart streptavidin. The strong bridging between avidin and biotin moieties on other molecules is a proven adaptable tool with broad biological applications. Under these circumstances, a biotin moiety is certainly an attractive choice for live-cell imaging, biosensing, and target delivery.

  2. Possible biotin deficiency in adults receiving long-term total parenteral nutrition.

    PubMed

    Innis, S M; Allardyce, D B

    1983-02-01

    Two adult patients receiving total parenteral nutrition on a long-term home basis presented with severe loss of hair. Both patients had extensive gut resection, consumed no biotin orally and received no biotin parenterally. Supplementation with Berroca-C, one ampule containing 200 micrograms biotin per day resulted in gradual regrowth of healthy hair. The patients now receive a parenteral solution containing biotin and have shown no recurrence of alopecia. It is suggested that biotin deficiency can occur in the adult when no preformed biotin is provided to the body and the contribution of this vitamin from intestinal microbial biosynthesis is compromised.

  3. Biotin determination in food supplements by an electrochemical magneto biosensor.

    PubMed

    Kergaravat, Silvina V; Gómez, Gabriel A; Fabiano, Silvia N; Laube Chávez, Tamara I; Pividori, María I; Hernández, Silvia R

    2012-08-15

    An electrochemical magneto biosensor for the rapid determination of biotin in food samples is reported. The affinity reaction was performed on streptavidin-modified magnetic microbeads as a solid support in a direct competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP) competes with free biotin in the sample for the binding sites of streptavidin on the magnetic microbeads. The modified magnetic beads were then easily captured by a magneto graphite-epoxy composite electrode and the electrochemical signal was based on the enzymatic activity of the HRP enzyme under the addition of H(2)O(2) as the substrate and o-phenilendiamine as cosubstrate. The response was electrochemically detected by square wave voltammetry. The limit of detection was 8.4×10(-8) mol L(--1) of biotin (20 μg L(--1)) with a dynamic range from 0.94 to 2.4×10(-7) mol L(--1). Biotin-fortified commercial dietary supplement and infant formula samples were evaluated obtaining good performances in the results. Total time of analysis was 40 min per 20 assays.

  4. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  5. Molecular origins of the slow streptavidin-biotin dissociation kinetics

    SciTech Connect

    Chilkoti, A.; Stayton, P.S.

    1995-11-01

    The association of streptavidin and avidin with biotin is among the strongest known noncovalent protein-ligand interactions (K{sub a} nearly equals 2.5 x 10{sup 13} M{sup -1}) and is controlled by an exceptionally slow off-rate. We have used this model system to elucidate the role of aromatic tryptophan side-chain binding contacts in the dissociation reaction coordinate and relatedly to the construction of the activation barrier and to the structure of the transition state. We have also conducted a transition state analysis of the temperature-dependent dissociation kinetics, which along with the independent estimation of the equilibrium biotin-binding free energies and enthalpies has provided thermodynamic profiles defining the enthalpic, entropic, and free energy barriers to dissociation for the mutants relative to wild-type streptavidin. The increased biotin off-rate for W79F, which contacts the valeric acid moiety of biotin, and for W120F, which partially caps the bicyclic ring system, is caused largely by free energy destabilization of the ligand-bound ground state relative to wild-type streptavidin. W79F displays an increased equilibrium binding enthalpy relative to wild-type, and thus streptavidin sacrifices potential binding enthalpy to minimize the entropic costs of biotin immobilization. 26 refs., 5 figs., 4 tabs.

  6. Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

    PubMed

    White, H B; Dennison, B A; Della Fera, M A; Whitney, C J; McGuire, J C; Meslar, H W; Sammelwitz, P H

    1976-08-01

    1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.

  7. Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

    PubMed Central

    White, H B; Dennison, B A; Della Fera, M A; Whitney, C J; McGuire, J C; Meslar, H W; Sammelwitz, P H

    1976-01-01

    1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen. PMID:962874

  8. MORPHOLOGICAL ABERRATION OF ARTHROBACTER GLOBIFORMIS CELLS DUE TO BIOTIN DEFICIENCY.

    PubMed

    CHAN, E C

    1964-03-01

    Chan, E. C. S. (University of New Brunswick, Fredericton, New Brunswick, Canada). Morphological aberration of Arthrobacter globiformis cells due to biotin deficiency. J. Bacteriol. 87:641-651. 1964.-Morphological aberration of Arthrobacter globiformis strain 425 was shown to occur during growth in a chemically defined medium without added biotin. Such aberrant cells could revert back to normal coccoid forms upon inoculation into fresh medium supplemented with the vitamin. This abnormal cellular development occurred even when there was good growth (turbidity) or increase in total cell mass. Light photomicrographs of negative and cell-wall stains of the organism at different times of the morphological growth cycle are presented in support of these observations. The relationship between cellular aberration and the biochemical role of biotin is briefly discussed.

  9. Marginal Maternal Biotin Deficiency in CD-1 Mice Reduces Fetal Mass of Biotin-dependent Carboxylases12

    PubMed Central

    Sealey, Wendy M.; Stratton, Shawna L.; Mock, Donald M.; Hansen, Deborah K.

    2005-01-01

    Marginal maternal biotin deficiency reduces hepatic activity of biotin-dependent carboxylases and causes high rates of fetal birth defects in mice. We tested the hypothesis that the decreased carboxylase activity observed in deficient dams and their offspring is mediated by decreased abundance of biotinylated carboxylases, decreased expression of their mRNAs, or both. During gestation, CD-1 mice were fed a diet that induced biotin deficiency or a biotin-sufficient diet. On gestational d 17, gravid uteri were removed, and each live fetus was examined grossly for defects. The expected high incidence of cleft palate (83%) in offspring was observed. In maternal and fetal liver, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and β-methylcrotonyl-CoA carboxylase abundances were determined by Western blotting; the content of mRNAs for most of these enzymes and holocarboxylase synthetase was determined by real-time RT-PCR. Biotin deficiency significantly reduced the abundance of the carboxylases in maternal and fetal liver; neither the content of mRNAs for the carboxylases nor holocarboxylase synthetase changed. This study provides evidence that the decrease in carboxylase activities is attributable to a decrease in the abundance of biotinylated carboxylases; further, this effect is more severe in fetuses than dams. PMID:15867267

  10. Compact Ho:YLF Laser

    NASA Technical Reports Server (NTRS)

    Hemmati, H.

    1988-01-01

    Longitudinal pumping by laser diodes increases efficiency. Improved holmium:yttrium lithium fluoride laser radiates as much as 56 mW of power at wavelength of 2.1 micrometer. New Ho:YLF laser more compact and efficient than older, more powerful devices of this type. Compact, efficient Ho:YLF laser based on recent successes in use of diode lasers to pump other types of solid-state lasers.

  11. Streptavidin and its biotin complex at atomic resolution

    SciTech Connect

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

    2011-09-01

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

  12. A streptavidin variant with slower biotin dissociation and increased mechanostability

    PubMed Central

    Chivers, Claire E.; Crozat, Estelle; Chu, Calvin; Moy, Vincent T.; Sherratt, David J.; Howarth, Mark

    2010-01-01

    Streptavidin binds biotin-conjugates with exceptional stability, but dissociation does occur and can be limiting in imaging, DNA amplification, and nanotechnology. We identified a mutant streptavidin, which we call traptavidin, showing ~10-fold slower biotin off-rate, increased mechanical strength, and improved thermostability; this resilience should find diverse applications. We show that the motor protein FtsK could strip proteins from DNA, rapidly displacing streptavidin from biotinylated DNA; traptavidin resisted displacement and thus indicated the force generated by FtsK translocation. PMID:20383133

  13. Biochemical Hyperthyroidism in a Newborn Baby Caused by Assay Interaction from Biotin Intake

    PubMed Central

    Bülow Pedersen, Inge; Laurberg, Peter

    2016-01-01

    We describe a case of biochemical neonatal thyrotoxicosis caused by biotin supplementation. Biotin may interact with thyroid function testing to imitate thyrotoxicosis with low thyroid-stimulating hormone and elevated triiodothyronine and thyroxine levels. PMID:27843813

  14. Biochemical Hyperthyroidism in a Newborn Baby Caused by Assay Interaction from Biotin Intake.

    PubMed

    Bülow Pedersen, Inge; Laurberg, Peter

    2016-09-01

    We describe a case of biochemical neonatal thyrotoxicosis caused by biotin supplementation. Biotin may interact with thyroid function testing to imitate thyrotoxicosis with low thyroid-stimulating hormone and elevated triiodothyronine and thyroxine levels.

  15. Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein.

    PubMed

    Seshagiri, P B; Adiga, P R

    1987-12-07

    By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin.

  16. Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies.

    PubMed Central

    Swamy, M J; Heimburg, T; Marsh, D

    1996-01-01

    Fourier-transform infrared studies have been carried out to investigate the secondary structure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondary structure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipid assemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondary structure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondary structure. The complexes of avidin with both biotin and membrane-bound DMBPE lipid assemblies display a large increase in thermal stability compared with the native protein. PMID:8842222

  17. Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies.

    PubMed

    Swamy, M J; Heimburg, T; Marsh, D

    1996-08-01

    Fourier-transform infrared studies have been carried out to investigate the secondary structure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondary structure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipid assemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondary structure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondary structure. The complexes of avidin with both biotin and membrane-bound DMBPE lipid assemblies display a large increase in thermal stability compared with the native protein.

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

    PubMed

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

    2017-09-01

    In order to improve the efficacy of photodynamic therapy (PDT), biotin was axially conjugated with silicon(IV) phthalocyanine (SiPc) skeleton to develop a new tumor-targeting photosensitizer SiPc-biotin. The target compound SiPc-biotin showed much higher binding affinity toward BR-positive (biotin receptor overexpressed) HeLa human cervical carcinoma cells than its precursor SiPc-pip. However, when the biotin receptors of HeLa cells were blocked by free biotin, >50% uptake of SiPc-biotin was suppressed, demonstrating that SiPc-biotin could selectively accumulate in BR-positive cancer cells via the BR-mediated internalization. The confocal fluorescence images further confirmed the target binding ability of SiPc-biotin. As a consequence of specificity of SiPc-biotin toward BR-positive HeLa cells, the photodynamic effect was also largely dependent on the BR expression level of HeLa cells. The photodynamic activities of SiPc-biotin against HeLa cells were dramatically reduced when the biotin receptors were blocked by the free biotin (IC50: 0.18μM vs. 0.46μM). It is concluded that SiPc-biotin can selectively damage BR-positive cancer cells under irradiation. Furthermore, the dark toxicity of SiPc-biotin toward human normal liver cell lines LO2 was much lower than that of its precursor SiPc-pip. The targeting photodynamic activity and low dark toxicity suggest that SiPc-biotin is a promising photosensitizer for tumor-targeting photodynamic therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Two New Antimetabolites of Biotin: α-Methyldethiobiotin and α-Methylbiotin

    PubMed Central

    Hanka, L. J.; Martin, D. G.; Reineke, L. M.

    1972-01-01

    Two new antimetabolites of biotin were isolated from culture filtrates of Streptomyces lydicus: β-methyldethiobiotin and β-methylbiotin. 14C-biotin or 14C-pimelic acid was not incorporated into either of these antimetabolites by the growing culture. Neither of the compounds could substitute for the biotin requirement in Saccharomyces cerevisiae. Both compounds had a strong and rather specific antimicrobial effect against mycobacteria. Their antimicrobial activities were reversed by biotin. Both compounds had an affinity for avidin. PMID:4680803

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2017-05-01

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

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

    PubMed

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

    2016-09-01

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

  3. Systematic evaluation of avidin-biotin interaction by fluorescence spectrophotometry.

    PubMed

    Sun, Shuguo; Huang, Xi; Ma, Meihu; Qiu, Ning; Cai, Zhaoxia; Luo, Zhang; Alies, Nichia Primadani

    2012-04-01

    The avidin-biotin interaction was evaluated systematically by fluorescence spectroscopy under different conditions of temperature, pressure, pH, metal ions, incubation time and initial avidin concentration. The binding constant was calculated according to the modified Stern-Volmer equation, which deduced the existence of static quenching mechanism. The data obtained revealed that avidin-biotin interaction exhibited temperature, pH, metal ions, incubation time and initial avidin concentration sensitivity. The binding constants decreased with increase in temperature, while the binding sites were independent of temperature. The values of thermodynamic parameter ΔH (-149.85 kJ mol(-1)) and ΔS (-284.26 J mol(-1) K(-1)) suggested hydrogen bonds and van der Waals played a major role in the reaction. The binding constants between avidin and biotin increased firstly and then decreased gradually with the increase of pH values. Metal ions can also affected the binding constants between avidin and biotin. The association kinetics firstly acquired by the combination of the change in fluorescence per unit time and the modified Stern-Volmer equation indicated that the reaction time required to reach equilibrium was 2200 s, and the average reaction rate for the binding process was very high in the first 180 s. Reaction of the avidin in the first 180 s was more than 40% of the total avidin involved in the whole process. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  4. Systematic evaluation of avidin-biotin interaction by fluorescence spectrophotometry

    NASA Astrophysics Data System (ADS)

    Sun, Shuguo; Huang, Xi; Ma, Meihu; Qiu, Ning; Cai, Zhaoxia; Luo, Zhang; Alies, Nichia Primadani

    2012-04-01

    The avidin-biotin interaction was evaluated systematically by fluorescence spectroscopy under different conditions of temperature, pressure, pH, metal ions, incubation time and initial avidin concentration. The binding constant was calculated according to the modified Stern-Volmer equation, which deduced the existence of static quenching mechanism. The data obtained revealed that avidin-biotin interaction exhibited temperature, pH, metal ions, incubation time and initial avidin concentration sensitivity. The binding constants decreased with increase in temperature, while the binding sites were independent of temperature. The values of thermodynamic parameter ΔH (-149.85 kJ mol-1) and ΔS (-284.26 J mol-1 K-1) suggested hydrogen bonds and van der Waals played a major role in the reaction. The binding constants between avidin and biotin increased firstly and then decreased gradually with the increase of pH values. Metal ions can also affected the binding constants between avidin and biotin. The association kinetics firstly acquired by the combination of the change in fluorescence per unit time and the modified Stern-Volmer equation indicated that the reaction time required to reach equilibrium was 2200 s, and the average reaction rate for the binding process was very high in the first 180 s. Reaction of the avidin in the first 180 s was more than 40% of the total avidin involved in the whole process.

  5. Measurement of posttransfusion red cell survival with the biotin label.

    PubMed

    Mock, Donald M; Widness, John A; Veng-Pedersen, Peter; Strauss, Ronald G; Cancelas, Jose A; Cohen, Robert M; Lindsell, Christopher J; Franco, Robert S

    2014-07-01

    The goal of this review is to summarize and critically assess information concerning the biotin method to label red blood cells (RBC) for use in studies of RBC and transfusion biology-information that will prove useful to a broad audience of clinicians and scientists. A review of RBC biology, with emphasis on RBC senescence and in vivo survival, is included, followed by an analysis of the advantages and disadvantages of biotin-labeled RBC (BioRBC) for measuring circulating RBC volume, posttransfusion RBC recovery, RBC life span, and RBC age-dependent properties. The advantages of BioRBC over (51)Cr RBC labeling, the current reference method, are discussed. Because the biotin method is straightforward and robust, including the ability to follow the entire life spans of multiple RBC populations concurrently in the same subject, BioRBC offers distinct advantages for studying RBC biology and physiology, particularly RBC survival. The method for biotin labeling, validation of the method, and application of BioRBCs to studies of sickle cell disease, diabetes, and anemia of prematurity are reviewed. Studies documenting the safe use of BioRBC are reviewed; unanswered questions requiring future studies, remaining concerns, and regulatory barriers to broader application of BioRBC including adoption as a new reference method are also presented. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    MedlinePlus

    ... in the SLC19A3 gene likely result in a protein with impaired ability to transport thiamine into cells, resulting in decreased absorption of the vitamin and leading to neurological dysfunction. In this disorder, ... protein. Others propose that biotin transporter proteins may interact ...

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

    PubMed

    Li, R; Kast, J

    2017-01-01

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

  8. Biotin deficiency blocks thymocyte maturation, accelerates thymus involution, and decreases nose-rump length in mice.

    PubMed

    Báez-Saldaña, Armida; Ortega, Enrique

    2004-08-01

    Biotin deficiency in experimental animals causes low body weight as well as several phenomena suggestive of an altered immune system. We reported previously that chronic biotin deficiency in mice decreases body weight and alters the number and proportion of lymphocyte subpopulations in the spleen. To further characterize the effects of biotin deficiency, we studied in detail the maturation of thymocytes and the status of biotin in the thymus, as well as the body length of biotin-deficient mice. Male Balb/cAnN mice were fed for up to 20 wk either standard control diet, a biotin-deficient diet, or a biotin-sufficient diet. At different times, nose-rump length, weight of the thymus, spleen and liver, total number of cells in the spleen and thymus, pyruvate carboxylase (PC) and propionyl CoA carboxylase (PCC) activity in thymus cells, and the proportion of distinct thymocyte subsets were determined. These variables did not differ between mice fed the control and biotin-sufficient diets. In contrast, biotin-deficient mice differed from biotin-sufficient mice in all of the analyzed variables. PC and PCC specific activities of thymocytes of mice fed the biotin-depleting diet decreased during the first 4 wk by 84.5%. The maturation of thymocytes in biotin-deficient mice was arrested at the double-negative stage. Our results suggest that biotin deficiency in mice causes an accelerated involution of the thymus and decreases nose-rump length, but these effects do not correlate in magnitude or in temporality with the sharp decrease in the activity of the biotin-dependent carboxylases. As such, the possibility that the aforementioned effects are not related directly to the prosthetic function of biotin should be considered.

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

    SciTech Connect

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

    1987-11-01

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

  10. Development of an egg-white bioassay for monitoring biotin levels in urine and serum.

    PubMed

    Zarogiannis, Sotirios; Liakopoulos, Vassilios; Hatzoglou, Chrissi; Vogiatzidis, Konstantinos; Salmas, Marios; Stefanidis, Ioannis; Gourgoulianis, Konstantinos; Molyvdas, Paschalis Adam; Lafis, Spiros

    2007-05-01

    This article reports on the development of a simple and cost-effective bioassay for the detection of biotin in urine and serum, based on the very selective binding of avidin and biotin. Avidin was allowed to react without isolating it from egg white. Egg white was treated with the dye HABA, which binds to avidin. Upon subsequent treatment with biotin, HABA is released due to the high affinity of biotin to avidin. The amount of HABA released is proportional to the amount of biotin used.

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

    PubMed

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

    2017-05-01

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

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

    PubMed

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

    2016-12-15

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

  13. Fluorescence enhancement of fluorescent unnatural streptavidin by binding of a biotin analogue with spacer tail and its application to biotin sensing.

    PubMed

    Zhu, Xianwei; Shinohara, Hiroaki

    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 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-(AC5)2-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-(AC5)2-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.

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

    PubMed

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

    2004-12-01

    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 hydrolysis and the HPLC/avidin-binding assay. These values are consistent with published values in that meat, fish, poultry, egg, dairy, and some vegetables are relatively rich sources of biotin. However, these biotin values disagreed substantially with published values for many foods. Assay values varied between 247 times greater than published values for a given food to as much as 36% less than the published biotin value. Among 51 foods assayed for which published values were available, only seven agreed within analytical variability (720%). We conclude that published values for biotin content of foods are likely to be inaccurate.

  15. Intestinal absorption and renal excretion of biotin in patients with biotinidase deficiency.

    PubMed

    Suormala, T; Wick, H; Bonjour, J P; Baumgartner, E R

    1985-05-01

    We have investigated four patients from three unrelated families with typical clinical and biochemical features of "late-onset" multiple carboxylase deficiency. All patients suffered from biotinidase deficiency (plasma biotinidase activities 1.4%-3% of normal). Intestinal absorption of biotin, measured in three of the patients using a single load of 1.5 micrograms/kg, was found to be normal. Deficient activities of the mitochondrial biotin-dependent carboxylases in lymphocytes of one of these patients increased from 25% of mean basal control values to 33%-36% within 45 min and to 46%-47% within 2 h of the 1.5 micrograms/kg biotin load. After a high biotin load of 100 micrograms/kg, the values normalised within 45 min in all three patients studied. These results indicate normal cellular transport of biotin and normal holocarboxylase synthesis. After cessation of biotin supplementation, the plasma and urinary biotin in patients decreased to subnormal levels. In one patient, available for more detailed studies, both plasma and urinary biotin declined about twice as fast as in controls (apparent half-life 12-14 h in the patient and 26 h in controls). These results point to increased excretion of free biotin in our patient. Renal loss of biotin is one of the factors contributing to the high biotin requirement observed in patients with biotinidase deficiency.

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

    PubMed

    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.

  17. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2015-06-23

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

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

    PubMed Central

    Ronquillo-Sánchez, M. D.; Camacho-Carranza, R.; Fernandez-Mejia, C.; Hernández-Ojeda, S.; 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 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. PMID:23984390

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

    PubMed Central

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

    2015-01-01

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

  2. Topics in Ho Morphophonology and Morphosyntax

    ERIC Educational Resources Information Center

    Pucilowski, Anna

    2013-01-01

    Ho, an under-documented North Munda language of India, is known for its complex verb forms. This dissertation focuses on analysis of several features of those complex verbs, using data from original fieldwork undertaken by the author. By way of background, an analysis of the phonetics, phonology and morphophonology of Ho is first presented. Ho has…

  3. Topics in Ho Morphophonology and Morphosyntax

    ERIC Educational Resources Information Center

    Pucilowski, Anna

    2013-01-01

    Ho, an under-documented North Munda language of India, is known for its complex verb forms. This dissertation focuses on analysis of several features of those complex verbs, using data from original fieldwork undertaken by the author. By way of background, an analysis of the phonetics, phonology and morphophonology of Ho is first presented. Ho has…

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

    PubMed Central

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

    2013-01-01

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

  5. NdHO, a novel oxyhydride

    SciTech Connect

    Wideroe, Marius; Fjellvag, Helmer; Norby, Truls; Willy Poulsen, Finn; Willestofte Berg, Rolf

    2011-07-15

    A new metal oxyhydride; neodymium oxyhydride, NdHO, has been synthesized from a reactant mixture of metal hydride (CaH{sub 2} or NdH{sub 3}) and neodymium oxide (Nd{sub 2}O{sub 3}). The unit cell dimensions decrease smoothly in the series from LaHO, CeHO, PrHO to NdHO, in line with the lanthanide contraction. The crystal structure of NdHO is described on the basis of Rietveld refinement on neutron powder diffraction data: Space group: P4/nmm (no. 129, D{sub 4h}{sup 7}). Axis lengths: a=7.8480(5) A, c=5.5601(8) A. Volume: V=342.46(6) A{sup 3}. The tetragonal structure is derived from the fluorite structure, showing complete ordering of hydride and oxide ions over the anion sublatttice. The formation of NdHO was further substantiated by Raman spectroscopy. - Graphical Abstract: View of the NdHO structure. Highlights: > CaH{sub 2} or NdH{sub 3} react with Nd{sub 2}O{sub 3} in reducing atmospheres forming a new oxyhydride, NdHO. > NdHO crystalises in tetragonal space group P4/nmm with axes a=b=7.8480(5) A and c=5.5601(8) A. > The structure is similar to the fluorite structure with complete ordering of hydride and oxide ions. > Unit cell dimensions decrease smoothly in the series from LaHO, CeHO, PrHO to NdHO. > The formation of NdHO is substantiated by Raman spectroscopy.

  6. Energetic methods to study bifunctional biotin operon repressor.

    PubMed

    Beckett, D

    1998-01-01

    Application of a broad range of approaches and techniques to analysis of the functional energetics of the biotin regulatory system has enabled dissection of each of the steps in the assembly of this transcriptional repression complex. Although the molecular details of the interactions are not yet completely understood, the studies described in this article have laid a solid foundation for future studies of the system. The application of kinetic and equilibrium methods to studies of binding of the allosteric effector has allow determination of the kinetic parameters governing the interaction of the protein and ligand. The kinetic parameters have, furthermore, been utilized to calculate the equilibrium parameters associated with the binding. The great advantage of using kinetic methods to study the binding process is the additional information provide about the mechanism of allosteric activation of the protein. Based on the initial observation of a kinetic time course that is consistent with the occurrence of a structural change concomitant with effector binding, additional measurements have been performed that have allowed formulation of a testable hypothesis concerning the nature and location of one locus: the structural change in the three-dimensional structure of BirA. Studies of assembly of the protein indicate the bio-5-AMP is an allosteric activator of dimerization of the protein. The dimerization is, however, weak. These results have been critical in analyzing site-specific DNA binding measurements. Application of the DNase I footprinting technique has allowed formulation of a model for association of holoBirA with bioO. Results of studies of binding of the protein to mutant operator templates, although not yielding the anticipated results, provide further insight into the mechanism of association of the protein and DNA. Two models for binding, the validity of which can be tested via the application of kinetic techniques, have been derived from these

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

    PubMed Central

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

    2017-01-01

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

  8. [The avidin-biotin system. Its application in virology].

    PubMed

    Popa, L M; Kappel, D; Marcheş, F; Borza, B

    1989-01-01

    The report briefly reviews the strategies of utilisation of the avidin-biotin system and its variants, as well as the applications of this system in different fields, especially those linked to the diagnosis and the treatment of some viral and bacterial infections. Some of the techniques using the system are presented, as well as its advantages and the perspectives to ameliorate the methodologies and to enlarge the field of its applications.

  9. Biotin and carnitine profiles in preterm infants in Japan.

    PubMed

    Tokuriki, Shuko; Hayashi, Hisako; Okuno, Takashi; Yoshioka, Kikuko; Okazaki, Shintaro; Kawakita, Akiko; Ohta, Genrei; Hata, Ikue; Shigematsu, Yosuke; Ohshima, Yusei

    2013-06-01

    Biotin plays an important role as a covalently bound coenzyme for carboxylases. Carnitine is essential in β-oxidation to transport long-chain fatty acids across the inner mitochondrial membrane. The present study was conducted to assess the risk of biotin and carnitine deficiencies in preterm infants who received enteral feeding with maternal milk and/or standard infant formula made in Japan. Forty-six infants were enrolled in the study. Urine and serum samples and dried blood spots were collected at 1 week and 1 month of age. Additionally, samples were collected at 40 and 44 weeks post-menstrual age (PMA) in preterm infants. Free carnitine and C5-OH acylcarnitine, which consist of 3-hydroxyisovalerylcarnitine as a major isomer, were measured in serum samples and dried blood spots using tandem mass spectrometry. Urine 3-hydroxyisovaleric acid (3-HIVA) was measured using gas chromatography/mass spectrometry. The free carnitine levels in preterm infants were significantly lower than those in term infants, but increased with PMA in serum samples and dried blood spots. C5-OH acylcarnitine and urinary 3-HIVA levels, which were very low in term infants, were increased with PMA in preterm infants. The present results may indicate chronic biotin deficiency in preterm infants fed maternal milk and/or standard infant formula. Analyses of carnitine profiles of dried blood spots and urine 3-HIVA are relatively non-invasive and useful for the early detection of biotin deficiency in preterm infants. © 2013 The Authors. Pediatrics International © 2013 Japan Pediatric Society.

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

    PubMed

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

    2017-01-01

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

  11. Biotin Protein Ligase Is a Target for New Antibacterials

    PubMed Central

    Feng, Jiage; Paparella, Ashleigh S.; Booker, Grant W.; Polyak, Steven W.; Abell, Andrew D.

    2016-01-01

    There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5′-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5′-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. PMID:27463729

  12. Structure and function of biotin-dependent carboxylases

    PubMed Central

    Tong, Liang

    2012-01-01

    Biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), geranyl-CoA carboxylase (GCC), pyruvate carboxylase (PC), and urea carboxylase (UC). They contain biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes. PMID:22869039

  13. Directed evolution of the substrate specificity of biotin ligase.

    PubMed

    Lu, Wei-Cheng; Levy, Matthew; Kincaid, Rodney; Ellington, Andrew D

    2014-06-01

    We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications.

  14. Single and multiple bonds in (strept)avidin-biotin interactions.

    PubMed

    Teulon, Jean-Marie; Delcuze, Yannick; Odorico, Michael; Chen, Shu-wen W; Parot, Pierre; Pellequer, Jean-Luc

    2011-01-01

    Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

  15. The Rhizobium etli bioMNY operon is involved in biotin transport.

    PubMed

    Guillén-Navarro, Karina; Araíza, Gisela; García-de los Santos, Alejandro; Mora, Yolanda; Dunn, Michael F

    2005-09-15

    Because Rhizobium etli CE3 is normally dependent on an external source of biotin and lacks orthodox biotin biosynthesis genes, we undertook an analysis of biotin uptake in this organism. By complementation of a Sinorhizobium meliloti bioM mutant we isolated an R. etli chromosomal region encoding homologs of the S. meliloti bioMNB genes, whose products have been implicated in intracellular biotin retention in that organism. Disruption of the R. etli bioM resulted in a mutant which took up biotin at a lower rate and accumulated significantly less biotin than the wild type. As in S. meliloti, the R. etli bioMN gene-products resemble the ATPase and permease components, respectively, of an ABC-type transporter. The bioB gene product is in fact similar to members of the BioY family, which has been postulated to function in biotin transport, and we refer to this gene as bioY. An R. etli bioY mutant exhibited lower biotin uptake than the wild-type, providing the first experimental evidence for a role of BioY in biotin transport. We show that the bioMNY operon is transcriptionally repressed by biotin. An analysis of the competitiveness of the wild-type strain versus the bioM mutant showed that the mutant had a diminished capacity to form nodules on bean plants.

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

    PubMed

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

    2017-08-21

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

  17. Arrested Embryos from the bio1 Auxotroph of Arabidopsis thaliana Contain Reduced Levels of Biotin 1

    PubMed Central

    Shellhammer, Joe; Meinke, David

    1990-01-01

    The bio1 auxotroph of Arabidopsis thaliana is a recessive embryonic lethal that forms normal plants in the presence of biotin. The purpose of this study was to determine whether aborted seeds produced by heterozygous plants grown without vitamin supplements contained reduced levels of biotin. Two methods were used to determine the biotin content of mutant and wild-type tissues: streptavidin binding in microtiter plates and growth of the biotin-requiring bacterium Lactobacillus plantarum. Total biotin was measured in extracts prepared from immature seeds prior to desiccation. Aborted seeds produced by heterozygous (bio1/BIO1) plants contained some biotin in the maternal seed coat but virtually no detectable biotin in the arrested embryo. This lack of biotin was not observed in arrested embryos from other mutants with similar patterns of abnormal development. These results are consistent with the model that bio1 tissues are defective in biotin synthesis. The alternative model of increased degradation is inconsistent with the recessive nature of the mutation and the ability of rescued plants to continue growing for several weeks following removal of supplemental biotin. PMID:16667573

  18. Biotin deficiency in a patient with short bowel syndrome during home parenteral nutrition.

    PubMed

    Khalidi, N; Wesley, J R; Thoene, J G; Whitehouse, W M; Baker, W L

    1984-01-01

    A 54-year-old woman with short bowel syndrome was supported with home parenteral nutrition. Six months after receiving 2200 kcal/day of balanced home parenteral nutrition without biotin, she developed biotin deficiency with complete hair loss, eczematous dermatitis, waxy pallor, lethargy, and hypersthesias . Blood and urine samples were collected prior to treatment. Serum zinc was 64 micrograms/dl (nl 50-150 micrograms/dl), and the triene/tetraene ratio was 0.068 (nl 0.4), thereby ruling out zinc and essential fatty acid deficiencies. Serum biotin was 332 pg/ml (nl 520 +/- 220 pg/ml), and urine biotin was 5.22 ng/mg of creatinine (nl 4.3-95 with a mean of 30.2 ng/mg creatinine). The same parenteral nutrition regimen was contained and oral biotin was administered (10 mg/day). After 3 wk, serum and urine biotin levels were 650 pg/ml and 35.6 ng/mg creatinine, respectively. New hair growth was evident and all of her other symptoms resolved. Intravenous biotin was then provided (5 mg/day) for a month after which serum and urine biotin levels were 1316 pg/ml and 178 ng/mg creatine, respectively. The patient has been subsequently maintained on an intravenous multivitamin product containing 60 micrograms biotin per daily dose and remains free of signs and symptoms of biotin deficiency.

  19. Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin.

    PubMed

    Limat, A; Suormala, T; Hunziker, T; Waelti, E R; Braathen, L R; Baumgartner, R

    1996-01-01

    In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2 x 10(-10) mol/l) or by pharmacological doses of biotin (10(-5) mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.

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

    PubMed

    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.

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

    PubMed Central

    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 yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae. PMID:16269718

  2. Covalent Immobilization of Biotin on Magnetic Nanoparticles: Synthesis, Characterization, and Cytotoxicity Studies.

    PubMed

    Islam, Md Rafiqul; Bach, Long Giang; Vo, Thanh-Sang; Lim, Kwon Taek

    2015-01-01

    A simple protocol for covalent immobilization of biotin onto the surface of Fe3O4 magnetic nanoparticles (MNPs) for improving the biocompatibility of original MNPs has been realized. MNPs were first prepared by co-precipitation method which was subsequently anchored with functionalized biotin. The as-synthesized MNPs were observed to be monocrystalline as evidenced from XRD and TEM images. The covalent grafting of biotin to MNPs was confirmed by FT-IR. The XPS analysis suggested the successful preparation of Biotin-f-MNPs. The as-synthesized Biotin-f-MNPs were found to be superparamagnetic character as recorded by SQUID. Cell viability studies revealed that the biocompatibility of MNPs was improved upon Biotin immobilization.

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

    PubMed

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K; Weiss, David S; Cronan, John E

    2015-06-09

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

  4. Shape coexistence in 153Ho

    NASA Astrophysics Data System (ADS)

    Pramanik, Dibyadyuti; Sarkar, S.; Saha Sarkar, M.; Bisoi, Abhijit; Ray, Sudatta; Dasgupta, Shinjinee; Chakraborty, A.; Krishichayan, Kshetri, Ritesh; Ray, Indrani; Ganguly, S.; Pradhan, M. K.; Ray Basu, M.; Raut, R.; Ganguly, G.; Ghugre, S. S.; Sinha, A. K.; Basu, S. K.; Bhattacharya, S.; Mukherjee, A.; Banerjee, P.; Goswami, A.

    2016-08-01

    The high-spin states in 153Ho have been studied by the La57(20Ne139,6 n ) reaction at a projectile energy of 139 MeV at the Variable Energy Cyclotron Centre (VECC), Kolkata, India, utilizing an earlier campaign of the Indian National Gamma Array (INGA) setup. Data from γ -γ coincidence, directional correlation, and polarization measurements have been analyzed to assign and confirm the spins and parities of the levels. We have suggested a few additions and revisions of the reported level scheme of 153Ho. The RF-γ time difference spectra have been useful to confirm the half-life of an isomer in this nucleus. From the comparison of experimental and theoretical results, it is found that there are definite indications of shape coexistence in this nucleus. The experimental and calculated lifetimes of several isomers have been compared to follow the coexistence and evolution of shape with increasing spin.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-05-01

    Biotin acts as a coenzyme for carboxylases regulating lipid and amino-acid metabolism. We investigated alterations of the biotin-dependent functions in obesity and the downstream effects of biotin restriction in adipocytes in vitro. Twenty-four monozygotic twin pairs discordant for body mass index (BMI). Mean within-pair difference (heavy-lean co-twin, Δ) of BMI was 6.0 kg m(-2) (range 3.1-15.2 kg m(-)(2)). Adipose tissue (AT) DNA methylation, gene expression of AT and adipocytes, and leukocytes (real-time quantitative PCR), serum biotin, C-reactive protein (CRP) and triglycerides were measured in the twins. Human adipocytes were cultured in low and control biotin concentrations and analyzed for lipid droplet content, mitochondrial morphology and mitochondrial respiration. The gene expression levels of carboxylases, PCCB and MCCC1, were upregulated in the heavier co-twins' leukocytes. ΔPCCB (r=0.91, P=0.0046) and ΔMCCC1 (r=0.79, P=0.036) correlated with ΔCRP within-pairs. Serum biotin levels were lower in the heavier (274 ng l(-1)) than in the lean co-twins (390 ng l(-1), P=0.034). ΔBiotin correlated negatively with Δtriglycerides (r=-0.56, P=0.045) within-pairs. In AT, HLCS and ACACB were hypermethylated and biotin cycle genes HLCS and BTD were downregulated (P<0.05). Biotin-dependent carboxylases were downregulated (ACACA, ACACB, PCCB, MCCC2 and PC; P<0.05) in both AT and adipocytes of the heavier co-twins. Adipocytes cultured in low biotin had decreased lipid accumulation, altered mitochondrial morphology and deficient mitochondrial respiration. Biotin-dependent functions are modified by adiposity independent of genetic effects, and correlate with inflammation and hypertriglyceridemia. Biotin restriction decreases lipid accumulation and respiration, and alters mitochondrial morphology in adipocytes.

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

    PubMed Central

    Riverón-Negrete, Leticia; Sicilia-Argumedo, Gloria; Álvarez-Delgado, Carolina; Alcántar-Fernández, Jonathan

    2016-01-01

    Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers. PMID:28105429

  8. A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

    PubMed

    Mayende, Lungisa; Swift, Rachel D; Bailey, Lisa M; Soares da Costa, Tatiana P; Wallace, John C; Booker, Grant W; Polyak, Steven W

    2012-01-01

    Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

    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 3H-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

  11. Biotin Transport and Accumulation by Cells of Lactobacillus plantarum II. Kinetics of the System

    PubMed Central

    Waller, James R.; Lichstein, Herman C.

    1965-01-01

    Waller, James R. (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Biotin transport and accumulation by cells of Lactobacillus plantarum. II. Kinetics of the system. J. Bacteriol. 90:853–856. 1965.—Bound biotin-saturated cells of Lactobacillus plantarum accumulated free biotin by a time-dependent process exhibiting substrate saturation phenomena in the presence and absence of glucose. Apparent Km and Vmax values determined in the presence and absence of glucose, respectively, from Lineweaver-Burk plots were found to be 31.5 and 7.72 mμm (Km) and 9.72 and 3.26 μμmoles per mg per min (Vmax). Free biotin transport per se appeared to be an energy-independent, mediated process, whereas the accumulation of large intracellular vitamin concentrations was energy-dependent. Internal free biotin was quantitatively converted to bound biotin. The rate and extent of bound biotin formation was slower than free biotin uptake, and dependent upon intracellular free biotin levels up to a saturating concentration. PMID:5847806

  12. Epigenetic synergies between biotin and folate in the regulation of pro-inflammatory cytokines and repeats.

    PubMed

    Xue, J; Zempleni, J

    2013-11-01

    The protein biotin ligase, holocarboxylase synthetase (HLCS), is a chromatin protein that interacts physically with the DNA methyltransferase DNMT1, the methylated cytosine-binding protein MeCP2 and the histone H3 K9-methyltransferase EHMT1, all of which participate in folate-dependent gene repression. Here we tested the hypothesis that biotin and folate synergize in the repression of pro-inflammatory cytokines and long-terminal repeats (LTRs), mediated by interactions between HLCS and other chromatin proteins. Biotin and folate supplementation could compensate for each other's deficiency in the repression of LTRs in Jurkat and U937 cells. For example, when biotin-deficient Jurkat cells were supplemented with folate, the expression of LTRs decreased by >70%. Epigenetic synergies were more complex in the regulation of cytokines compared with LTRs. For example, the abundance of TNF-α was 100% greater in folate- and biotin-supplemented U937 cells compared with biotin-deficient and folate-supplemented cells. The NF-κB inhibitor curcumin abrogated the effects of folate and biotin in cytokine regulation, suggesting that transcription factor signalling adds an extra layer of complexity to the regulation of cytokine genes by epigenetic phenomena. We conclude that biotin and folate synergize in the repression of LTRs and that these interactions are probably mediated by HLCS-dependent epigenetic mechanisms. In contrast, synergies between biotin and folate in the regulation of cytokines need to be interpreted in the context of transcription factor signalling.

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

    PubMed Central

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

    2009-01-01

    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 the biotin label. The first quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second enumerated biotin-labeled RBCs by flow cytometry after incubation with fluorescein-conjugated avidin. RCV measurements made using the two-biotin quantitation techniques were validated against both (14C)cyanate and 51Cr as reference methods. Both biotin techniques produced RCV values that agreed well with the reference methods and with each other, producing correlation coefficients averaging ≥ 0.93. Sequential repetitive measurements in the same animal also agreed with the (14C)cyanate method and each other (average difference < 10%). These results establish biotin-labeled RBCs as an accurate method for performing RCV measurements in sheep. This biotin method can be applied in studies that model neonatal erythropoiesis. PMID:18596580

  14. Red cell volume can be accurately determined in sheep using a nonradioactive biotin label.

    PubMed

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

    2008-11-01

    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 the biotin label. The first one quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second one enumerated biotin-labeled RBCs by flow cytometry after incubation with fluorescein-conjugated avidin. RCV measurements made using the two biotin quantitation techniques were validated against both (14C)cyanate and 51Cr as reference methods. Both biotin techniques produced RCV values that agreed well with the reference methods and with each other, producing correlation coefficients averaging >or =0.93. Sequential repetitive measurements in the same animal also agreed with the (14C)cyanate method and each other (average difference <10%). These results establish biotin-labeled RBCs as an accurate method for performing RCV measurements in sheep. This biotin method can be applied in studies that model neonatal erythropoiesis.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    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 additional regulatory role. Included among these properties are the order of substrate binding and linkage between oligomeric state and ligand binding. PMID:20499837

  17. Selectivity in Post-translational Biotin Addition to Five Human Carboxylases*

    PubMed Central

    Ingaramo, Maria; Beckett, Dorothy

    2012-01-01

    Human holocarboxylase synthetase (HCS) catalyzes linkage of the vitamin biotin to the biotin carboxyl carrier protein (BCCP) domain of five biotin-dependent carboxylases. In the two-step reaction, the activated intermediate, bio-5′-AMP, is first synthesized from biotin and ATP, followed by covalent linkage of the biotin moiety to a specific lysine residue of each carboxylase BCCP domain. Selectivity in HCS-catalyzed biotinylation to the carboxylases was investigated in single turnover stopped flow and quench flow measurements of biotin transfer to the minimal biotin acceptor BCCP fragments of the carboxylases. The results demonstrate that biotinylation of the BCCP fragments of the mitochondrial carboxylases propionyl-CoA carboxylase, pyruvate carboxylase, and methylcrotonoyl-CoA carboxylase is fast and limited by the bimolecular association rate of the enzyme with substrate. By contrast, biotinylation of the acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2) fragments, both of which are accessible to HCS in the cytoplasm, is slow and displays a hyperbolic dependence on substrate concentration. The correlation between HCS accessibility to biotin acceptor substrates and the kinetics of biotinylation suggests that mitochondrial carboxylase sequences evolved to produce fast association rates with HCS in order to ensure biotinylation prior to mitochondrial import. In addition, the results are consistent with a role for HCS specificity in dictating biotin distribution among carboxylases. PMID:22123817

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

  20. Dietary biotin requirement for maximum growth of juvenile grass shrimp, Penaeus monodon.

    PubMed

    Shiau, S Y; Chin, Y H

    1998-12-01

    A feeding trial was conducted to estimate the minimal dietary biotin requirement for juvenile grass shrimp, Penaeus monodon. Purified diets with eight levels (0, 0.2, 0.5, 1.0, 3.0, 6.0, 10.0 and 20.0 mg/kg) of supplemental biotin were fed to P. monodon (mean weight 0. 26 +/- 0.01 g) for 8 wk. Each diet was fed to three replicate groups of shrimp. Shrimp fed diets supplemented with biotin (0.2-20.0 mg/kg) had significantly (P < 0.05) higher weight gain, feed efficiency and protein efficiency ratio than those fed the unsupplemented control diet. Weight gain was high in shrimp fed 3. 0-10.0 mg biotin/kg diet and lowest in shrimp fed biotin concentration in shrimp generally increased as dietary biotin supplementation increased. Highest hepatopancreatic pyruvate carboxylase and acetyl CoA carboxylase activity were in shrimp fed diets with 10 and 20 mg biotin/kg and 3. 0 mg biotin/kg, respectively. Weight gain percentage and protein efficiency ratio of the shrimp analyzed by broken-line regression indicated that the minimal dietary biotin concentration in growing P. monodon is 2.0-2.4 mg/kg.

  1. Characterization of the cDNA and gene coding for the biotin synthase of Arabidopsis thaliana.

    PubMed Central

    Weaver, L M; Yu, F; Wurtele, E S; Nikolau, B J

    1996-01-01

    Biotin, an essential cofactor, is synthesized de novo only by plants and some microbes. An Arabidopsis thaliana expressed sequence tag that shows sequence similarity to the carboxyl end of biotin synthase from Escherichia coli was used to isolate a near-full-length cDNA. This cDNA was shown to code for the Arabidopsis biotin synthase by its ability to complement a bioB mutant of E. coli. Site-specific mutagenesis indicates that residue threonine-173, which is highly conserved in biotin synthases, is important for catalytic competence of the enzyme. The primary sequence of the Arabidopsis biotin synthase is most similar to biotin synthases from E. coli, Serratia marcescens, and Saccharomyces cerevisiae (about 50% sequence identity) and more distantly related to the Bacillus sphaericus enzyme (33% sequence identity). The primary sequence of the amino terminus of the Arabidopsis biotin synthase may represent an organelle-targeting transit peptide. The single Arabidopsis gene coding for biotin synthase, BIO2, was isolated and sequenced. The biotin synthase coding sequence is interrupted by five introns. The gene sequence upstream of the translation start site has several unusual features, including imperfect palindromes and polypyrimidine sequences, which may function in the transcriptional regulation of the BIO2 gene. PMID:8819873

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

    PubMed Central

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

    2015-01-01

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

  3. Five patients with a biotin-responsive defect in holocarboxylase formation: evaluation of responsiveness to biotin therapy in vivo and comparative biochemical studies in vitro.

    PubMed

    Suormala, T; Fowler, B; Duran, M; Burtscher, A; Fuchshuber, A; Tratzmüller, R; Lenze, M J; Raab, K; Baur, B; Wick, H; Baumgartner, R

    1997-05-01

    Biochemical studies in five patients with a defect in biotin-responsive holocarboxylase synthesis are reported. The age of onset (2 d to 6 y) as well as the severity of illness varied considerably. In all patients diagnosis was established by the finding of organic aciduria typical for multiple carboxylase deficiency in a catabolic state. In four patients the response to biotin therapy was evaluated by measurement of mitochondrial carboxylase activities in lymphocytes and by monitoring urinary organic acid excretion. In three patients clinical symptoms disappeared with 10-20 mg biotin/d, whereas normalization of the biochemical parameters required higher doses (20-40 mg/d). The fourth patient required a dose of 100 mg biotin/d before her skin rash disappeared. She remains mentally retarded and shows slightly elevated urinary organic acid excretion. Carboxylase activities were clearly deficient in fibroblasts grown in the commonly used medium which contains 10 nmol/L biotin (contributed by FCS in medium) in two patients. Fibroblasts of the other three patients became deficient only in a low biotin medium (0.1 nmol/L). Reactivation of deficient carboxylase activities in relation to time and biotin concentration correlated well with the severity and age of onset of illness in four patients. In one patient, however, carboxylase reactivation followed a more complex pattern requiring the longest incubation time but only a moderately increased biotin concentration of 19 nmol/L compared with 3-5 nmol/L in normal cells and 34-4000 nmol/L in the other four patients. The results in the five patients are in accordance with a primary defect of holocarboxylase synthetase due to a decreased affinity for biotin, in one patient combined with a decreased Vmax.

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

    PubMed

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

    2017-04-01

    It is important to assess pediatric patients for nutritional deficiency when they are receiving specific interventions, such as enteral feeding. We focused on measurement of C0 and 3-hydroxyisovalerylcarnitine (C5-OH) with tandem mass spectrometry (MS/MS), which is performed as part of the newborn mass screening. The purpose of this study was to investigate the usefulness of MS/MS for screening carnitine and biotin deficiencies. Forty-two children (24 boys, 18 girls) were enrolled between December 2013 and December 2015. Blood tests, including measurement of serum free carnitine via the enzyme cycling method, and acylcarnitine analysis on MS/MS of dried blood spot (DBS), were performed for the evaluation of nutrition status. Median patient age was 2 years (range, 2 months-14 years). Mean serum free carnitine was 41.8 ± 19.2 μmol/L. In six of the 42 patients, serum free carnitine was <20 μmol/L (range, 4.0-18.7 μmol/L). C0 and C5-OH measured on MS/MS of DBS were 33.8 ± 20.2 nmol/mL and 0.48 ± 0.22 nmol/mL, respectively. There was a strong positive correlation (r = 0.89, P < 0.001) between serum free carnitine and C0 measured on the same day. In one patient on hydrolyzed formula, C5-OH was >1.00 nmol/L. Therapy-resistant eczema was improved by treatment with additional biotin and a non-hydrolyzed formula. C0 and C5-OH, measured on MS/MS of DBS, were useful for screening carnitine and biotin deficiencies. © 2016 Japan Pediatric Society.

  5. Tm:YLF Pumped Ho:YAG and Ho:LuAG Lasers

    NASA Technical Reports Server (NTRS)

    Barnes, Norman P.; Reichle, Donald J.; Walsh, Brian M.; Axenson, Theresa J.

    2004-01-01

    Room temperature Ho:YAG and Ho:LuAG lasers pumped by a Tm:YLF laser demonstrated a 3.4 mJ threshold and 0.41 slope efficiency, incident optical to laser output energy. Results for numerous rod lengths, Ho concentrations, and output mirror reflectivities are presented.

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

    PubMed Central

    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 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 ∼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 Km for BCCP was ∼5.2 µM and ∼420 nM for biotin. MtBPL has low affinity (Kb = 1.06×10−6 M) for biotin relative to EcBirA but their Km 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. PMID:18509457

  7. Marginal biotin deficiency can be induced experimentally in humans using a cost-effective outpatient design.

    PubMed

    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

    2012-01-01

    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 white for a period of 28 d. In study design 1, participants (n = 4; 3 women) received avidin in capsules with a biotin binding capacity of 7 times the estimated dietary biotin intake of a typical self-selected diet. In study design 2, participants (n = 2; 2 women) received double the amount of avidin capsules (14 times the estimated dietary biotin intake). In study design 3, participants (n = 5; 3 women) consumed egg-white beverages containing avidin with a biotin binding capacity of 7 times the estimated dietary biotin intake. Established indices of biotin status [lymphocyte propionyl-CoA carboxylase activity; urinary excretion of 3-hydroxyisovaleric acid, 3-hydroxyisovaleryl carnitine (3HIA-carnitine), and biotin; and plasma concentration of 3HIA-carnitine] indicated that study designs 1 and 2 were not effective in inducing marginal biotin deficiency, but study design 3 was as effective as previous inpatient study designs that induced deficiency by egg-white beverage. Marginal biotin deficiency can be induced experimentally by using a cost-effective outpatient design by avidin delivery in egg-white beverages. This design should be useful to the broader nutritional research community.

  8. Biotin limitation in Sinorhizobium meliloti strain 1021 alters transcription and translation.

    PubMed

    Heinz, Elke B; Streit, Wolfgang R

    2003-02-01

    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 starvation response in this microbe. A gene coding for a protein-methyl carboxyl transferase (pcm) exhibited 13.6-fold-higher transcription under biotin-limiting conditions than cells grown in the presence of 40 nM biotin. Consistent with this observation, biotin-limiting conditions resulted in a significantly decreased survival of pcm mutant cells compared to parental cells or cells grown in the presence of 40 nM biotin. Further studies indicated that the autoinducer synthase gene, sinI, was transcribed at a 4.5-fold-higher level in early stationary phase in biotin-starved cells than in biotin-supplemented cells. Lastly, we observed that open reading frame smc02283, which codes for a putative copper resistance protein (CopC), was 21-fold down-regulated in response to biotin starvation. (ii) In a second approach, proteome analysis identified 10 proteins which were significantly down-regulated under the biotin-limiting conditions. Among the proteins identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry were the pi subunit of the RNA polymerase and the 50S ribosomal protein L7/L12 (L8) subunit, indicating that biotin-limiting conditions generally affect transcription and translation in S. meliloti.

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

    PubMed

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

    2012-03-01

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

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

    PubMed Central

    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 probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources. PMID:12039741

  11. Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport.

    PubMed

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

    2002-06-01

    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 probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.

  12. Level structure of 154Ho

    NASA Astrophysics Data System (ADS)

    Moon, Chang-Bum; Komatsubara, Tetsuro; Furuno, Kohei

    2013-10-01

    The excited states of the odd-odd 154Ho nucleus have been studied by using in-beam γ-ray spectroscopy with the 141Pr (16O, 3n) 154Ho reaction at Elab=75 MeV. The beam was provided by the 12UD Pelletron accelerator at the University of Tsukuba. In this work, the complicated decay pattern of low energy transitions just above the T1/2=3.10 min isomer have been established. In addition, a number of new states and γ-ray transitions, especially those associated with energetically favored band termination, have been observed for the first time in 154Ho. A negative collective band and its signature partner built on the 11- level are interpreted as being based on the πh11/2⊗νi13/2 configuration. A positive band built on the 10+ level is based on the πh11/2⊗νh9/2 configuration while another positive band built on the 9+ level is being associated with the πh11/2⊗νf7/2 configuration. An energetically favored level Jπ=19- can be interpreted as being attributed to the πh11/2⊗νi13/2 configuration coupled to the 8+ state in neighboring core 152Dy, namely, a four-quasiparticle alignment based on the [πh11/2νi13/2]11-⊗[ν(h9/2f7/2)]8- configuration. Another energetically favored state at Jπ=27- is assigned the six-quasiparticle [π(h11/2)3]27/2-⊗[ν(f7/2h9/2i13/2)]27/2- configuration.

  13. Ferroelectricity and competing interactions in Ho-deficient non-stoichiometric orthorhombic HoMnO{sub 3}

    SciTech Connect

    Wang, J. X.; Yan, Z. B.; Xie, Y. L.; Zhou, X. H.; Liu, J.-M.

    2015-05-07

    We investigate the consequences of the Ho-deficient non-stoichiometry in orthorhombic HoMnO{sub 3} in terms of microscopic mechanisms for ferroelectricity modulation. It is suggested that the Ho-deficiency (then Mn excess) results in Ho-vacancies and then Mn occupation of the Ho-site with increasing non-stoichiometry. The Ho-deficiency enhances the Mn-Mn symmetric exchange striction by suppressing the independent Ho-Ho interaction, and thus benefits to the induced Ho spin ordering against the independent Ho spin ordering. The symmetric Ho-Mn exchange striction is thus enhanced by this induced Ho spin ordering, leading to remarkably enhanced ferroelectric polarization as observed. This work presents an alternative scheme to modulate the multiferroicity in rare-earth manganites of strong 4f-3d coupling.

  14. Ferroelectricity and competing interactions in Ho-deficient non-stoichiometric orthorhombic HoMnO3

    NASA Astrophysics Data System (ADS)

    Wang, J. X.; Yan, Z. B.; Xie, Y. L.; Zhou, X. H.; Liu, J.-M.

    2015-05-01

    We investigate the consequences of the Ho-deficient non-stoichiometry in orthorhombic HoMnO3 in terms of microscopic mechanisms for ferroelectricity modulation. It is suggested that the Ho-deficiency (then Mn excess) results in Ho-vacancies and then Mn occupation of the Ho-site with increasing non-stoichiometry. The Ho-deficiency enhances the Mn-Mn symmetric exchange striction by suppressing the independent Ho-Ho interaction, and thus benefits to the induced Ho spin ordering against the independent Ho spin ordering. The symmetric Ho-Mn exchange striction is thus enhanced by this induced Ho spin ordering, leading to remarkably enhanced ferroelectric polarization as observed. This work presents an alternative scheme to modulate the multiferroicity in rare-earth manganites of strong 4f-3d coupling.

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

    PubMed

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

    2016-02-21

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

  16. The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding.

    PubMed

    Streaker, Emily D; Gupta, Aditi; Beckett, Dorothy

    2002-12-03

    The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP. Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization. The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex. It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface. In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form. Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics. However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

  17. [Effects of biotin on blood glucose regulation in type 2 diabetes rat model].

    PubMed

    Xiang, Xuesong; Liu, Yan; Zhang, Xuesong; Zhang, Wenzong; Wang, Zhu

    2015-03-01

    To explore the effect of biotin on blood glucose regulation in rats and its possible mechanism. According to initial body weight and blood glucose, we randomly divided the 90 Wistar rats into 5 groups: the normal control group, model group, biotin low-dose group (0. 6mg/kg BW), biotin medium-dose group (3. 0 mg/kg BW) and biotin high-dose group (6. 0 mg/kg BW). After 2 months, the rats with HFS feed were injected with STZ (25 mg/kg BW) to manufacture diabetic rat model. After the OGTT experiment at 10th week, the blood glucose, insulin, liver/muscle glycogen and other biochemical indexes were detected. The GCK, PCK1 mRNA expression were measured with RT-PCR method. Biotin has a certain improvement on postprandial glucose in diabetic rats. Compared with the model group, the AUC and the 30min postprandial blood glucose of biotin high-dose group were significantly decreased (P <0. 05). Biotin can affect some key enzyme gene in glucose metabolism, such as GCK, PCK1. The possible mechanism of the decreasing biotin blood sugar in diabetic rats may by promoting the synthesis of glycogenand reducing gluconeogenesis.

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

    SciTech Connect

    Said, H.M.; Redha, R.

    1987-01-01

    Transport of biotin was examined in rat intestine using the everted sac technique. Transport of 0.1 ..mu..M 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/sup -1/ min/sup -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/sup -1/ x 25 min/sup -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/sup +/ 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/sup +/, energy, and temperature dependent.

  19. Design and synthesis of metabolically stable chelate-biotin conjugates for pretargeted tumor radioimmunotherapy

    SciTech Connect

    Gustavson, L.M.; Su, F.M.; Reno, J.M.

    1995-12-01

    The use of radiolabeled chelate biotin conjugates for targeting antibody-avidin complexes prelocalized on tumors offers the advantage of delivering the radioactivity on a small molecule which exhibits fast tumor localization and rapid whole body clearance. To maximize efficacy, the radiolabeled biotin conjugate should exhibit high serum stability, rapid renal excretion, and uncompromised avidin binding capacity. we determined that the Y-90 labeled 2-(benzylamidocaproyl-biotin)-tetraatzacyclododecane-N, N`, N``, N```- tetraacetic acid conjugate, DOTA-LC-biotin, incubated in serum for 15 minutes, was 98% degraded to a fragment which no longer binds avidin. The synthesis of standards DOTA-benzylamine and 6- amninocaproamido benzyl-DOTA permitted the identification of the site of cleavage as the biotin carboxylamide. To prevent the enzymatic cleavage of the biotin-amide functionality, we designed four amide stabilized DOTA-biotin analogs. The analogs retained favorable biodistribution and avidin binding and showed excellent serum stability. The design of stable chelate biotin conjugates resulted in improved therapeutic efficacy in mice and a Phase I clinical trial has been initiated with a N-methyl biotinamide stabilized conjugate.

  20. Design of a reversible biotin analog and applications in protein labeling, detection, and isolation.

    PubMed

    Ying, Lai-Qiang; Branchaud, Bruce P

    2011-08-14

    To expand the applicability of the biotin-(strept)avidin system, a biotin analog with reversible binding under non-denaturing conditions has been designed, and its applications in protein labeling, detection, and isolation have been evaluated. This journal is © The Royal Society of Chemistry 2011

  1. Response of sows and litters to added dietary biotin in environmentally regulated facilities.

    PubMed

    Hamilton, C R; Veum, T L

    1984-07-01

    A 3-yr study was conducted to evaluate the effects of biotin on sow longevity, reproductive performance and piglet performance to weaning utilizing 161 sows and 414 litters. Sows and gilts were fed a basal corn-soybean meal diet (without any antibiotic or chemotherapeutic compounds) during gestation and lactation containing either 0 or .55 ppm added biotin. The basal diet contained .17 ppm total dietary biotin based on microbiological assay. Results indicated sow culling rates and weight gains, number of live pigs at birth, pig weights at birth and weaning, and the interval from weaning to rebreeding were similar for both treatment groups. However, sows fed the diet with added biotin weaned more (P less than .05) pigs/litter overall and at gestation-lactation period 1 than did sows fed the basal diet without added biotin, although biotin did not increase (P greater than .10) the number of pigs weaned at gestation-lactation periods 2 through 5. The incidence of dermatitis, hair loss and soundness of feet and legs did not appear to be affected by adding biotin to the diet. Thus, the addition of .55 ppm biotin to a corn-soybean meal diet fed during gestation and lactation did not improve any of the criteria measured except number of pigs weaned overall.

  2. Biotin as a probe of the surface of Ascaris suum developmental stages.

    PubMed

    Hill, D E; Fetterer, R H; Urban, J F

    1990-06-01

    Sulfo-NHS-biotin (aqueous soluble) and NHS-biotin (organic soluble) labeled similar SDS-2ME (sodium dodecyl sulfate/beta-mercaptoethanol) soluble cuticular proteins of second stage larvae (L2) and third stage Ascaris suum larvae (L3). Comparable analysis of biotin-labeled fourth stage larvae (L4), young adults, and mature adult Ascaris suum revealed strong labeling of several SDS-2ME soluble cuticular proteins with NHS-biotin, while sulfo-NHS-biotin appeared to strongly label a single SDS-2ME soluble cuticular protein. Both biotin probes labeled only cuticular proteins, since no evidence of internal labeling was observed in any developmental stage examined by either electroblot analysis or by electron microscopy. Our data suggest a greater cuticular permeability to the organic soluble biotin reagent in the later developmental stages (greater than L3) of A. suum than to the aqueous soluble biotin reagent, and may indicate the presence of a hydrophobic barrier in the cuticle of the later stages of the parasite.

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

    PubMed Central

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

    2012-01-01

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

  4. In Vivo Biotinylation of Bacterial Magnetic Particles by a Truncated Form of Escherichia coli Biotin Ligase and Biotin Acceptor Peptide ▿

    PubMed Central

    Maeda, Yoshiaki; Yoshino, Tomoko; Matsunaga, Tadashi

    2010-01-01

    Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation. PMID:20622127

  5. [Establishment of a novel biotin-inducible eukaryotic gene regulation system].

    PubMed

    Ye, Lingling; Hong, Liu; Li, Shichong; Wang, Qiwei; Lan, Sanchun; Chen, Zhaolie

    2014-08-01

    To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-03-20

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

  8. Effect of dietary biotin supplementation on sow reproductive performance and soundness and pig growth and mortality.

    PubMed

    Watkins, K L; Southern, L L; Miller, J E

    1991-01-01

    A 3-yr study was conducted to evaluate the effect of dietary biotin supplementation on the reproductive performance of 90 sows and gilts, and on the pre-weaning growth and mortality of 223 litters. Corn-soybean meal-based diets supplemented with either 0 or 440 micrograms/kg d-biotin were fed to sows throughout their reproductive cycle. Biotin supplementation had no beneficial effect (P greater than .10) on 107-d sow weight, sow weight at weaning, weaning to estrus interval, foot lesion score, hair loss score, structural soundness score, number of pigs born, number and percentage of pigs born alive or number and percentage of pigs alive at 21 d of age. Biotin supplementation had no effect (P greater than .10) on pig growth or mortality to 21 d of age. These data do not support the concept that biotin supplementation of sow diets is needed.

  9. A sandwich-type electrochemical immunosensor based on the biotin- streptavidin-biotin structure for detection of human immunoglobulin G

    PubMed Central

    Li, Yueyun; Zhang, Yihe; Jiang, Liping; Chu, Paul K.; Dong, Yunhui; Wei, Qin

    2016-01-01

    A sandwich-type immunosensor is designed and fabricated to detect the human immunoglobulin G (HIgG) using polyaniline and tin dioxide functionalized graphene (GS-SnO2-PAN) as the platform and biotin-functionalized amination magnetic nanoparticles composite (B-Fe3O4@APTES) as the label. GS-SnO2-PAN is used as the sensing agent to capture the primary anti-HIgG (Ab1) and SnO2 reduces the stack of GS. The B-Fe3O4@APTES with a large surface area and excellent biocompatibility captures second antibody (Ab2) efficiently based on the highly selective recognition of streptavidin to biotinylated antibody. The B-Fe3O4@APTES has better electro-catalytic activity in the reduction of hydrogen peroxide (H2O2) and the “biotin-streptavidin-biotin” (B-SA-B) strategy leads to signal amplification. Under optimal conditions, the immunosensor has a wide sensitivity range from 1 pg/L to 10 ng/L and low detection limit of 0.33 pg/L (S/N = 3) for HIgG. The immunosensor has high sensitivity, fast assay rate, as well as good reproducibility, specificity, and stability especially in the quantitative detection of biomolecules in serum samples. PMID:26948273

  10. Increasing Accuracy and Increasing Tension in Ho

    NASA Astrophysics Data System (ADS)

    Freedman, Wendy L.

    2017-01-01

    The Hubble Constant, Ho, provides a measure of the current expansion rate of the universe. In recent decades, there has been a huge increase in the accuracy with which extragalactic distances, and hence Ho, can be measured. While the historical factor-of-two uncertainty in Ho has been resolved, a new discrepancy has arisen between the values of Ho measured in the local universe, and that estimated from cosmic microwave background measurements, assuming a Lambda cold dark matter model. I will review the advances that have led to the increase in accuracy in measurements of Ho, as well as describe exciting future prospects with the James Webb Space Telescope (JWST) and Gaia, which will make it feasible to measure extragalactic distances at percent-level accuracy in the next decade.

  11. The hypotriglyceridemic effect of biotin supplementation involves increased levels of cGMP and AMPK activation.

    PubMed

    Aguilera-Méndez, Asdrúbal; Fernández-Mejía, Cristina

    2012-01-01

    In addition to its role as a carboxylase cofactor, biotin modifies gene expression and has manifold effects on systemic processes. Several studies have shown that biotin supplementation reduces hypertriglyceridemia. We have previously reported that this effect is related to decreased expression of lipogenic genes. In the present work, we analyzed signaling pathways and posttranscriptional mechanisms involved in the hypotriglyceridemic effects of biotin. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg of free biotin/kg diet, respectively for 8 weeks after weaning. The abundance of mature sterol regulatory element-binding protein (SREBP-1c), fatty-acid synthase (FAS), total acetyl-CoA carboxylase-1 (ACC-1) and its phosphorylated form, and AMP-activated protein kinase (AMPK) were evaluated in the liver. We also determined the serum triglyceride concentrations and the hepatic levels of triglycerides and cyclic GMP (cGMP). Compared to the control group, biotin-supplemented mice had lower serum and hepatic triglyceride concentrations. Biotin supplementation increased the levels of cGMP and the phosphorylated forms of AMPK and ACC-1 and decreased the abundance of the mature form of SREBP-1c and FAS. These data provide evidence that the mechanisms by which biotin supplementation reduces lipogenesis involve increased cGMP content and AMPK activation. In turn, these changes lead to augmented ACC-1 phosphorylation and decreased expression of both the mature form of SREBP-1c and FAS. Our results demonstrate for the first time that AMPK is involved in the effects of biotin supplementation and offer new insights into the mechanisms of biotin-mediated hypotriglyceridemic effects.

  12. Pharmacological concentrations of biotin reduce serum triglycerides and the expression of lipogenic genes.

    PubMed

    Larrieta, Elena; Velasco, Fidel; Vital, Paz; López-Aceves, Teresita; Lazo-de-la-Vega-Monroy, María Luisa; Rojas, Alberto; Fernandez-Mejia, Cristina

    2010-10-10

    Besides its role as a carboxylase prosthetic group, biotin regulates gene expression and has a wide repertoire of effects on systemic processes. Several studies have shown that pharmacological concentrations of biotin reduce hypertriglyceridemia. The molecular mechanisms by which pharmacological concentrations of biotin affect lipid metabolism are largely unknown. The present study analyzed the effects of pharmacological doses of biotin on triglyceridemia, insulin sensitivity and on mRNA expression of various lipogenic genes. Three-week-old male BALB/cAnN Hsd mice were fed a biotin-control or a biotin-supplemented diet (1.76 or 97.7mg of free biotin/kg diet, respectively) over a period of eight weeks. Serum triglyceride concentrations, insulin and glucose tolerance and mRNA abundance of various lipogenic genes were investigated. The biotin-supplemented group showed 35% less serum triglycerides than control mice. In the liver, we found a significant (P<0.05) reduction of mRNA levels of SREBP1-c, glucose transporter-2, phosphofructokinase-1, pyruvate kinase, acetyl-CoA carboxylase and fatty acid synthase, while glucose-6-phosphate dehydrogenase expression increased. No changes in glucokinase, stearoyl-CoA desaturase-1, FoxO1 or PPAR-gamma expression were observed. In adipose tissue, we found a decreased expression of SREBP1c, glucose-6-phosphate deshydrogenase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase-1, phosphofructokinase-1 and PPAR-gamma, but no changes in FoxO1 expression. Moreover, the group fed a biotin-supplemented diet showed a significant decrease in adipose tissue weight. No differences in insulin sensitivity or serum insulin concentrations were observed between groups. Our results indicate that pharmacological concentrations of biotin decrease serum tryglyceride concentrations and lipogenic gene expression in liver and adipose tissues. Copyright 2010 Elsevier B.V. All rights reserved.

  13. Antioxidant status of serum, muscle, intestine and hepatopancreas for fish fed graded levels of biotin.

    PubMed

    Feng, Lin; Zhao, Shu; Chen, Gangfu; Jiang, Weidan; Liu, Yang; Jiang, Jun; Hu, Kai; Li, Shuhong; Zhou, Xiaoqiu

    2014-04-01

    Lipid peroxidation, protein oxidation and antioxidant activities of muscle, intestine, hepatopancreas and serum in juvenile Jian carp (Cyprinus carpio var. Jian) were investigated after feeding graded levels of biotin (0.010, 0.028, 0.054, 0.151, 0.330, 1.540 and 2.680 mg kg(-1) diet) for 63 days. Both malondialdehyde and protein carbonyl content in all studied tissues and serum were the lowest in fish fed diets containing 0.151-0.330 mg biotin kg(-1) diet and then increased in fish fed the diet with 2.680 mg biotin kg(-1) diet (P < 0.05). Similarly, glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in serum significantly decreased with biotin levels up to 0.151 mg kg(-1) diet (P < 0.05). Conversely, capacities of anti-hydroxyl radical (AHR) and anti-superoxide anion (ASA) in the detected tissues and serum significantly improved with biotin levels up to 0.054-1.540 mg kg(-1) diet and then decreased in 2.680 mg biotin kg(-1) diet group for muscle and intestinal AHR as well as hepatopancreas ASA (P < 0.05). Activities of superoxide dismutase in all studied tissues and serum significantly elevated with biotin levels up to 0.330 mg kg(-1) diet and then decreased when fish fed the diet with 2.680 mg biotin kg(-1) diet, except intestine (P < 0.05). Meanwhile, activities of catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase and total thiol content in all studied tissues and serum showed the upward trend with biotin supplementations (P < 0.05). These results indicated that biotin improved antioxidant status and depressed lipid peroxidation and protein oxidation in all studied tissues and serum.

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

    PubMed Central

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

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

  15. Design and solid phase synthesis of new DOTA conjugated (+)-biotin dimers planned to develop molecular weight-tuned avidin oligomers.

    PubMed

    Pratesi, Alessandro; Ginanneschi, Mauro; Melani, Fabrizio; Chinol, Marco; Carollo, Angela; Paganelli, Giovanni; Lumini, Marco; Bartoli, Mattia; Frediani, Marco; Rosi, Luca; Petrucci, Giorgio; Messori, Luigi; Papini, Anna Maria

    2015-04-07

    Chemical modifications of the biotin carrier in pretargeted avidin–biotin radionuclide therapy may be of paramount importance for tuning the amount of the radioactivity delivered to cancer cells by labelled biotins. We report here the synthesis of a collection of new synthetic DOTA-constructs bearing two (+)-biotin molecules (bis-biotins), designed for the creation of multimeric Av units (tetramers) bonded to the antibody. All the syntheses were carried out following the solid phase strategy and growing the molecules on a Rink Amide resin. The biotin heads are connected through spacers containing PEG or non-PEG residues. Molecular modelling calculations suggested that the Av cross-linking ability of the bis-biotins depends mainly on the spacers length, with the best results being expected for arms affording distances in the range of 10–25 Å between the biotin carboxylate atoms, in the fully extended conformation. SEC-HPLC MALLS analysis of the products of our Av/bis-biotin reaction mixtures have confirmed this hypothesis. The bis-biotin 16, where the non-PEG linker ensured a distance of 26.7 Å between the biotin moieties, gave about 50% of Av oligomers while the shorter analogue 18 (19.5 Å) afforded 100% of an Av polymer containing about 21 protein units. Remarkably, the solubility of both the bis-biotins, i.e.16 and 18, in aqueous solutions was good and they showed excellent stability against the action of peptidases.

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

    PubMed

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

    2015-01-01

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

  17. Thermochemistry of HO2 + HO2 → H2O4: Does HO2 Dimerization Affect Laboratory Studies?

    PubMed

    Sprague, Matthew K; Irikura, Karl K

    2015-07-09

    Self-reaction is an important sink for the hydroperoxy radical (HO2) in the atmosphere. It has been suggested (Denis, P. A.; Ornellas, F. R. J. Phys. Chem. A, 2009, 113 (2), 499-506) that the minor product hydrogen tetroxide (HO4H) may act as a reservoir of HO2. Here, we compute the thermochemistry of HO2 self-reactions to determine if either HO4H or the cyclic hydrogen-bound dimer (HO2)2 can act as reservoirs. We computed electronic energies using coupled-cluster calculations in the complete basis set limit, CCSD(T)/CBS[45]//CCSD(T)/cc-pVTZ. Our model chemistry includes corrections for vibrational anharmonicity in the zero-point energy and vibrational partition functions, core-valence correlation, scalar relativistic effects, diagonal Born-Oppenheimer, spin-orbit splitting, and higher-order corrections. We compute the Gibbs energy of dimerization to be (-20.1 ± 1.6) kJ/mol at 298.15 K (2σ uncertainty), and (-32.3 ± 1.5) kJ/mol at 220 K. For atmospherically relevant [HO2] = 10(8) molecules per cm(3), our thermochemistry indicates that dimerization will be negligible, and thus H2O4 species are atmospherically unimportant. Under conditions used in laboratory experiments ([HO2] > 10(12) molecules per cm(3), 220 K), H2O4 formation may be significant. We compute two absorption spectra that could be used for laboratory detection of HO4H: the OH stretch overtone (near-IR) and electronic (UV) spectra.

  18. Theoretical Studies of the HO/HO2 Catalytic Cycle for Ozone Destruction

    NASA Technical Reports Server (NTRS)

    Walch, Stephen P.; Langhoff, Steve R. (Technical Monitor)

    1996-01-01

    Recently it has been determined that the HO/HO2 catalytic cycle accounts for nearly one-half of the total ozone depletion in the lower stratosphere. The catalytic cycle is: (1) HO + O3 yields HO2 + O2; (2) HO2 + O3 yields HO + O2 + O2. The net reaction is 2O3 yields 3O2. The rate limiting step in this process is the reaction of HO2 with ozone. There is a problem extending the experimental measurement of the rate of this reaction over the range 233-400 K down to stratospheric temperatures of 210-220 K. Therefore we have undertaken a project to determine the temperature dependence of the rate constant for this reaction in the low temperature region. The first step in this project, which is described in this poster, is the determination of the relevant potential energy surfaces. The calculations use CASSCF/derivative methods to define the pathways followed by CASSCF/ACPF to determine the energetics. The HO + O3 reaction is found to proceed through an HO4 complex, which is unstable with respect to HO2 + O2. The HO2 +O3 reaction is more complex. One pathway, which has been characterized, is the formation of an HO5 complex which decomposes to HO3 + O2 and subsequently to HO + O2 + O2. Another pathway, which is believed to also play a role, is hydrogen abstraction to give O2 + HO3 and subsequent decomposition of HO3 to HO + O2. Isotopic labeling experiments indicate that the later pathway is dominant. However, so far attempts to locate the saddle point for this pathway have not been successful. We have also characterized the potential energy surfaces for a number of species involved in these reactions, including HO3 and triplet O4. The triplet O4 species is probably involved in the reaction of vibrationally excited O2 with ground state O2 leading to O3 + O. The latter reaction is believed to be important as an additional source of stratospheric ozone.

  19. Theoretical Studies of the HO/HO2 Catalytic Cycle for Ozone Destruction

    NASA Technical Reports Server (NTRS)

    Walch, Stephen P.; Langhoff, Steve R. (Technical Monitor)

    1996-01-01

    Recently it has been determined that the HO/HO2 catalytic cycle accounts for nearly one-half of the total ozone depletion in the lower stratosphere. The catalytic cycle is: (1) HO + O3 yields HO2 + O2; (2) HO2 + O3 yields HO + O2 + O2. The net reaction is 2O3 yields 3O2. The rate limiting step in this process is the reaction of HO2 with ozone. There is a problem extending the experimental measurement of the rate of this reaction over the range 233-400 K down to stratospheric temperatures of 210-220 K. Therefore we have undertaken a project to determine the temperature dependence of the rate constant for this reaction in the low temperature region. The first step in this project, which is described in this poster, is the determination of the relevant potential energy surfaces. The calculations use CASSCF/derivative methods to define the pathways followed by CASSCF/ACPF to determine the energetics. The HO + O3 reaction is found to proceed through an HO4 complex, which is unstable with respect to HO2 + O2. The HO2 +O3 reaction is more complex. One pathway, which has been characterized, is the formation of an HO5 complex which decomposes to HO3 + O2 and subsequently to HO + O2 + O2. Another pathway, which is believed to also play a role, is hydrogen abstraction to give O2 + HO3 and subsequent decomposition of HO3 to HO + O2. Isotopic labeling experiments indicate that the later pathway is dominant. However, so far attempts to locate the saddle point for this pathway have not been successful. We have also characterized the potential energy surfaces for a number of species involved in these reactions, including HO3 and triplet O4. The triplet O4 species is probably involved in the reaction of vibrationally excited O2 with ground state O2 leading to O3 + O. The latter reaction is believed to be important as an additional source of stratospheric ozone.

  20. Indicators of marginal biotin deficiency and repletion in humans: validation of 3-hydroxyisovaleric acid excretion and a leucine challenge123

    PubMed Central

    Mock, Donald M; Henrich, Cindy L; Carnell, Nadine; Mock, Nell I

    2006-01-01

    Background The results of clinical studies have provided evidence that marginal biotin deficiency is more common than was previously thought. A previous study of 10 subjects showed that the urinary excretion of biotin and 3-hydroxyisovaleric acid (3HIA) are early and sensitive indicators of marginal biotin deficiency. Objective Marginal biotin deficiency was experimentally induced and corrected to assess the utility of 3 indicators of biotin status: urinary excretion of biotin and 3HIA and the increase in 3HIA excretion after leucine loading. Design Eleven healthy adults consumed an egg white diet for 28 d. Blood and 24-h urine samples were collected before the start of the diet and twice weekly thereafter. In 5 subjects, an oral leucine challenge was performed weekly for 4 wk. After depletion, biotin status was restored with a general diet with or without a supplement containing 80 μg biotin. Urinary excretion of biotin, bisnorbiotin, and biotin sulfoxides was determined by avidin-binding assay after HPLC. Excretion of 3HIA, an indicator of reduced activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase (EC 6.4.1.4), was measured by gas chromatography–mass spectrometry. Results 3HIA excretion increased significantly with time on the egg white diet (P < 0.0001), as did 3HIA excretion in response to the leucine challenge (P < 0.002); the excretion of both biotin and bisnorbiotin decreased significantly with time (P < 0.0001). In most subjects, biotin status returned to normal after 1 wk of a general diet. Conclusions Excretion of 3HIA and of biotin are early and sensitive indicators of biotin deficiency. 3HIA excretion after a leucine challenge is at least as sensitive. PMID:12399279

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

    PubMed

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

    2015-09-01

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

  2. Half-sandwich ruthenium(II) biotin conjugates as biological vectors to cancer cells.

    PubMed

    Babak, Maria V; Plażuk, Damian; Meier, Samuel M; Arabshahi, Homayon John; Reynisson, Jóhannes; Rychlik, Błażej; Błauż, Andrzej; Szulc, Katarzyna; Hanif, Muhammad; Strobl, Sebastian; Roller, Alexander; Keppler, Bernhard K; Hartinger, Christian G

    2015-03-23

    Ruthenium(II)-arene complexes with biotin-containing ligands were prepared so that a novel drug delivery system based on tumor-specific vitamin-receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru(II) -biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012-2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half-sandwich ruthenium(II)-biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A Francisella virulence factor catalyses an essential reaction of biotin synthesis.

    PubMed

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

    2014-01-01

    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 Escherichia 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 subclade 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 reaction 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.

  4. Fatty liver and kidney syndrome in chicks. II. Biochemical role of biotin.

    PubMed

    Hood, R L; Johnson, A R; Fogerty, A C; Pearson, J A

    1976-12-01

    The role of biotin-dependent enzymes in the fatty liver and kidney syndrome of young chicks was studied. Under conditions of a marginal deficiency of dietary biotin, the level of biotin in the liver has differing effects on the activities of two biotin-dependent enzymes, pyruvate carboxylase and acetyl-CoA carboxylase. The activity of acetyl-CoA carboxylase is increased, but when the dietary deficiency of biotin produces biotin levels which are below 0-8 mug/g of liver, the activity of pyruvate carboxylase may be insufficient to completely metabolize pyruvate via gluconeogenesis. There is an increase in liver size and in the activities of enzymes involved in alternate pathways for the removal of pyruvate. Blood lactate accumulates and there is increased synthesis of fatty acids, and an accumulation of palmitoleic acid; these steps are accomplished by increased activities of at least the following enzymes: acetyl-CoA carboxylase, malate dehydrogenase (decarboxylating) (NADP+) and the desaturase enzyme. When the biotin level is below 0-35 mug/g of liver and the chick is subjected to a stress, physiological defence mechanisms of the chick may be inadequate to maintain homeostasis and they finally collapse, resulting in accumulation of triacylglycerol in the liver and blood; the chick is unable to maintain blood glucose levels and death occurs, often only a few hours after the imposition of the stress.

  5. Biotin deficiency and liver metabolism in relation to fatty liver and kidney syndrome.

    PubMed

    Pearce, J; Balnave, D

    1978-07-01

    Varying degrees of biotin deficiency were induced by adding freeze-dried, raw egg white to the diet of broiler chicks. Aspects of liver metabolism were studied with reference to fatty liver and kidney syndrome. Mortality was low with 11.8 g egg white/kg diet, or less, but with 17.7 g/kg or more, mortality was very high. High mortality was observed with less than 0.33 microgram biotin/g liver. Associated with low concentrations of liver biotin were substantial increases in liver weight and lipid content in starved birds. The increased liver lipid content was not observed in birds fed ad libitum. The increased liver lipid content in biotin-deficient, starved birds was not reflected in the specific activities of hepatic lipogenic enzymes or hepatic lipogenesis in vivo measured by the incorporation of tritium from 3H-labelled water into liver lipid. Biotin deficiency affected the specific activities of the biotin-requiring enzymes, pyruvate carboxylase and acetyl CoA carboxylase, differently; the latter was unaffected whereas the former decreased concomitantly with liver biotin concentration.

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

    PubMed Central

    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 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. PMID:24031727

  7. Development and characterization of a mouse with profound biotinidase deficiency: a biotin-responsive neurocutaneous disorder.

    PubMed

    Pindolia, Kirit; Jordan, Megan; Guo, Caiying; Matthews, Nell; Mock, Donald M; Strovel, Erin; Blitzer, Miriam; Wolf, Barry

    2011-02-01

    Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. DEVELOPMENT AND CHARACTERIZATION OF A MOUSE WITH PROFOUND BIOTINIDASE DEFICIENCY: A BIOTIN-RESPONSIVE NEUROCUTANEOUS DISORDER

    PubMed Central

    Pindolia, Kirit; Jordan, Megan; Guo, Caiying; Matthews, Nell; Mock, Donald M.; Strovel, Erin; Blitzer, Miriam; Wolf, Barry

    2010-01-01

    Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states. PMID:21051254

  9. Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells.

    PubMed

    Yagi, Takashi; Terada, Nobuo; Baba, Takeshi; Ohno, Shinichi

    2002-01-01

    A peroxidase-conjugated avidin-biotin complex was used to detect endogenous biotin-containing proteins in mouse cerebellum. By this method, Bergmann glial cells were found to be strongly labelled in the adult mouse cerebellum. Developmentally, cells in the granular layer, probably astrocytes, appeared to be labelled around postnatal 10-day (P10). Their labelling decreased after P20, although the positive-labelling remained in the Bergmann glial cells up to the adult stage. The findings were confirmed by using a Alexa Fluor 488-conjugated streptavidin technique. The labelling was not affected by routine hydrogen peroxide treatment, but it was eliminated by avidin-biotin blocking. By another transblot method, the reactive proteins in the mouse cerebellum were found to be 120 kDa (the strongest one) and 75 kDa. For electron microscopy, a gold-conjugated anti-biotin antibody was immunoreacted to the mitochondria of Bergmann glial cells. These results suggest that endogenous biotin-containing proteins are abundant in the Bergmann glial cells. Therefore, the avidin-biotin complex method is useful for detecting Bergmann glial cells, probably because of the difference of biotin metabolism in the cerebellar glial cells.

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

    PubMed

    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.

  11. [Prospective study of biotin treatment in patients with erythema due to gefitinib or erlotinib].

    PubMed

    Ogawa, Yoshikazu; Kiba, Takayoshi; Nakano, Kikuo; Fujiwara, Keiichi; Taniguchi, Hitoshi; Hosokawa, Atsuko; Nakashima, Toshihisa; Kimoto, Shizue; Kajiume, Sayoko; Okada, Yuuko; Ichiba, Yasunori

    2014-04-01

    Gefitinib anderlotinib, which are epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs), have been usedfor the treatment of inoperable andrecurrent non-small cell lung cancer(NSCLC)patients. These drugs are known to cause a skin rash, one of the major side effects, at a high frequency. Biotin is a water-soluble vitamin, andit belongs to the vitamin B family. It is well known that biotin deficiency increases the risk of skin dermatitis. We administered biotin to four patients with skin rash, all of whom were treatedwith either gefitinib or erlotinib andwere unable to be treatedby a steroid ointment alone. In all patients, administration of biotin reduced the skin rash. Surprisingly, in 2 patients in whom EGFR-TKI therapy was discontinued because of the skin rash, the administration of biotin allowed for long-term gefitinib or erlotinib treatment. Biotin may be considereduseful for the treatment of skin rash causedby EGFR-TKIs. Further trials may be needed to confirm the value of biotin in this setting.

  12. Highly stabilized and photoluminescence enhancement of ZnS:Mn{sup 2+} nanoparticles in biotin matrix

    SciTech Connect

    Keshari, Ashish K.; Pandey, Avinash C.

    2009-03-15

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

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

    PubMed

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

    2016-12-01

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

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

    PubMed Central

    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

  15. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    EPA Science Inventory

    An ELISA assay for heme oxygenase (HO-l )

    Abstract

    A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  16. Registration of "HoCP 00-950" Sugarcane

    USDA-ARS?s Scientific Manuscript database

    HoCP 00-950 sugarcane was selected from progeny of the cross HoCP 93-750 x HoCP 92-676 made at Canal Point, Florida. HoCP 00-950 was developed through cooperative research by the Agricultural Research Service of the United States Department of Agriculture, the Louisiana Agricultural Experiment Stati...

  17. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    EPA Science Inventory

    An ELISA assay for heme oxygenase (HO-l )

    Abstract

    A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  18. Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heat-stressed quail.

    PubMed

    Sahin, N; Sahin, K; Onderci, M; Gursu, M F; Cikim, G; Vijaya, J; Kucuk, O

    2005-08-01

    1. The effects of chromium picolinate and biotin supplementation alone and in combination on performance, carcase characteristics, malondialdehyde (MDA), vitamin C, vitamin E, glucose and cholesterol levels were evaluated in Japanese quail exposed to high ambient temperature. 2. Two hundred and forty quails (10d old) were assigned randomly to 4 dietary treatments at room temperature (22 degrees C; thermoneutral, TN) or ambient (34 degrees C for 8 h/d; heat stress, HS). Both TN and HS were fed either on a basal (control) diet or the basal diet supplemented with 400 microg of Cr/kg (Cr group), 0.5 mg of biotin/kg of diet (biotin group) or both (Cr + Biotin group). 3. Supplementing the diet of heat-stressed quails with chromium picolinate improved live weight gain, feed intake, feed efficiency and carcase traits. Biotin supplementation during TN and HS conditions did not have any beneficial effects on body weight gain, feed intake, feed efficiency or carcase traits. 4. Either in combination or alone, chromium picolinate increased serum concentrations of vitamins C and E, but decreased MDA, glucose and cholesterol concentrations in birds kept at high ambient temperature. There was no difference in vitamins C and E and MDA concentrations between birds given chromium picolinate and birds receiving chromium picolinate plus biotin, while glucose and cholesterol levels were significantly lower in all groups. The lowest concentrations of cholesterol and glucose were found in the combination group under both TN and HS conditions. An interaction between diet and temperature was detected for glucose and cholesterol concentrations. 5. Excretion rates for zinc, iron and chromium were lower in TN groups than in the corresponding HS groups. Supplementing diet with chromium picolinate and chromium picolinate plus biotin decreased excretion of minerals while biotin alone did not effect excretion of minerals. 6. Chromium supplementation, but not biotin supplementation, attenuated the

  19. Outcomes of oral biotin treatment in patients with biotinidase deficiency - Twenty years follow-up.

    PubMed

    Szymańska, Edyta; Średzińska, Małgorzata; Ługowska, Agnieszka; Pajdowska, Magdalena; Rokicki, Dariusz; Tylki-Szymańska, Anna

    2015-12-01

    Biotinidase deficiency (BTD) is an inborn error of biotin metabolism inherited as an autosomal recessive trait. Due to the, biotinidase deficiency, biotin is not recycled. Individuals with BTD usually exhibit neurological and cutaneous abnormalities unless treated with biotin. Supplementation with biotin may either ameliorate or if early introduced even prevent symptoms when introduced presymptomatically. Since 1991, 22 Polish patients from 19 families have been diagnosed with BTD. In 16 children the diagnosis had been suspected on the basis of clinical signs: skin lesions, hyperventilation, seizures, spasticity, and laboratory investigation (elevated lactate and metabolites on urine organic acids profile). The defect was enzymatically (serum biotinidase activity measurement) and genetically (tested for mutations in the BTD gene) confirmed afterwards. All patients were treated with biotin. Urine organic acids analysis (GC/MS) for 3-hydroxizovaleric acid was used for patients' monitoring. Neurological, audiological and ophthalmological evaluation has been conducted once a year. In 5 symptomatic patients a progressive optic nerve atrophy had already been noted at the time of treatment initiation. In these patients sensorineural hearing loss has also been diagnosed despite biotin supplementation. Asymptomatic patients treated with biotin supplementation presented no signs or symptoms of BTD. Supplementation with biotin slows the progression of BTD in symptomatic patients, but does not reverse nerve atrophy. Nonetheless, introduction of the treatment with biotin during presymptomatic stage of the disease prevents the onset of symptoms including optic atrophy and hearing loss. Homozygosity for the p.Leu215Phe mutation in BTD gene seems to be frequent in patients from the North-Eastern region of Poland and is connected with the hearing loss. Since the prognosis for individuals diagnosed with BTD is good, provided they are treated before symptoms occur, it is justified to

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

    PubMed Central

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

    2011-01-01

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

  1. Ho:YLF pumped HBr laser.

    PubMed

    Botha, L R; Bollig, C; Esser, M J D; Campbell, R N; Jacobs, C; Preussler, D R

    2009-10-26

    A Ho:YLF laser pumped HBr molecular laser was developed that produced up to 2.5 mJ of energy in the 4 micron wavelength region. The Ho:YLF laser was fiber pumped using a commercial Tm:fibre laser. The Ho:YLF laser was operated in a single longitudinal mode via injection seeding with a narrow band diode laser which in turn was locked to one of the HBr transitions. The behavior of the HBr laser was described using a rate equation mathematical model and this was solved numerically. Good agreement both qualitatively and quantitatively between the model and experimental results was obtained.

  2. Lactate administration and fatty liver and kidney syndrome development in biotin-deficient chicks.

    PubMed

    Balnave, D; Pearce, J

    1979-01-01

    Two experiments were carried out to determine whether administration of lactate to biotin-deficient chicks induced fatty liver and kidney syndrome (FLKS). 2. The results suggest that increased serum lactate concentrations are a consequence of the syndrome rather than a contributory factor in its incidence. 3. The increase in liver lipids of birds affected by FLKS was not associated with an increase in the specific activity of the hepatic lipogenic enzyme acetyl CoA carboxylase accept when birds developed FLKS spontaneously in experiment 2. 4. Some biotin-deficient chicks did not show physical symptoms of deficiency although mean liver biotin concentrations were low (0.31 microgram/g liver).

  3. Comparison of biotin production by recombinant Sphingomonas sp. under various agitation conditions.

    PubMed

    Saito; Honda; Kawabe; Mukumoto; Shimizu; Kobayashi

    2000-06-01

    Biotin production by fermentation of recombinant Sphingomonas sp./pSP304 was investigated. A complex medium containing 60g/l of glycerol and 30g/l of yeast extract was suitable for biotin production. Biotin was produced in the late logarithmic or stationary phase after glycerol starvation. The optimum pH value for biotin production was 7.0. When the dissolved oxygen concentration (DO) was controlled at a constant level, the biotin concentration produced after 120h was significantly lower than that obtained in a test tube culture. Therefore, a batchwise jar-fermentor culture with a constant agitation speed and without DO control was conducted for investigating the effect of agitation conditions on biotin production. Six types of impeller were tested: turbine-blade type, turbo-lift type, rotating mesh type (EGSTAR((R))), screw with draft tube type, Maxblend((R))type, and anchor type. With some impellers, agitation speed was also changed. Both the maximum cell concentration and biotin production varied depending on agitation conditions. Relatively high cell concentrations were attained with four of the impeller types, turbine-blade type, rotating mesh type, Maxblend((R)) type, and anchor type. Among these impellers, the turbine-blade impeller with sintered sparger was suitable for biotin production. After 120h, the cell concentration reached an OD(660) of 43 and a biotin concentration of 66mg/l was obtained, which was comparable with the results from the test tube culture. Morphological variation was also observed depending on the agitation conditions: oval-shaped, rod-shaped, and elongated-shaped cells. Biotin production was relatively high in slightly long rod-shape cells but low in elongated cells. The difference in morphology appeared to depend on the shear stress. It was found that biotin production was strongly correlated with cell length and the oxygen transfer coefficient (k(L)a); cell lengths in the range 4-7µm and k(L)a values in the range 1.5-2.0/min were

  4. Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme

    PubMed Central

    Berkovitch, Frederick; Nicolet, Yvain; Wan, Jason T.; Jarrett, Joseph T.; Drennan, Catherine L.

    2006-01-01

    The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-l-methionine and dethiobiotin has been determined to 3.4 angstrom resolution. This structure addresses how “AdoMet radical” or “radical SAM” enzymes use Fe4S4 clusters and S-adenosyl-l-methionine to generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. The structure places the substrates between the Fe4S4 cluster, essential for radical generation, and the Fe2S2 cluster, postulated to be the source of sulfur, with both clusters in unprecedented coordination environments. PMID:14704425

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

    PubMed Central

    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 distinct variants at the holocarboxylase synthetase locus. PMID:3920902

  6. Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

    PubMed

    Hou, Shuai; Shi, Lei; Lei, Haixin

    2016-01-01

    RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

  7. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes.

    PubMed Central

    Chollet, A; Kawashima, E H

    1985-01-01

    Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA. Images PMID:4000941

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

    PubMed

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

    2017-09-26

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

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

    PubMed

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

    2013-10-01

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

  10. Local Measurement of Tropospheric HO(x)

    NASA Technical Reports Server (NTRS)

    Crosley, David R.

    1994-01-01

    In March of 1992 a workshop sponsored by NASA and NSF was held at SRI International to assess the current ability to measure atmospheric OH and HO2. The measurement techniques reviewed during the workshop for detection of OH included five laser-induced fluorescence schemes, five laser-based adsorption techniques, and four non-laser methods. Six instruments or instrument concepts for HO2 detection, including chemical amplification, conversion to OH with subsequent OH detection, or direct spectroscopic detection of the HO2 were also discussed. The conclusions from the workshop identify several measurement techniques for OH and HO2 that are ready for field tests. These have the ability to measure the radicals with sufficient sensitivity and accuracy to form meaningful comparison with atmospheric model predictions. The workshop conclusions also include recommendations for informal and formal intercomparison protocols.

  11. LiHo(PO3)4

    PubMed Central

    Ben Zarkouna, Emna; Driss, Ahmed; Férid, Mokhtar

    2009-01-01

    Lithium holmium(III) polyphosphate(V), LiHo(PO3)4, belongs to the type I of polyphosphates with general formula ALn(PO3)4, where A is a monovalent cation and Ln is a trivalent rare earth cation. In the crystal structure, the polyphosphate chains spread along the b-axis direction, with a repeat period of four tetra­hedra and 21 inter­nal symmetry. The Li and Ho atoms are both located on twofold rotation axes and are surrounded by four and eight O atoms, leading to a distorted tetra­hedral and dodeca­hedral coordination, respectively. The HoO8 polyhedra are isolated from each other, the closest Ho⋯Ho distance being 5.570 (1) Å. PMID:21581738

  12. HO-1 expression control in the rat glomerulus.

    PubMed

    Detsika, Maria G; Duann, Pu; Lianos, Elias A

    2015-05-08

    The differential localization of HO-1 in renal cells under conditions of injury, and the demonstration that exaggerated HO-1 expression can have detrimental rather than beneficial effects, raises the question of whether HO-1 expression in these cells is subject to control. The present study identifies a unique HO-1 expression pattern in the renal glomerulus indicative of presence of HO-1 expression control following prolonged HO-1 induction. HO-1 and HO-2 expression in response to the natural HO substrate/inducer Fe(++) protoporphyrin (PP) IX (hemin) was assessed in normal rat glomeruli. Following 18 h incubations with hemin (0-200 μM), HO-1 expression increased in a concentration-dependent manner and via a hemopexin (HPX) independent mechanism with no effect on HO-2. In incubations with higher hemin concentrations (400 μM), likely to be encountered in hemolytic disorders, HO-1 expression, decreased. This was preceded by a prolonged and sustained increase in HO-1 protein and was independent of the Fe(++) moiety as incubations with Cobalt protoporphyrin (CoPP) resulted in an identical expression pattern. The decrease of HO-1 protein could not be accounted for by proteasomal degradation since it was not reversed in co-incubations with hemin and the proteasome inhibitor, MG132, at concentrations sufficient to increase HO-1 glomerular content when used alone. Moreover, in the presence of MG132, a decrease of HO-1 expression also occurred at 100 and 200 μM hemin. The effect of MG132 was mimicked by two additional mechanistically different approaches which also raised HO-1 content: a) co-incubations of hemin with ZnPP which increased HO-1 protein when used alone, and b) glomerular HO-1 over expression achieved by SB transposon mediated transgenesis. In contrast, the decrease in HO-1 levels observed at high hemin concentrations was reversed in co-incubations with hemin and SnPP, which reduced HO-1 content when used alone. Expression of NF-E2 related factor 2 (Nrf2

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

    ERIC Educational Resources Information Center

    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)

  14. HIGH-DOSE BIOTIN TREATMENT FOR SECONDARY PROGRESSIVE MULTIPLE SCLEROSIS MAY INTERFERE WITH THYROID ASSAYS

    PubMed Central

    Minkovsky, Alissa; Lee, Mark N.; Dowlatshahi, Mitra; Angell, Trevor E.; Mahrokhian, Lilian S.; Petrides, Athena K.; Melanson, Stacy E.F.; Marqusee, Ellen; Woodmansee, Whitney W.

    2016-01-01

    Objective To review cases and increase awareness in clinicians treating patients who may be taking biotin. Methods We describe the presentation and workup of a woman with secondary progressive multiple sclerosis on high dose biotin with laboratory studies suggestive of thyrotoxicosis. Results Plasma samples showed laboratory evidence of elevated thyroid hormone levels with elevated free thyroxine >7.8 ng/dl (reference interval (RI) 0.9-1.7 ng/dl) and decreased thyroid stimulating hormone <0.02 uIU/ml (RI 0.50-5.70 uIU/ml). Laboratory values normalized when biotin was withheld prior to repeat testing. Conclusions Our case report demonstrates that ingestion of high dose biotin in multiple sclerosis patients can cause interference with laboratory assessment of thyroid function. This interference causes laboratory values suggestive of thyrotoxicosis and can lead to unnecessary evaluation. Clinicians should be aware of the risk of laboratory interference in this patient demographic. PMID:27917400

  15. HIGH-DOSE BIOTIN TREATMENT FOR SECONDARY PROGRESSIVE MULTIPLE SCLEROSIS MAY INTERFERE WITH THYROID ASSAYS.

    PubMed

    Minkovsky, Alissa; Lee, Mark N; Dowlatshahi, Mitra; Angell, Trevor E; Mahrokhian, Lilian S; Petrides, Athena K; Melanson, Stacy E F; Marqusee, Ellen; Woodmansee, Whitney W

    2016-01-01

    To review cases and increase awareness in clinicians treating patients who may be taking biotin. We describe the presentation and workup of a woman with secondary progressive multiple sclerosis on high dose biotin with laboratory studies suggestive of thyrotoxicosis. Plasma samples showed laboratory evidence of elevated thyroid hormone levels with elevated free thyroxine >7.8 ng/dl (reference interval (RI) 0.9-1.7 ng/dl) and decreased thyroid stimulating hormone <0.02 uIU/ml (RI 0.50-5.70 uIU/ml). Laboratory values normalized when biotin was withheld prior to repeat testing. Our case report demonstrates that ingestion of high dose biotin in multiple sclerosis patients can cause interference with laboratory assessment of thyroid function. This interference causes laboratory values suggestive of thyrotoxicosis and can lead to unnecessary evaluation. Clinicians should be aware of the risk of laboratory interference in this patient demographic.

  16. Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

    PubMed

    Gorokhovatsky, Andrey Yu; Rudenko, Natalia V; Marchenkov, Victor V; Skosyrev, Vitaly S; Arzhanov, Maxim A; Burkhardt, Nils; Zakharov, Mikhail V; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2003-02-01

    Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

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

    ERIC Educational Resources Information Center

    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)

  18. [Adaptation of the avidin-biotin system for identifying and quantifying Sendai virus antigens].

    PubMed

    Popa, L M; Marcheş, F; Repanovici, R; Iliescu, R; Muţiu, A; Cajal, N

    1989-01-01

    The avidin-biotin system was adapted in view of the identification and dosage of the Sendai parainfluenza virus and of its antigens, using the method of double antibodies (biotinylated and nonbiotinylated) in ELISA type tests.

  19. Severe holocarboxylase synthetase deficiency with incomplete biotin responsiveness resulting in antenatal insult in samoan neonates.

    PubMed

    Wilson, Callum J; Myer, Michael; Darlow, Brian A; Stanley, Thorsten; Thomson, Glen; Baumgartner, E Regula; Kirby, Denise M; Thorburn, David R

    2005-07-01

    We describe 7 Polynesian babies with a unique severe form of holocarboxylase synthetase deficiency characterized by antenatal growth retardation, subependymal cysts, only partial response to biotin, and a poor outcome.

  20. Biotin-conjugated fusogenic liposomes for high-quality cell purification.

    PubMed

    Hersch, Nils; Wolters, Benjamin; Ungvari, Zoltan; Gautam, Tripti; Deshpande, Dhruva; Merkel, Rudolf; Csiszar, Anna; Hoffmann, Bernd; Csiszár, Agnes

    2016-01-01

    Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.

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

    PubMed

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

    2016-03-01

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

  2. Biotin and carnitine deficiency due to hypoallergenic formula nutrition in infants with milk allergy.

    PubMed

    Hayashi, Hisako; Tokuriki, Shuko; Okuno, Takashi; Shigematsu, Yosuke; Yasushi, Akiba; Matsuyama, Go; Sawada, Ken; Ohshima, Yusei

    2014-04-01

    Amino acid formulas and hydrolyzed formulas given to infants in Japan with milk allergies theoretically contain little, if any, biotin and carnitine. We assessed biotin and carnitine insufficiency in six infants with milk allergy who were fed amino acid formulas and/or hydrolyzed formulas, by measuring urine 3-hydroxyisovaleric acid (3-HIA) and serum free carnitine (C0), respectively. All patients presented with elevated urine 3-HIA and lowered serum C0 compared with post-menstrual age-matched infants who were fed breast milk or standard infant formulas. Supplementation with biotin and L-carnitine immediately improved the insufficiency. Care should be taken to avoid biotin and carnitine deficiency in allergic infants fed amino acid or hydrolyzed formulas.

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

    NASA Astrophysics Data System (ADS)

    Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

    2007-04-01

    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 5 mol 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).

  4. An improved smaller biotin ligase for BioID proximity labeling

    PubMed Central

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

    2016-01-01

    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

  5. A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

    PubMed

    Martin, Harry; Murray, Colleen; Christeller, John; McGhie, Tony

    2008-10-01

    A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

  6. [Biotidinase deficiency: a disease with neurologic and cutaneous expression susceptible to biotin].

    PubMed

    de Parscau, L; Beaufrère, B; Vianey-Liaud, C; Rolland, M O; Langue, J; Divry, P; Guibaud, P

    1989-01-01

    The authors report 2 familial cases of biotin deficiency. The first neurological signs appeared at the age of 2 years in a boy. The diagnosis was established in his sister in the neonatal period. A review of 41 published cases summarizes the neurologic signs (seizures, ataxia, hypotonia and later, developmental delay and deafness) and the cutaneous signs (rash, alopecia). An early treatment with biotin cures or prevents the clinical signs of the disease in most cases.

  7. Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms

    PubMed Central

    Subramanya, Sandeep B.; Subramanian, Veedamali S.; Kumar, Jeyan S.; Hoiness, Robert

    2011-01-01

    The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5′-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5′-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding. PMID:21148397

  8. Thermodynamic analysis of small ligand binding to the Escherichia coli repressor of biotin biosynthesis.

    PubMed

    Xu, Y; Johnson, C R; Beckett, D

    1996-04-30

    BirA is the transcriptional repressor of biotin biosynthesis and a biotin holoenzyme synthetase. It catalyzes synthesis of biotinyl-5'-AMP from the substrates biotin and ATP. The adenylate is the activated intermediate in the biotin transfer reaction as well as the positive allosteric effector for site-specific DNA binding. The affinity of BirA for the adenylate is considerably greater than its affinity for biotin, and both binding reactions are coupled to changes in the conformation of the protein. The temperature dependencies of the two binding interactions have been determined using kinetic techniques. Van't Hoff analysis of the equilibrium dissociation constants derived from the kinetic data indicate that while the two binding processes are characterized by large negative enthalpies, the entropic contributions are small for both. Binding enthalpies have also been determined by isothermal titration calorimetry. Consistent with the results of the van't Hoff analyses, the calorimetric enthalpies are large and negative. The greater precision of the calorimetric measurements allowed more accurate estimation of the entropic contributions to the binding processes, which are of opposite sign for the two ligands. In addition, the heat capacity changes associated with the two binding reactions are small. The measured thermodynamic parameters for binding of biotin and bio-5'-AMP to BirA have been utilized to dissect out structural contributions to the binding energetics. Results of these calculations indicate equivalent contributions of burial of polar and apolar surface area to both binding processes. The total loss of solvent accessible surface area is, however, greater for biotin binding. The analysis indicates furthermore that although both binding reactions are coupled to losses in configurational entropy, the magnitude of the conformational change is significantly larger for biotin binding.

  9. Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms.

    PubMed

    Subramanya, Sandeep B; Subramanian, Veedamali S; Kumar, Jeyan S; Hoiness, Robert; Said, Hamid M

    2011-03-01

    The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5'-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5'-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.

  10. Intake and urinary amounts of biotin in Japanese elementary school children, college students, and elderly persons.

    PubMed

    Shibata, Katsumi; Tsuji, Tomiko; Fukuwatari, Tsutomu

    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 consecutive days were recorded. A 24-hour urine sample was collected on the fourth day, and the urine biotin was measured. The urine biotin at the fourth day was 57.8, 50.9, 81.0, 66.2, and 82.3 nmol/day in boys, girls, male students, female students, and elderly persons, respectively. The average intake of biotin for 4 consecutive days was 35, 31, 28, 26, and 32 μg/day in boys, girls, male students, female students, and elderly persons, respectively.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors. PMID:26954237

  13. A case of holocarboxylase synthetase deficiency with insufficient response to prenatal biotin therapy.

    PubMed

    Yokoi, Kyoko; Ito, Tetsuya; Maeda, Yasuhiro; Nakajima, Yoko; Kurono, Yukihisa; Sugiyama, Naruji; Togari, Hajime

    2009-11-01

    Holocarboxylase synthetase (HCS) deficiency is an inborn error of biotin metabolism, leading to a multiple carboxylases deficiency. As the affected fetus sometimes presents with enlargement of the cerebral ventricles and intrauterine growth retardation (IUGR), prenatal administration of biotin has been attempted in some pregnancies. We present herein the case of a Japanese neonate with HCS deficiency who received maternal administration of biotin (10mg/day) from 33 weeks' gestation. After biotin administration, the fetal body weight increased and gestation was continued to full term. However, lactic acidemia and metabolic acidosis were observed after birth. To evaluate the effects of prenatal therapy, we collected serum samples and measured the acylcarnitine profiles using high-performance liquid chromatography electrospray ionization tandem mass spectrometry. At birth, levels of propionylcarnitine and 3-hydroxyisovalerylcarnitine had already increased. At 2h after birth, these levels of acylcarnitines were further increased. At 3.5h after the start of biotin, these chemical findings were slightly improved. In conclusion, we considered that prenatal biotin therapy at 10mg/day may have been inadequate to avoid neonatal acidotic crisis in this case.

  14. Contribution of Cysteine Desulfurase (NifS Protein) to the Biotin Synthase Reaction of Escherichia coli

    PubMed Central

    Kiyasu, Tatsuya; Asakura, Akira; Nagahashi, Yoshie; Hoshino, Tatsuo

    2000-01-01

    The contribution of cysteine desulfurase, the NifS protein of Klebsiella pneumoniae and the IscS protein of Escherichia coli, to the biotin synthase reaction was investigated in in vitro and in vivo reaction systems with E. coli. When the nifS and nifU genes of K. pneumoniae were coexpressed in E. coli, NifS and NifU proteins in complex (NifU/S complex) and NifU monomer forms were observed. Both the NifU/S complex and the NifU monomer stimulated the biotin synthase reaction in the presence of l-cysteine in an in vitro reaction system. The NifU/S complex enhanced the production of biotin from dethiobiotin by the cells growing in an in vivo reaction system. Moreover, the IscS protein of E. coli stimulated the biotin synthase reaction in the presence of l-cysteine in the cell-free system. These results strongly suggest that cysteine desulfurase participates in the biotin synthase reaction, probably by supplying sulfur to the iron-sulfur cluster of biotin synthase. PMID:10781558

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

    PubMed

    Andreoni, Alessandra; Nardo, Luca; Rigler, Rudolf

    2016-09-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Shibata, Katsumi; Tsuji, Tomiko; Fukuwatari, Tsutomu

    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 consecutive days were recorded. A 24-hour urine sample was collected on the fourth day, and the urine biotin was measured. The urine biotin at the fourth day was 57.8, 50.9, 81.0, 66.2, and 82.3 nmol/day in boys, girls, male students, female students, and elderly persons, respectively. The average intake of biotin for 4 consecutive days was 35, 31, 28, 26, and 32 μg/day in boys, girls, male students, female students, and elderly persons, respectively. PMID:24812517

  19. Contribution of cysteine desulfurase (NifS protein) to the biotin synthase reaction of Escherichia coli.

    PubMed

    Kiyasu, T; Asakura, A; Nagahashi, Y; Hoshino, T

    2000-05-01

    The contribution of cysteine desulfurase, the NifS protein of Klebsiella pneumoniae and the IscS protein of Escherichia coli, to the biotin synthase reaction was investigated in in vitro and in vivo reaction systems with E. coli. When the nifS and nifU genes of K. pneumoniae were coexpressed in E. coli, NifS and NifU proteins in complex (NifU/S complex) and NifU monomer forms were observed. Both the NifU/S complex and the NifU monomer stimulated the biotin synthase reaction in the presence of L-cysteine in an in vitro reaction system. The NifU/S complex enhanced the production of biotin from dethiobiotin by the cells growing in an in vivo reaction system. Moreover, the IscS protein of E. coli stimulated the biotin synthase reaction in the presence of L-cysteine in the cell-free system. These results strongly suggest that cysteine desulfurase participates in the biotin synthase reaction, probably by supplying sulfur to the iron-sulfur cluster of biotin synthase.

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

    PubMed

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

    2017-08-15

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

  1. [The effects of biotin on the metabolism of ammonia and amino acids in urease-induced hyperammonemic rats].

    PubMed

    Nagamine, T; Saito, S; Yamada, S; Sekiguchi, T; Kobayashi, S; Nakano, M

    1989-07-01

    The effects of oral and intraperitoneal administration of biotin in urease-induced hyperammonemic rats, as well as the influence of biotin deficiency, have been studied. Biotin deficiency was produced by feeding standard diet MF (Oriental Yeast Co.) supplemented with dry egg-white (egg-white group). Egg-white + biotin group had free access to 0.0014% of biotin solution at all time. Following an intraperitoneal injection of urease, 25 U/kg (B.W.), plasma ammonia levels in egg-white + biotin group were lower than in egg-white group, especially there was significance (p less than 0.05) at 8 hours after the urease injection. Similarly, plasma ammonia levels in biotin-injected rats, in which 1 mg of biotin had been injected intraperitoneally prior to the experiment, were significantly low compared with saline-injected controls at 4 and 6 hours after urease administration. Results of plasma amino acid analysis, 9 hours after the urease injection indicated that Fischer's molar ratio (Leu + Ileu + Val/Tyr + Phe) was significantly higher in the biotin-injected rats than the saline-injected control. It suggests that biotin might decrease blood ammonia by facilitating the detoxification mechanism as follow: L-glutamate + NH3----L-glutamine.

  2. Intramitochondrial accumulation of cationic Atto520-biotin proceeds via voltage-dependent slow permeation through lipid membrane.

    PubMed

    Antonenko, Yuri N; Nechaeva, Natalya L; Baksheeva, Victoria E; Rokitskaya, Tatyana I; Plotnikov, Egor Y; Kotova, Elena A; Zorov, Dmitry B

    2015-06-01

    Conjugation to penetrating cations is a general approach for intramitochondrial delivery of physiologically active compounds, supported by a high membrane potential of mitochondria having negative sign on the matrix side. By using fluorescence correlation spectroscopy, we found here that Atto520-biotin, a conjugate of a fluorescent cationic rhodamine-based dye with the membrane-impermeable vitamin biotin, accumulated in energized mitochondria in contrast to biotin-rhodamine 110. The energy-dependent uptake of Atto520-biotin by mitochondria, being slower than that of the conventional mitochondrial dye tetramethyl-rhodamine ethyl ester, was enhanced by the hydrophobic anion tetraphenylborate (TPB). Atto520-biotin also exhibited accumulation in liposomes driven by membrane potential resulting from potassium ion gradient in the presence valinomycin. The induction of electrical current across planar bilayer lipid membrane by Atto520-biotin proved the ability of the compound to permeate through lipid membrane in a cationic form. Atto520-biotin stained mitochondria in a culture of L929 cells, and the staining was enhanced in the presence of TPB. Therefore, the fluorescent Atto520 moiety can serve as a vehicle for intramitochondrial delivery of hydrophilic drugs. Of importance for biotin-streptavidin technology, binding of Atto520-biotin to streptavidin was found to cause quenching of its fluorescence similar to the case of fluorescein-4-biotin.

  3. Preparation and characterization of salmon calcitonin-biotin conjugates.

    PubMed

    Cetin, Meltem; Youn, Yu Seok; Capan, Yilmaz; Lee, Kang Choon

    2008-01-01

    This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.

  4. The Unimolecular Decomposition and H Abstraction Reactions by HO and HO2 from n-Butanol

    NASA Astrophysics Data System (ADS)

    Moc, Jerzy; Black, Gráinne; Simmie, John M.; Curran, Henry J.

    2009-08-01

    By using correlated ab initio (MP2, CCSD(T)) and multi-level (G3, CBS-QB3) methods we have studied unimolecular and bimolecular reactions of n-butanol in the gas phase. The specific processes investigated include H2O elimination and hydrogen abstraction by the hydroxy (HO) and hydroperoxy (HO2) radicals from this alcohol.

  5. Therapeutic potential of HO-1 in autoimmune diseases.

    PubMed

    Li, Bao-Zhu; Guo, Biao; Zhang, Hai-Yan; Liu, Juan; Tao, Sha-Sha; Pan, Hai-Feng; Ye, Dong-Qing

    2014-10-01

    Heme oxygenase-1 (HO-1), the inducible isoform of heme oxygenase (HO), has raised a lot of concerns in recent years due to its multiple functions. HO-1 was found to be a pivotal cytoprotective, antioxidant, anti-apoptotic, immunosuppressive, as well as anti-inflammatory molecule. Recent studies have clarified its significant functions in many diseases with substantial findings. In autoimmune diseases, HO-1 may have promising therapeutic potential. Here, we briefly reviewed recent advances in this field, aiming at hopefully exploring the potential therapeutic roles of HO-1, and design HO-1-based strategies for the treatment of autoimmune diseases.

  6. Estimation of human absorbed dose for (166)Ho-PAM: comparison with (166)Ho-DOTMP and (166)Ho-TTHMP.

    PubMed

    Vaez-Tehrani, Mahdokht; Zolghadri, Samaneh; Yousefnia, Hassan; Afarideh, Hossein

    2016-10-01

    In this study, the human absorbed dose of holmium-166 ((166)Ho)-pamidronate (PAM) as a potential agent for the management of multiple myeloma was estimated. (166)Ho-PAM complex was prepared at optimized conditions and injected into the rats. The equivalent and effective absorbed doses to human organs after injection of the complex were estimated by radiation-absorbed dose assessment resource and methods proposed by Sparks et al based on rat data. The red marrow to other organ absorbed dose ratios were compared with these data for (166)Ho-DOTMP, as the only clinically used (166)Ho bone marrow ablative agent, and (166)Ho-TTHMP. The highest absorbed dose amounts are observed in the bone surface and bone marrow with 1.11 and 0.903 mGy MBq(-1), respectively. Most other organs would receive approximately insignificant absorbed dose. While (166)Ho-PAM demonstrated a higher red marrow to total body absorbed dose ratio than (166)Ho-1,4,7,10-tetraazacyclo dodecane-1,4,7,10 tetra ethylene phosphonic acid (DOTMP) and (166)Ho-triethylene tetramine hexa (methylene phosphonic acid) (TTHMP), the red marrow to most organ absorbed dose ratios for (166)Ho-TTHMP and (166)Ho-PAM are much higher than the ratios for (166)Ho-DOTMP. The result showed that (166)Ho-PAM has significant characteristics than (166)Ho-DOTMP and therefore, this complex can be considered as a good agent for bone marrow ablative therapy. In this work, two separate points have been investigated: (1) human absorbed dose of (166)Ho-PAM, as a potential bone marrow ablative agent, has been estimated; and (2) the complex has been compared with (166)Ho-DOTMP, as the only clinically used bone marrow ablative radiopharmaceutical, showing significant characteristics.

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

    PubMed Central

    Chakravartty, Vandana

    2012-01-01

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

  8. Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli†

    PubMed Central

    Zeczycki, Tonya N.; Menefee, Ann L.; Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Surinya, Kathy H.; Wallace, John C.; Attwood, Paul V.; St. Maurice, Martin; Cleland, W. Wallace

    2011-01-01

    The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3− deprotonation (Glu305 and Arg301) and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3−, Lys245, Glu218 and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate, but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5 and 4-fold increase in kcat for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO3− by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO2 and PO43−. PO43− then acts as the base to deprotonate the tethered biotin at the N1-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO2 to give carboxybiotin. The formation of a distinct salt

  9. Murine monoclonal anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining.

    PubMed

    Cassano, W F

    1989-02-24

    To improve the sensitivity of conventional immunoassay methods using avidin-biotin-enzyme complex reagents, we have developed several murine monoclonal antibodies to the egg white avidin glycoprotein. These anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays by selectively enlarging the avidin-biotin-enzyme complex through bridging avidin to a second layer of avidin-biotin-enzyme complex, thereby increasing the signal from substrate conversion. Six hybridomas producing murine monoclonal antibodies which bind avidin-biotin-enzyme complexes were isolated and cloned. Two of these anti-avidin antibodies, WC19.10 and WC19.7, have been shown to produce a four-fold enhancement of signal obtained in avidin-biotin-enzyme ELISAs and qualitative enhancement of immunohistologic staining procedures.

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

    PubMed

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

    2013-02-01

    The Arabidopsis SUC5 protein represents a classical sucrose/H(+) symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  11. Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes.

    PubMed

    Saha, Sounik; Majumdar, Ritankar; Hussain, Akhtar; Dighe, Rajan R; Chakravarty, Akhil R

    2013-07-28

    Iron(III) complexes [FeL(B)] (1-4) of a tetradentate phenolate-based ligand (H3L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido [3,2-a:2',3'-c]-phenazine (dppza in 1), (N-dipyrido[3,2-a:2',3'-c]-phenazino)amidobiotin (dppzNB in 2), dipyrido [3,2-a:2',3'-c]-phenazine-11-carboxylic acid (dppzc in 3) and 2-((2-biotinamido)ethyl) amido-dipyrido[3,2-a:2',3'-c]-phenazine (dppzCB in 4) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near -0.7 V versus saturated calomel electrode in dimethyl sulfoxide-0.1 M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

  12. Biotin and glutathione targeting of solid nanoparticles to cross human brain endothelial cells.

    PubMed

    Veszelka, Szilvia; Mészáros, Mária; Kiss, Lóránd; Kóta, Zoltán; Páli, Tibor; Hoyk, Zsófia; Bozsó, Zsolt; Fülöp, Lívia; Tóth, András; Rákhely, Gábor; Deli, Mária A

    2017-07-27

    Background The blood-brain barrier restricts drug penetration to the central nervous system. Targeted nanocarriers are new potential tools to increase the brain entry of drugs. Ligands of endogenous transporters of the blood-brain barrier can be used as targeting vectors for brain delivery of nanoparticles. Objective We tested biotin-labeled solid nanoparticles for the first time and compared to biotinylated glutathione-labeled nanoparticles in brain endothelial cells. Method Neutravidin coated fluorescent polystyrene nanoparticles were derivatized with biotin and biotinylated glutathione. As a human in vitro blood-brain barrier model hCMEC/D3 brain endothelial cells were used. Cell viability by MTT test, uptake and transfer of the nanoparticles across the endothelial monolayers were measured. The uptake of the nanoparticles was visualized by confocal microscopy. Results The tested nanoparticles caused no change in cell viability. The uptake of biotin- and glutathione-labeled nanoparticles by brain endothelial cells was time-dependent and significantly higher compared to non-labeled nanoparticles. The penetration of the glutathione-labeled nanoparticles across the endothelial monolayer was higher than the biotin-targeted ones. Biotin- and glutathione-targeted nanoparticles were visualized in hCMEC/D3 cells. We verified that hCMEC/D3 express mRNA for sodium-dependent multivitamin transporter (SMVT/SLC5A6) responsible for the blood-brain barrier transport of biotin. Conclusion Biotin as a ligand increased the uptake and the transfer of nanoparticles across brain endothelial cells. Biotinylated glutathione could further increase nanoparticle permeability through endothelial monolayers supporting its use as a brain targeting vector. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    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 10 d before harvesting. The abundance of 1,009 proteins from whole cell extracts was quantified by using high-throughput immunoblots. The abundance of 44 proteins changed by at least 25% in biotin-deficient and biotin-supplemented cells compared with physiological controls. One-third of these proteins participate in cell signaling. Specifically, proteins associated with receptor tyrosine kinase-mediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells compared with controls. This was associated with increased DNA-binding activities of transcription factors Fos and Jun, and increased expression of a reporter gene driven by AP1-binding elements in biotin-deficient cells compared with physiological controls. The abundance of selected signaling proteins was not paralleled by the abundance of mRNA, suggesting that biotin affects expression of these genes at a posttranscriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. This study is consistent with the existence of clusters of biotin-responsive proteins in distinct biological processes, including signaling by Fos/Jun; the latter might mediate pro-inflammatory and anti-apoptotic effects of biotin deficiency. PMID:15987846

  15. Measurement of acylcarnitine substrate to product ratios specific to biotin-dependent carboxylases offers a combination of indicators of biotin status in humans.

    PubMed

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

    2012-09-01

    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 propionylcarnitine:methylmalonylcarnitine (Pc : MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine : malonylcarnitine (Ac : Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring, we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc : MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc : MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001). The mean change in the Ac : Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes.

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

    PubMed Central

    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 propionylcarnitine:methylmalonylcarnitine (Pc : MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine : malonylcarnitine (Ac : Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring, we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc : MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc : MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001). The mean change in the Ac : Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes. PMID:22833654

  17. Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli. Sulfide transfer from cysteine to biotin.

    PubMed

    Bui, B T; Escalettes, F; Chottard, G; Florentin, D; Marquet, A

    2000-05-01

    We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.

  18. Temperature changes under Ho:YLF irradiation

    NASA Astrophysics Data System (ADS)

    Zezell, Denise M.; Cecchini, Silvia C. M.; Pinotti, Marcos; Eduardo, Carlos d. P.

    1996-04-01

    A prototype of Er:Tm:Ho:LiYF4 (Ho:YLF) laser, emitting at 2,065 micrometers , 1,25 J 5 Hz, with pulsewidth of 250 microsecond(s) was developed for biomedical applications. In order to verify the possibility of using this laser for cavities preparation in vivo, temperature rise in the pulp chamber must be known. Temperature changes were measured during Ho:YLF irradiation with 500 mJ/pulse, 30 pulses/position with energy density of 2079 J/cm2/pulse. Two groups of teeth were used: group I with pulp chamber empty and group II with pulp chamber filled with phase change material. In both cases, there were no temperature rise above 3,8 degree(s)C in the pulp chamber.

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

    PubMed

    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 and fermented foods tested, it was found that wide distributions of biotin content were observed in powdered milk, natto, sake (rice wine), beer, edible oil and sea weed. Since powdered milk is important for child health and development, 14 kinds of powdered and special milks for use in children's diseases were intensively measured. We found that several special milk powders for children with allergies contained low levels of free biotin. Use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase, and administration of free biotin improved their symptoms dramatically. Therefore, it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people.

  20. Characterization of mutant holocarboxylase synthetase (HCS): a Km for biotin was not elevated in a patient with HCS deficiency.

    PubMed

    Aoki, Y; Suzuki, Y; Li, X; Sakamoto, O; Chikaoka, H; Takita, S; Narisawa, K

    1997-12-01

    Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. A deficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant, L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site. Km and Vmax values for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apo-carboxyl carrier protein as a substrate. A Km for biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. The Vmax for the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.

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

    PubMed Central

    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, interpretation of urinary 3HIA excretion rates is problematic, because renal function is altered by pregnancy per se. In a recent pilot study, activity of PCC in peripheral blood lymphocytes was decreased in 18 of 22 pregnant women. In 4 of 4 pregnant women with decreased PCC activity, biotin supplementation caused increased PCC activity by a mean of 95%. Taken together, such studies provide evidence that a substantial proportion of pregnant women are marginally biotin deficient. In mice, degrees of biotin deficiency that are metabolically similar to those seen in pregnant women are very teratogenic. Moreover, in mice, a marginal degree of biotin deficiency in the dam causes a much more severe degree of deficiency in the fetus. These observations further raise concerns that biotin deficiency does occur and does cause human birth defects. PMID:19056637

  2. Water dependence of the HO2 self reaction: kinetics of the HO2-H2O complex.

    PubMed

    Kanno, Nozomu; Tonokura, Kenichi; Tezaki, Atsumu; Koshi, Mitsuo

    2005-04-14

    Transient absorption spectra and decay profiles of HO2 have been measured using cw near-IR two-tone frequency modulation absorption spectroscopy at 297 K and 50 Torr in diluent of N2 in the presence of water. From the depletion of the HO2 absorption peak area following the addition of water, the equilibrium constant of the reaction HO2 + H2O <--> HO2-H2O was determined to be K2 = (5.2 +/- 3.2) x 10(-19) cm3 molecule(-1) at 297 K. Substituting K2 into the water dependence of the HO2 decay rate, the rate coefficient of the reaction HO2 + HO2-H2O was estimated to be (1.5 +/- 0.1) x 10(-11) cm3 molecule(-1) s(-1) at 297 K and 50 Torr with N2 as the diluent. This reaction is much faster than the HO2 self-reaction without water. It is suggested that the apparent rate of the HO2 self-reaction is enhanced by the formation of the HO2-H2O complex and its subsequent reaction. Results are discussed with respect to the kinetics and atmospheric chemistry of the HO2-H2O complex. At 297 K and 50% humidity, the concentration ratio of [HO2-H2O]/[HO2] was estimated from the value of K2 to be 0.19 +/- 0.11.

  3. 75 FR 52534 - Su Van Ho: Debarment Order

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-26

    ... Salmonella bacteria, with verification of such exportation or destruction by FDA. Mr. Ho concealed and... with Salmonella bacteria. As a result of his conviction, on June 10, 2010, FDA sent Mr. Ho a notice by...

  4. Chemistry of HO x radicals in the upper troposphere

    NASA Astrophysics Data System (ADS)

    Jaeglé, Lyatt; Jacob, Daniel J.; Brune, William H.; Wennberg, Paul O.

    Aircraft observations from three recent missions (STRAT, SUCCESS, SONEX) are synthesized into a theoretical analysis of the factors controlling the concentrations of HO x radicals (HO x=OH+peroxy) and the larger reservoir family HO y (HO y=HO x+2H 2O 2+2CH 3OOH+HNO 2+HNO 4) in the upper troposphere. Photochemical model calculations capture 66% of the variance of observed HO x concentrations. Two master variables are found to determine the variance of the 24 h average HO x concentrations: the primary HO x production rate, P(HO x), and the concentration of nitrogen oxide radicals (NO x=NO+NO 2). We use these two variables as a coordinate system to diagnose the photochemistry of the upper troposphere and map the different chemical regimes. Primary HO x production is dominated by the O( 1D)+H 2O reaction when [H 2O]>100 ppmv, and by photolysis of acetone (and possibly other convected HO x precursors) under drier conditions. For the principally northern midlatitude conditions sampled by the aircraft missions, the HO x yield from acetone photolysis ranges from 2 to 3. Methane oxidation amplifies the primary HO x source by a factor of 1.1-1.9. Chemical cycling within the HO x family has a chain length of 2.5-7, while cycling between the HO x family and its HO y reservoirs has a chain length of 1.6-2.2. The number of ozone molecules produced per HO y molecule consumed ranges from 4 to 12, such that ozone production rates vary between 0.3 and 5 ppbv d -1 in the upper troposphere. Three chemical regimes (NO x-limited, transition, NO x-saturated) are identified to describe the dependence of HO x concentrations and ozone production rates on the two master variables P(HO x) and [NO x]. Simplified analytical expressions are derived to express these dependences as power laws for each regime. By applying an eigenlifetime analysis to the HO x-NO x-O 3 chemical system, we find that the decay of a perturbation to HO y in the upper troposphere (as from deep convection) is represented

  5. Synthesis of fluorine-18-labeled biotin derivatives: Biodistribution and infection localization

    SciTech Connect

    Shoup, T.M.; Fischman, A.J.; Jaywook, S.

    1994-10-01

    Recently there has been much interest in the exploitation of the high binding affinity of avidin/biotin as a means of targeting drugs and radionuclides for in vivo applications. We are interested in broadening the application of the avidin/biotin complex to PET. To this end we set out to prepare {sup 18}F-labeled biotin analogs. Two {sup 18}F biotin derivatives, [3aS-(3a{alpha},4{beta},6a{alpha})]-hexa-hydro-2-oxo-1H-thieno[3,4-d]imidazole-4-(N-3-(1-[{sup 18F}]fluoropropyl))pentanamide (1) and [3aS-(3a{alpha},4{beta},6a{alpha})]-tetrahydro - 4 - 5-(1-[{sup 18}F]fluoropentyl)-1H-thieno[3,4-d]imidazol-2(3H)-F(2) were prepared with high specific activity (NCA) and evaluated for their potential in infection localization. Compound 1 binds to avidin and the biodistribution of these derivatives were studied in Escherichia coli infected rats. Half of the infected rats were treated with avidin 24 hr prior to intravenous injection of the {sup 18}F-labeled biotin analogs. Biotin 1, without avidin pretreatment, showed a selectivity of 6.08 {plus_minus} 1.12 for infection compared to normal muscle. With avidin pretreatment, selectivity increased slightly, giving an infection to normal muscle ratio of 6.39 {plus_minus} 0.96. In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection to normal muscle ratio of 0.58 {plus_minus} 0.07. This lack of selectivity illustrates the importance of the side-chain amide group in infection localization. There was some defluorination of 1 and 2, as evidenced by increased {sup 18}F bone uptake after 60 min: 2.94 {plus_minus} 0.37 and 1.17 {plus_minus} 0.21%IG/g {plus_minus}s.d., respectively. Biotin derivatives could be radiofluorinated with high specific activity. Biotin 1, is a potential positron tomography tracer for infection imaging. 16 refs., 2 figs., 6 tabs.

  6. Enhanced growth of lactobacilli and bioconversion of isoflavones in biotin-supplemented soymilk by electroporation.

    PubMed

    Ewe, Joo-Ann; Wan-Abdullah, Wan-Nadiah; Alias, Abdul Karim; Liong, Min-Tze

    2012-08-01

    This study aimed at utilizing electroporation to further enhance the growth of lactobacilli and their isoflavone bioconversion activities in biotin-supplemented soymilk. Strains of lactobacilli were treated with different pulsed electric field strength (2.5, 5.0 and 7.5 kV/cm) for 3, 3.5 and 4 ms prior to inoculation and fermentation in biotin-soymilk at 37°C for 24 h. Electroporation triggered structural changes within the cellular membrane of lactobacilli that caused lipid peroxidation (p < 0.05) and alteration of membrane fluidity (p < 0.05). This was due to the application of electric potential difference across membrane that induced pores formation and subsequently increased membrane permeability. Reversible permeabilized cells resumed growth to >9 log CFU/ml after fermentation in biotin-soymilk (p < 0.05). Lactobacilli cells treated at electric field strength of 7.5 kV/cm for 3.5 ms also showed enhanced β-glucosidase activity (p < 0.05) compared to lower doses and control, leading to increased bioconversion of isoflavones glucosides to aglycones in biotin-soymilk (p < 0.05). Results from this study show that electroporation could be used to produce biotin-soymilk with increased bioactive aglycones.

  7. Purification of biotin-binding protein from chicken egg yolk and comparison with avidin.

    PubMed

    Murthy, C V; Adiga, P R

    1984-05-17

    A simple alternative procedure for the purification in higher yields of the biotin-binding protein from the chicken egg yolk in a ligand-free form is described. The isolated protein was homogeneous by the criteria of polyacrylamide gel electrophoresis, gel filtration chromatography, immuno-double diffusion and immuno-electrophoresis. The protein had an Mr of 72 000 +/- 2000 and was a homotetramer of subunit Mr of 18 000 +/- 1000. It bound [14C]biotin in the molar ratio of 1:4 with an association constant (Ka) of 0.58 X 10(12) M-1. The yolk biotin-binding protein and avidin exhibited qualitatively similar spectral changes on interaction with biotin and p- hydroxyazobenzoic acid, but quantitatively these changes were more pronounced with avidin. Despite the lack of gross immunological cross-reactivity between the two biotin-binders, evidence based on immunological techniques for some degree of common conformational characteristics restricted to or around the ligand-binding sites of the two proteins was adduced. The mixed subunits of the two proteins failed to form hetero-oligomers on reconstitution.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Terahertz spectra of biotin based on first principle, molecular mechanical, and hybrid simulations.

    PubMed

    Bykhovski, Alexei; Woolard, Dwight

    2013-07-01

    Terahertz (THz) absorption of biotin was simulated using the first principle and the density functional theory (DFT) both in the harmonic approximation and with corrections for the anharmonicity. Anharmonicity corrections were calculated using two different approaches. First, the perturbation theory-based first principle calculations were performed to include third- and fourth-order anharmonicity corrections in atomic displacements to harmonic vibrational states. Second, the atom-centered density matrix propagation molecular dynamics model that provides a good energy conservation was used to calculate the atomic trajectories, velocities, and a dipole moment time history of biotin at low and room temperatures. Predicted low-THz lines agree well with the experimental spectra. The influence of the polyethylene (PE) matrix embedment on the THz spectra of biotin at the nanoscale was studied using the developed hybrid DFT/molecular mechanical approach. While PE is almost transparent at THz frequencies, additional low-THz lines are predicted in the biotin/PE system, which reflects a dynamic interaction between biotin and a surrounding PE cavity.

  10. A Step Closer to Membrane Protein Multiplexed Nanoarrays Using Biotin-Doped Polypyrrole

    PubMed Central

    2015-01-01

    Whether for fundamental biological research or for diagnostic and drug discovery applications, protein micro- and nanoarrays are attractive technologies because of their low sample consumption, high-throughput, and multiplexing capabilities. However, the arraying platforms developed so far are still not able to handle membrane proteins, and specific methods to selectively immobilize these hydrophobic and fragile molecules are needed to understand their function and structural complexity. Here we integrate two technologies, electropolymerization and amphipols, to demonstrate the electrically addressable functionalization of micro- and nanosurfaces with membrane proteins. Gold surfaces are selectively modified by electrogeneration of a polymeric film in the presence of biotin, where avidin conjugates can then be selectively immobilized. The method is successfully applied to the preparation of protein-multiplexed arrays by sequential electropolymerization and biomolecular functionalization steps. The surface density of the proteins bound to the electrodes can be easily tuned by adjusting the amount of biotin deposited during electropolymerization. Amphipols are specially designed amphipathic polymers that provide a straightforward method to stabilize and add functionalities to membrane proteins. Exploiting the strong affinity of biotin for streptavidin, we anchor distinct membrane proteins onto different electrodes via a biotin-tagged amphipol. Antibody-recognition events demonstrate that the proteins are stably immobilized and that the electrodeposition of polypyrrole films bearing biotin units is compatible with the protein-binding activity. Since polypyrrole films show good conductivity properties, the platform described here is particularly well suited to prepare electronically transduced bionanosensors. PMID:24476392

  11. A step closer to membrane protein multiplexed nanoarrays using biotin-doped polypyrrole.

    PubMed

    Della Pia, Eduardo Antonio; Holm, Jeppe V; Lloret, Noemie; Le Bon, Christel; Popot, Jean-Luc; Zoonens, Manuela; Nygård, Jesper; Martinez, Karen Laurence

    2014-02-25

    Whether for fundamental biological research or for diagnostic and drug discovery applications, protein micro- and nanoarrays are attractive technologies because of their low sample consumption, high-throughput, and multiplexing capabilities. However, the arraying platforms developed so far are still not able to handle membrane proteins, and specific methods to selectively immobilize these hydrophobic and fragile molecules are needed to understand their function and structural complexity. Here we integrate two technologies, electropolymerization and amphipols, to demonstrate the electrically addressable functionalization of micro- and nanosurfaces with membrane proteins. Gold surfaces are selectively modified by electrogeneration of a polymeric film in the presence of biotin, where avidin conjugates can then be selectively immobilized. The method is successfully applied to the preparation of protein-multiplexed arrays by sequential electropolymerization and biomolecular functionalization steps. The surface density of the proteins bound to the electrodes can be easily tuned by adjusting the amount of biotin deposited during electropolymerization. Amphipols are specially designed amphipathic polymers that provide a straightforward method to stabilize and add functionalities to membrane proteins. Exploiting the strong affinity of biotin for streptavidin, we anchor distinct membrane proteins onto different electrodes via a biotin-tagged amphipol. Antibody-recognition events demonstrate that the proteins are stably immobilized and that the electrodeposition of polypyrrole films bearing biotin units is compatible with the protein-binding activity. Since polypyrrole films show good conductivity properties, the platform described here is particularly well suited to prepare electronically transduced bionanosensors.

  12. Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Park, Sae Woong; Casalena, Dominick E; Wilson, Daniel J; Dai, Ran; Nag, Partha P; Liu, Feng; Boyce, Jim P; Bittker, Joshua A; Schreiber, Stuart L; Finzel, Barry C; Schnappinger, Dirk; Aldrich, Courtney C

    2015-01-22

    Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.

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

    PubMed

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

    2016-02-23

    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. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; Lin, Yi; St Maurice, Martin

    2014-11-15

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

  15. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption

    PubMed Central

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B.; Kapadia, Rubina

    2013-01-01

    The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health. PMID:23104561

  16. The role of biotin and oxamate in the carboxyl transferase reaction of pyruvate carboxylase

    PubMed Central

    Lietzan, Adam D.; Lin, Yi; St. Maurice, Martin

    2014-01-01

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes. PMID:25157442

  17. Determination of Anticyclic Citrullinated Peptide Based on Biotin-Streptavidin-Amplified Time-Resolved Fluoroimmunoassay.

    PubMed

    Sheng, Huiming; Hu, Zhi-Gang; Liu, Jie; Yuan, Fenghong; Li, Mei; Zou, Yaohong; Chen, Yu

    2015-11-01

    A rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) based on the biotin-streptavidin amplification system was developed for the determination of anticyclic citrullinated peptide (anti-CCP). Europium-labeled streptavidin derivatives combined with europium and anhydride of diethylene triamine pentaacetic acid were used to label streptavidin, biotin was coupled with rabbit anti-human IgG to form a biotin-anti-human IgG bridge between streptavidin-europium and the anti-CCP antibody in the immunoassay. The anti-CCP assay was carried out by measuring the fluorescence of Eu(3+) -streptavidin at 615 nm. The presented method produced a wide linear range from 0.58 to 9,463 U/ml, while it was only 591.4-18.48 U/ml when using an ELISA kit, and featured a detection limit up to 0.5 U/ml for anti-CCP. The values determined by the biotin-streptavidin-TRFIA and ELISA correlated well (R(2) = 0.8927). The method was applied to determine anti-CCP in serum samples with satisfied recoveries of 96.45-104.63%. The assay results obtained by the present method showed that biotin-streptavidin-amplified TRFIA improve the traditional ELISA kit for anti-CCP detection. Therefore, it offers a better alternative immunoassay in rheumatoid arthritis management. © 2014 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26903199

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

    This study is a different and new application of molecular imprinted polymers (MIPs) based on sensor technologies. In this study, semi-synthetic biotin imprinted polymeric shell has been decorated onto the surface of avidin crosslinked Au/Ag nanoclusters using bis (2-2'-bipyridyl) MATyr-MATrp-ruthenium(II) (MATyr-Ru-MATrp) as photosensitive monomer. The synthesized nanoclusters have been used the recognition of biotin by flourometric method. Synthesis of the photosensitive monomers has been realized by AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method. This method provides a strategy for the preparation of photosensitive ruthenium based aminoacid monomers and oligomers, aminoacid monomer-protein crosslinking using photosensitation and conjugation approach on micro and nano-structures by ruthenium-chelate based monomers. The affinity constant ( K a) of biotin imprinted Au/Ag nanoclusters has been determined using the Scatchard method and found to be 3.89 × 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.

  20. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

    PubMed

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B; Kapadia, Rubina; Said, Hamid M

    2013-01-01

    The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

  1. Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Park, Sae Woong; Casalena, Dominick; Wilson, Daniel; Dai, Ran; Nag, Partha; Liu, Feng; Boyce, Jim P.; Bittker, Joshua; Schreiber, Stuart; Finzel, Barry C.; Schnappinger, Dirk; Aldrich, Courtney C.

    2014-01-01

    SUMMARY Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts. PMID:25556942

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

    PubMed

    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.

  3. Spectroscopic properties of Ho3+ ions doped in tellurite glass.

    PubMed

    Rai, S B; Singh, Anant Kumar; Singh, Shiv Kumar

    2003-12-01

    Spectroscopic properties of Ho3+ doped tellurite glass (1 mol.% of Ho3+) have been studied. The absorption and fluorescence spectra have been recorded and analysed using the Judd-Offelt theory. The analysis indicates that Ho doped tellurite glasses can show lasing on the 5F4 (5S2)-5I8 transition (548.0 nm).

  4. Relationship between kinetic properties of mutant enzyme and biochemical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency.

    PubMed

    Sakamoto, O; Suzuki, Y; Li, X; Aoki, Y; Hiratsuka, M; Suormala, T; Baumgartner, E R; Gibson, K M; Narisawa, K

    1999-12-01

    Holocarboxylase synthetase (HCS) deficiency is a metabolic disorder that causes a biotin-responsive multiple carboxylase deficiency. We analyzed the kinetic properties of seven mutant HCS proteins. Two of these enzymes harbored mutations within the putative biotin-binding region of HCS and showed elevated Km values for biotin compared with that of the wild-type form (Km mutant; Gly581Ser: 45 times, delThr610: 3 times). The remaining five mutations (Arg183Pro, Leu216Arg, Leu237Pro, Val333Glu, and Val363Asp) were located outside the biotin-binding region. The enzymes containing these mutations showed normal or low Km values for biotin (non-Km mutant). Symptoms of patients who have the non-Km, mutants, as well as those of patients who have the Km, mutants, responded to biotin therapy. This is probably because the Km value for biotin of normal HCS is higher than the physiologic concentration of biotin in human cells. The Vmax values of all mutant HCS proteins were considerably decreased, but to a variable degree. The responsiveness to biotin supplementation of propionyl-CoA carboxylase activity in cultured cells bearing the mutations correlated well with the degree of reduction in the Vmax of HCS. Patients who have mutant HCS proteins with lower Vmax showed poorer clinical and biochemical responses to biotin therapy. These observations suggest that the reduction of Vmax is an essential factor for pathophysiology and prognosis of HCS deficiency under treatment with large amounts of biotin. The determination of HCS genotype can be valuable for characterizing the clinical phenotype in HCS deficient patients.

  5. Kinetics of the reaction HO2 + NO2 + M yields HO2NO2 + M

    NASA Technical Reports Server (NTRS)

    Sander, S. P.; Peterson, M. E.

    1984-01-01

    The flash photolysis/ultraviolet absorption technique was used to measure the rate constants for the reaction HO2 + NO2 + M yields HO2NO2 + M over the pressure range 50-700 torr and temperature range 229-362 K using He, O2, and N2 as diluent gases. The data were fit to the expression derived by Troe (1979) and co-workers for describing the pressure and temperature dependence of reactions in the falloff region. By combining these data with recent measurements of the rate constant for HO2NO2 thermal decomposition values of 73.8 + or - 2 eu for the standard entropy and -12.6 + or - kcal/mol for the standard enthalpy of formation of HO2NO2 were obtained. A significant enhancement in the rate constant was observed when water vapor was added to the system.

  6. Three-dimensional structures of avidin and the avidin-biotin complex.

    PubMed

    Livnah, O; Bayer, E A; Wilchek, M; Sussman, J L

    1993-06-01

    The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.

  7. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin

    PubMed Central

    Howarth, Mark; Ting, Alice Y

    2009-01-01

    This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin’s applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d. PMID:18323822

  8. Construction of heparinylated multilayer films on Tisbnd O via streptavidin/biotin interaction

    NASA Astrophysics Data System (ADS)

    Weng, Y. J.; Jing, F. J.; Chen, J. Y.; Huang, N.

    2012-06-01

    Construction of heparinylated multilayer films on Tisbnd O via streptavidin/biotin interaction was conducted in the present study. An organic layer of 3-aminopropylphosphonic acid (APP) was first introduced on Tisbnd O by self-assembling, and then biotin was immobilized by photochemical methods. So streptavidin and biotinylated heparin were assembling through biorecognition, and a desired 6-layer heparinylated multilayer was obtained through layer-by-layer driven by streptavidin/biotin interaction. The in vitro platelet adhesion and activation were investigated by a static platelet adhesion test. The clotting time was examined by activated partial thromboplastin time (APTT). Results show that the heparinylated multilayer coated Tisbnd O can significantly decrease platelet adhesion and activation, and prolong clotting time of APTT compared to untreated Tisbnd O, which indicates the heparinylated multilayer coated Tisbnd O displays more excellent anticoagulation performance than that of the bare Tisbnd O.

  9. Familial Uncombable Hair Syndrome: Ultrastructural Hair Study and Response to Biotin.

    PubMed

    Boccaletti, V; Zendri, E; Giordano, G; Gnetti, L; De Panfilis, G

    2007-01-01

    We report a family affected to the fourth generation by uncombable hair syndrome. This syndrome is characterized by unruly, dry, blond hair with a tangled appearance. The family pedigree strongly supports the hypothesis of autosomal dominant inheritance; some members of the family had, apart from uncombable hair, minor signs of atopy and ectodermal dysplasia, such as abnormalities of the nails. The diagnosis was confirmed by means of extensive scanning electron microscopy. A trial with oral biotin 5 mg/day was started on two young patients with excellent results as regards the hair appearance, although scanning electron microscopy did not show structural changes in the hair. After a 2-year-period of follow-up, hair normality was maintained without biotin, while nail fragility still required biotin supplementation for control.

  10. Irreversibility of Symptoms with Biotin Therapy in an Adult with Profound Biotinidase Deficiency.

    PubMed

    Ferreira, Patrick; Chan, Alicia; Wolf, Barry

    2017-02-21

    We report a 36-year-old woman who exhibited progressive optic atrophy at 13 years old, then stroke-like episodes and spastic diplegia in her 20s. Biotinidase deficiency was not readily considered in the differential diagnosis, and the definitive diagnosis was not made until pathological variants of the biotinidase gene (BTD) were found by exome sequencing. Profound biotinidase deficiency was confirmed by enzyme analysis. Unfortunately, her symptoms did not resolve or improve with biotin treatment. Biotin therapy is essential for all individuals with profound biotinidase deficiency and for preventing further damage in those who already exhibit irreversible neurological damage. Newborn screening for the disorder would have avoided years of clinical symptoms that now appear to be irreversible with biotin treatment.

  11. Detection of Protein Carbonyls by Means of Biotin Hydrazide-Streptavidin Affinity Methods.

    PubMed

    Hensley, Kenneth

    2015-01-01

    Oxidative posttranslational protein modifications occur as a normal process of cell biology and to a greater extent during pathogenic conditions. The detection and quantitation of protein oxidation has posed a continuing challenge to bioanalytical chemists because of the following reasons: The products of oxidative protein damage are chemically diverse; protein oxidation generally occurs at low background levels; and the complexity of biological samples introduces high background noise when standard techniques such as immunolabeling are applied to "dirty" tissue extracts containing endogenous immunoglobulins or small molecular weight, chemically reactive compounds has been developed which circumvents these difficulties by incorporating a biotin label at sites of protein carbonylation. Biotin hydrazide-labeled proteins are detectable using standard streptavidin-coupled detection techniques such as peroxidase-catalyzed chemiluminescence of immunoblots. Advantages of the biotin hydrazide-labeling technique are its sensitivity and its lack of reliance upon antibodies that inevitably suffer from nonspecific background noise and contaminating endogenous immunoglobulins.

  12. Effect of biotin supplementation on claw horn growth in young, clinically healthy cattle

    PubMed Central

    da Silva, Luiz Antônio Franco; Franco, Leandro Guimarães; Atayde, Ingrid Bueno; da Cunha, Paulo Henrique Jorge; de Moura, Maria Ivete; Goulart, Daniel Silva

    2010-01-01

    The effects of orally administered biotin supplementation on the growth of claw horn in young, clinically healthy cattle were analyzed. Twelve, 1-year-old Girolando cattle were randomly assigned to receive either 12.5 mg of diluted powdered biotin (GI) or a control treatment (GII) for 40 consecutive days. Cattle in the GI group showed an average hoof growth of 11.3 ± 0.72 mm, while those in GII had an average hoof growth of 7.2 ± 0.78 mm. The results confirmed the positive effect of biotin supplementation on the growth of angle and length of the dorsal hoof wall, hoof sole length, and on resistance to wearing, in young cattle extensively managed. PMID:20808571

  13. Use of chromium picolinate and biotin in the management of type 2 diabetes: an economic analysis.

    PubMed

    Fuhr, Joseph P; He, Hope; Goldfarb, Neil; Nash, David B

    2005-08-01

    This paper addresses the potential economic benefits of chromium picolinate plus biotin (Diachrome) use in people with Type 2 diabetes (T2DM). The economic model was developed to estimate the impact on health care systems' costs by improved HbA1C levels with chromium picolinate plus biotin (Diachrome). Lifetimes cost savings were estimated by adjusting a benchmark from the literature, using a price index to adjust for inflation. The cost of diabetes is highly dependent on the HbA1C level with higher initial levels and higher annual increments increasing the cost. Improvement in glycemic control has proven to be cost-effective in delaying the onset and progression of T2DM, reducing the risk for diabetes-associated complications and lowering utilization and cost of care. Chromium picolinate plus biotin (Diachrome) showed greater improvement of glycemic control in poorly controlled T2DM patients (HbA(1C) > or = 10%) compared to their better controlled counterparts (HbA(1C) < 10%). This improvement was additive to that achieved by oral hypoglycemic medications and correlates to calculated levels of cost savings. Average 3-year cost savings for chromium picolinate plus biotin (Diachrome) use could range from 1,636 dollars for a poorly controlled patient with diabetes without heart diseases or hypertension, to 5,435 dollars for a poorly controlled patient with diabetes, heart disease, and hypertension. Average 3-year cost savings was estimated to be between 3.9 billion dollars and 52.9 billion dollars for the 16.3 million existing patients with diabetes. Chromium picolinate plus biotin (Diachrome) use among the 1.17 million newly diagnosed patients with T2DM each year could deliver lifetime cost savings of 42 billion dollars, or 36,000 dollars per T2DM patient. Affordable, safe, and convenient, chromium picolinate plus biotin (Diachrome) could prove to be a cost-effective complement to existing pharmacological therapies for controlling T2DM.

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

    PubMed Central

    Kuwahara, Takashi; Kaneda, Shinya; Shimono, Kazuyuki

    2016-01-01

    Background: We have previously demonstrated that Candida albicans requires multivitamins (MVs) or lipid to increase rapidly in parenteral nutrition (PN) solutions. In this study, in detail, the effects of vitamins on the growth of C. albicans in PN solutions without lipid were investigated. Methods: In the 1st experiment, a commercial PN solution without lipid was supplemented with water-soluble vitamins (SVs: vitamins B1, B2, B6, B12 and C, folic acid, nicotinamide, biotin and panthenol), water-insoluble vitamins (IVs: vitamins A, D, E and K) or both (MVs). In the 2nd experiment, the test solutions were prepared by supplementing the PN solution with one of each or all of the SVs. In the 3rd experiment, another commercial peripheral PN (PPN) solution without lipid was supplemented with SVs, nicotinic acid, biotin or both nicotinic acid and biotin. In each of the experiments, a specified number of C. albicans organisms was added to each test solution, and all of the test solutions were allowed to stand at room temperature (23-26ºC). The number of C. albicans was counted at 0, 24, 48 and 72 hours after the addition of the organism. Results: In the 1st experiment, the C. albicans increased rapidly in the PN solution supplemented with the SVs, but increased slowly without the SVs, regardless of the addition of the IVs. In the 2nd experiment, the C. albicans increased rapidly in the PN solution supplemented with the SVs or biotin, but increased slowly with each of the other water-soluble vitamins. In the 3rd experiment, the C. albicans increased rapidly in the PPN solution supplemented with the SVs or biotin, but increased slowly with the addition of nicotinic acid. Conclusions: These results suggested that adding MVs or SVs to PN solutions without lipid promotes the growth of C. albicans, and that this effect is mostly attributable to biotin. PMID:27648003

  15. Chronic alcohol exposure negatively impacts the physiological and molecular parameters of the renal biotin reabsorption process

    PubMed Central

    Subramanian, Veedamali S.; Subramanya, Sandeep B.

    2011-01-01

    Normal body homeostasis of biotin is critically dependent on its renal recovery by kidney proximal tubular epithelial cells, a process that is mediated by the sodium-dependent multivitamin transporter (SMVT; a product of the SLC5A6 gene). Chronic ethanol consumption interferes with the renal reabsorption process of a variety of nutrients, including water-soluble vitamins. To date, however, there is nothing known about the effect of chronic alcohol feeding on physiological and molecular parameters of the renal biotin reabsorption process. We addressed these issues using rats and transgenic mice carrying the human SLC5A6 (P1P2) 5′-regulatory region as an in vivo model systems of alcohol exposure, and cultured human renal proximal tubular epithelial HK-2 cells chronically exposed to alcohol as an in vitro model of alcohol exposure. The [3H]biotin uptake results showed that chronic ethanol feeding in rats leads to a significant inhibition in carrier-mediated biotin transport across both renal brush border and basolateral membrane domains. This inhibition was associated with a marked reduction in the level of expression of SMVT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Furthermore, studies with transgenic mice carrying the SLC5A6 5′-regulatory region showed that chronic alcohol feeding leads to a significant decrease in promoter activity. Studies with HK-2 cells chronically exposed to alcohol again showed a marked reduction in carrier-mediated biotin uptake, which was associated with a significant reduction in promoter activity of the human SLC5A6 5′-regulatory region. These findings demonstrate for the first time that chronic ethanol feeding inhibits renal biotin transport and that this effect is, at least in part, being exerted at the transcriptional level. PMID:21209005

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

    PubMed

    Kuwahara, Takashi; Kaneda, Shinya; Shimono, Kazuyuki

    2016-01-01

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

  17. Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

    SciTech Connect

    Chou, C.; Yu, L; Tong, L

    2009-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton from bicarbonate, and Arg{sup 338} is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-{angstrom} shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.

  18. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

    SciTech Connect

    Suzuki, Y.; Aoki, Y.; Ishida, Y.

    1994-09-01

    Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.

  19. Chronic alcohol exposure negatively impacts the physiological and molecular parameters of the renal biotin reabsorption process.

    PubMed

    Subramanian, Veedamali S; Subramanya, Sandeep B; Said, Hamid M

    2011-03-01

    Normal body homeostasis of biotin is critically dependent on its renal recovery by kidney proximal tubular epithelial cells, a process that is mediated by the sodium-dependent multivitamin transporter (SMVT; a product of the SLC5A6 gene). Chronic ethanol consumption interferes with the renal reabsorption process of a variety of nutrients, including water-soluble vitamins. To date, however, there is nothing known about the effect of chronic alcohol feeding on physiological and molecular parameters of the renal biotin reabsorption process. We addressed these issues using rats and transgenic mice carrying the human SLC5A6 (P1P2) 5'-regulatory region as an in vivo model systems of alcohol exposure, and cultured human renal proximal tubular epithelial HK-2 cells chronically exposed to alcohol as an in vitro model of alcohol exposure. The [(3)H]biotin uptake results showed that chronic ethanol feeding in rats leads to a significant inhibition in carrier-mediated biotin transport across both renal brush border and basolateral membrane domains. This inhibition was associated with a marked reduction in the level of expression of SMVT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Furthermore, studies with transgenic mice carrying the SLC5A6 5'-regulatory region showed that chronic alcohol feeding leads to a significant decrease in promoter activity. Studies with HK-2 cells chronically exposed to alcohol again showed a marked reduction in carrier-mediated biotin uptake, which was associated with a significant reduction in promoter activity of the human SLC5A6 5'-regulatory region. These findings demonstrate for the first time that chronic ethanol feeding inhibits renal biotin transport and that this effect is, at least in part, being exerted at the transcriptional level.

  20. Infrared Identification of HO2 and HO3 Radicals in Irradiated Ices

    NASA Astrophysics Data System (ADS)

    Cooper, P. D.; Moore, M. H.; Hudson, R. L.

    2006-05-01

    Absorption bands at 1259 and 1142 cm-1 in H2O-O2 ice mixtures irradiated with 0.8 MeV protons at 9 K, have been assigned to the HO3 and HO2 radicals respectively. The assignment of these bands is based upon isotopic shift measurements when 18O2 is used. These bands are also observed in irradiated H2O-O3 and pure H2O2 ices. In each case, they are formed by the following reactions: H + O3 → HO3 H + O2 → HO2 While neither radical has been observed in ice using infrared spectroscopy before, the HO2 radical is known to form in irradiated water ice, but remarkably, the lesser known HO3 is the more prominent spectral feature. These species are probably formed in the surfaces of icy outer solar system bodies where O2 and O3 have already been observed. Their ability to remain trapped in ice is unknown and their reactivity at typical surface temperatures may prevent signficant concentrations being produced. However, even as intermediate species, due to their reactivity they may constitute an important component of the oxidant population in such ices. If they can remain trapped, they may provide a potential energy source for organic reactions or even microbial life in places such as Europa's sub-surface ocean.

  1. Kinetics of Reactions of HO2 with HO2 and with organics

    NASA Astrophysics Data System (ADS)

    Tyndall, G. S.; Orlando, J. J.; Tang, Y.; Hasson, A. S.; Scaldaferri, M. C.; Pimentel, A. S.; Wallington, T. J.

    2009-12-01

    Reactions of HO2 radicals with organics are in general assumed to be quite slow. However, it has been known for a number of years that HO2 adds reversibly to HCHO, in a reaction which can lead to the production of formic acid, particularly at low temperature. We have studied the reaction of HO2 with a number of carbonyls as a function of temperature using both time resolved diode laser absorption and FTIR analysis. In the time resolved studies, HO2 is produced by flash photolysis and detected in the overtone of the OH stretching mode by diode laser spectroscopy. The self reaction of HO2 was first studied as a function of temperature and added complexing gases (H2O, CH3OH). The rate coefficient of the self reaction was found to have a moderate negative temperature dependence, in agreement with older studies of this reaction. The addition of carbonyls (HCHO, CH3CHO, CF3CHO, acetone) led to an increase in the loss rate of HO2, enabling kinetic and thermodynamic information to be obtained about the equilibria. Complementary experiments were conducted to examine the products in an environmental chamber using FTIR detection of reactants and products. The reaction mechanisms and environmental impacts of the results will be discussed.

  2. Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

    PubMed Central

    Gitlin, G; Bayer, E A; Wilchek, M

    1987-01-01

    Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site. PMID:3109401

  3. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    PubMed Central

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-01-01

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin. PMID:3355517

  4. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    PubMed

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-02-15

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

  5. Genetic Analysis of the System that Reduces Biotin-d-Sulfoxide in Escherichia coli

    PubMed Central

    Dykhuizen, Daniel

    1973-01-01

    Four genes of Escherichia coli whose products are needed to reduce biotin-d-sulfoxide to biotin have been mapped: bisA next to chlA, bisB next to chlE, bisC linked to xyl, and bisD next to chlG. A defective λ transducing phage, λdbis5, which carries all the bacterial genes between the λ attachment site and chlE, was isolated and shown to have lost the phage genes from int through Q. PMID:4579877

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. Uncombable hair syndrome: observations on response to biotin and occurrence in siblings with ectodermal dysplasia.

    PubMed

    Shelley, W B; Shelley, E D

    1985-07-01

    Three children are reported with uncombable hair syndrome, consisting of slow-growing, straw-colored scalp hair that could not be combed flat. The hairs appeared normal on light microscopy but on scanning electron microscopy were triangular in cross section, with canal-like longitudinal depressions. Oral biotin, 0.3 mg three times a day, produced significant improvement after 4 months in one patient, with increased growth rate and with strength and combability of the hair, although the triangular shape remained. The other two patients were unique in having associated ectodermal dysplasia. Their hair slowly improved in appearance and combability over 5 years without biotin therapy.

  9. Some biochemical observations on gluconeogenesis from propionate in hepatocytes isolated from normal and biotin-deficients rats.

    PubMed

    Djabal, A; Mangeot, M; Cherruau, B; Lemonnier, A

    1985-01-01

    Propionate and pyruvate added to isolated normal and biotin-deficient adult rat hepatocytes increase the production of glucose. This production decreases about 30% on biotin deficiency. Malonate inhibits gluconeogenesis from propionate showing the metabolic transformation of propionyl-CoA via the Krebs cycle. Neither glucagon nor dibutyryl-cyclic AMP significantly stimulate gluconeogenesis.

  10. Preliminary evidence of the contribution of the intestinal microflora to biotin supply in zebrafish Danio rerio (Hamilton-Buchanan).

    PubMed

    Yossa, Rodrigue; Sarker, Pallab K; Vandenberg, Grant W

    2011-12-01

    A study was conducted to preliminarily assess the contribution of the intestinal microflora to biotin supply in zebrafish. Biotin and avidin were added to three isonitrogenous and isocaloric purified diets to provide molar avidin: biotin ratios of 0:0 (basal diet), 0:1 (biotin-supplemented diet), and 120:0. Another diet was made by supplementing the antibiotic succinylsulfathiazole (1%, wt/wt) to the basal diet. A fifth diet was the Zeigler commercial diet for zebrafish. Each diet was fed to a triplicate group of fish (mean initial mass 0.266 g) for 8 weeks. The condition factor, feed conversion ratio (FCR), percentage weight gain, and survival were similar in fish groups fed the commercial and the biotin-supplemented diets, but energy conversion efficiency and whole-body biotin content were highest in the fish fed the commercial diet (p<0.05). Reduced growth and survival, and increased FCR were noted in fish fed basal diet compared with those fed biotin-supplemented diet. The supplementation of avidin in diet led to lower survival and condition factor, and higher FCR than that observed with basal diet. Intestinal microbial synthesis is assumed to be a significant source of biotin to the zebrafish, as fish fed the antibiotic-supplemented diet showed the lowest growth, health condition, and feed utilization.

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

    PubMed

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

    2013-11-01

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

  12. The influence of valproic acid and carbamazepine treatment on serum biotin and zinc levels and on biotinidase activity.

    PubMed

    Castro-Gago, Manuel; Pérez-Gay, Laura; Gómez-Lado, Carmen; Castiñeiras-Ramos, Daisy E; Otero-Martínez, Santiago; Rodríguez-Segade, Santiago

    2011-12-01

    We determined the serum concentration of biotin, zinc, antiepileptic drugs, and biotinidase enzyme activity in 20 children treated with valproic acid, in 10 children treated with carbamazepine, and in 75 age- and sex-matched healthy controls. There were no significant differences in the serum levels of biotin, and biotinidase enzyme activity between the patients treated with valproic acid, the patients treated with carbamazepine, and the control group. Zinc serum levels were lower in the patients treated with valproic acid and with carbamazepine than in the control group, but within the normal range. Hair loss was observed in 3 patients treated with valproic acid, with normal serum levels of biotin, zinc, and biotinidase activity, and the alopecia disappeared with the oral administration of biotin (10 mg/d) in 3 months. These results suggest that the treatment with valproic acid does not alter the serum levels of biotin, zinc, and biotinidase enzyme activity.

  13. HO-1/CO system in tumor growth, angiogenesis and metabolism - Targeting HO-1 as an anti-tumor therapy.

    PubMed

    Loboda, Agnieszka; Jozkowicz, Alicja; Dulak, Jozef

    2015-11-01

    Heme oxygenase-1 (HO-1, hmox-1) catalyzes the rate-limiting step in the heme degradation processes. Out of three by-products of HO-1 activity, biliverdin, iron ions and carbon monoxide (CO), the latter was mostly shown to mediate many beneficial HO-1 effects, including protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis. Mounting evidence suggests that HO-1/CO systemmay be of special benefit in protection inmany pathological conditions, like atherosclerosis or myocardial infarction. By contrast, the augmented expression of HO-1 in tumor tissues may have detrimental effect as HO-1 accelerates the formation of tumor neovasculature and provides the selective advantage for tumor cells to overcome the increased oxidative stress during tumorigenesis and during treatment. The inhibition of HO-1 has been proposed as an anti-cancer therapy, however, because of non-specific effects of known HO-1 inhibitors, the discovery of ideal drug lowering HO-1 expression/activity is still an open question. Importantly, in several types of cancer HO-1/CO system exerts opposite activities, making the possible treatment more complicated. All together indicates the complex role for HO-1/CO in various in vitro and in vivo conditions.

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

    PubMed Central

    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 (3HIA-carnitine) increases in response to the decreased activity of the biotin-dependent enzyme methylcrotonyl-coenzyme A carboxylase and might reflect biotin status. Objective: We sought to determine whether the plasma concentration of 3HIA-carnitine increases significantly in marginal biotin deficiency. Design: We experimentally induced marginal, asymptomatic biotin deficiency in 10 healthy adults (8 women) by having the subjects consume undenatured egg white for 28 d; biotin status was then repleted. Plasma concentrations of 3HIA-carnitine were measured on days 0, 14, 28, 35, and 50 by liquid chromatography–mass spectroscopy. Results: The mean plasma 3HIA-carnitine concentration increased with depletion (P < 0.0001) and decreased with repletion (P < 0.0001). Plasma 3HIA-carnitine concentrations were greater than the upper limit of normal concentrations in 7 of 10 subjects by day 14 and in 9 of 10 subjects by day 28 and decreased to within normal limits in 9 of 10 subjects by day 50. Conclusions: These studies provide evidence that 3HIA-carnitine is an early and sensitive indicator of marginal biotin deficiency. The ease of sample collection, small sample volume requirement, and stability of 3HIA-carnitine during storage suggest that plasma 3HIA-carnitine concentration is likely to be a useful indicator of marginal biotin deficiency for larger population studies. PMID:20943794

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  17. Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column.

    PubMed

    Hayakawa, Kou; Katsumata, Noriyuki; Hirano, Masahiko; Yoshikawa, Kazuyuki; Ogata, Tsutomu; Tanaka, Toshiaki; Nagamine, Takeaki

    2008-06-15

    A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.

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

    SciTech Connect

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

    2007-04-01

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

  19. The Absolute Free Energy of Binding of Avidin/Biotin Revisited

    PubMed Central

    General, Ignacio J.; Dragomirova, Ralitsa; Meirovitch, Hagai

    2012-01-01

    The binding of biotin to avidin is one of the strongest in nature with absolute free energy of binding, ΔA0 = −20.4 kcal/mol. Therefore, this complex became a target for a large number of computational studies, which all, however, are based on approximate techniques or simplified models and have led to a wide range of results Therefore, ΔA0 is calculated here by rigorous statistical mechanical methods and models that consider long-range electrostatics. (1) We apply our method, “hypothetical scanning molecular dynamics with thermodynamic integration” (HSMD-TI) to avidin-biotin modeled by periodic boundary conditions with Particle Mesh Ewald (PME). (2) We apply the double decoupling method (DDM) to this system modeled by the Spherical Solvent Boundary Potential (SSBP) and the Generalized Solvent Boundary Potential (GSBP). The corresponding results for neutral biotin, ΔA0=−29.1±0.8 and −25.2±0.5 kcal/mol are significantly lower than the experimental value; we also provide the result for a charged biotin, ΔA0 = −32.6±0.8 kcal/mol. It is plausible to suggest that this disagreement with the experiment may stem from ignoring the (positive) contribution of a mobile loop that changes its structure upon ligand binding. PMID:22300239

  20. Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression

    PubMed Central

    Woong Park, Sae; Klotzsche, Marcus; Wilson, Daniel J.; Boshoff, Helena I.; Eoh, Hyungjin; Manjunatha, Ujjini; Blumenthal, Antje; Rhee, Kyu; Barry, Clifton E.; Aldrich, Courtney C.; Ehrt, Sabine; Schnappinger, Dirk

    2011-01-01

    In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens. PMID:21980288

  1. Aspects of lipid and carbohydrate metabolism, dietary biotin and fatty liver and kidney syndrome in broilers.

    PubMed

    Balnave, D; Pearce, J

    1976-11-01

    Two experiments were performed to investigate the biochemical changes associated with FLKS. A diet reported to induce FLKS was fed with or without supplementary biotin to broilers. In experiment 1 various stresses were applied to the birds. 2. In experiment 1 mortality from FLKS was 6% and in experiment 2 nil. Stress had little effect on the induction of the syndrome. 3. There were no significant differences due to diet in any of the variables examined in apparently normal birds. 4. Birds affected by FLKS showed the typical changes of increased liver and kidney weights and lipid contents but hepatic enzyme activities did not differ significantly from those of normal birds except that malate dehydrogenase (decarboxylating) (NADP) activity was significantly decreased. 5. Despite the low content of biotin in the unsupplemented diet (57 mug/kg) liver biotin content was not low in birds fed on this diet. 6. The results suggest that the incidence of FLKS is not related solely to dietary biotin content.

  2. Fatty liver and kidney syndrome in chicks. I. Effect of biotin in diet.

    PubMed

    Pearson, J A; Johnson, A R; Hood, R L; Fogerty, A C

    1976-12-01

    Fatty liver and kidney syndrome, a disorder of young chicks, was studied under laboratory conditions. Affected chicks had enlarged livers (hepatomegaly), an increased content of lipid in the liver, and an increased level of palmitoleic acid in the liver lipids. The disorder was observed mainly in chicks from young parent flocks, and was associated either with commerical diets which were subsequently found to be low in biotin, or with specially formulated low-biotin diets. A third factor, imposition of stress, was required to initiate the disorder. There was evidence of increased lipogenesis causing an increase of triacylglycerols in the liver lipids and an increased production of saturated fatty acids, particularly palmitic acid. Increased levels of palmitoleic acid resulted from an increased desaturation of palmitic acid. Under stress, affected chicks had low blood glucose levels, suggesting that gluconeogenesis was impaired. Since biotin-dependent enzymes are involved in both gluconeogenesis and lipogenesis, it would appear that the relevant enzymes respond differently to a deficiency of biotin.

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

    PubMed

    Alfadhel, Majid; Al-Bluwi, Amal

    2017-01-01

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

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

    PubMed

    Li, Ru; Huang, Jiqing; Kast, Juergen

    2015-05-01

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

  5. Biotin-Binding Proteins in the Defense of Mushrooms against Predators and Parasites

    PubMed Central

    Bleuler-Martinez, Silvia; Schmieder, Stefanie; Aebi, Markus

    2012-01-01

    Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi. PMID:23001676

  6. Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine.

    PubMed

    Streaker, Emily D; Beckett, Dorothy

    2006-08-01

    Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate "promiscuous" biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5'-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5'-AMP. This reactivity may be important for its selective biotinylation in vivo.

  7. Inborn errors of biotin metabolism. Clinical and laboratory features of eight cases.

    PubMed

    Coşkun, T; Tokatli, A; Ozalp, I

    1994-01-01

    There are two genetically determined biotin-dependent disorders. The first is holocarboxylase synthetase (HCS) deficiency and the second biotinidase deficiency. HCS catalyzes the reaction in which active holocarboxylases are synthesized from inactive apocarboxylases. Biotin is required for this synthesis. Biotinidase facilitates the release and recycling of free biotin. Deficiency of either HCS or biotinidase is characterized by certain neurological, cutaneous and biochemical abnormalities. In this paper, six patients with biotinidase and two patients with HCS deficiency are described. Among the most common neurological findings were hypotonia (6/8), seizures (2/6) and optic atrophy (2/8). Dermatitis and conjunctivitis were present in three and four patients, respectively. All patients had low blood pH bicarbonate levels. Serum lactate was increased in all and pyruvate in six cases. Two patients with biotinidase deficiency presented earlier than the mean age of onset previously reported in the literature. Detection of eight cases during the past few years at a single metabolic unit indicates that biotinidase deficiency is not rare in Turkey, where the frequency of some other metabolic disorders has also been reported to be high. We suggest that biotin-dependent disorders should be considered in all infants with neurological symptoms, particularly those with jerks, even if other signs such as alopecia, seborrheic dermatitis and acidosis are not evident, regardless of the age of presentation.

  8. Measurement of Post-Transfusion Red Cell Survival with the Biotin Label

    PubMed Central

    Mock, Donald M; Widness, John A; Veng-Pedersen, Peter; Strauss, Ronald G; Cancelas, Jose A; Cohen, Robert M; Lindsell, Christopher J; Franco, Robert S

    2014-01-01

    The goal of this review is to summarize and critically assess information concerning the biotin method to label red blood cells (RBC) for use in studies of RBC and transfusion biology — information that will prove useful to a broad audience of clinicians and scientists. A review of RBC biology, with emphasis on RBC senescence and in vivo survival is included, followed by an analysis of the advantages and disadvantages of biotin labeled RBC (BioRBC) for measuring circulating RBC volume, post-transfusion RBC recovery, RBC lifespan, and RBC age-dependent properties. The advantages of BioRBC over 51Cr RBC labeling, the current reference method, are discussed. Because the biotin method is straightforward and robust, including the ability to follow the entire lifespans of multiple RBC populations concurrently in the same subject, BioRBC offers distinct advantages for studying RBC biology and physiology, particularly RBC survival. The method for biotin labeling, validation of the method, and application of BioRBCs to studies of sickle cell disease, diabetes, and anemia of prematurity are reviewed. Studies documenting the safe use of BioRBC are reviewed; unanswered questions requiring future studies, remaining concerns, and regulatory barriers to broader application of BioRBC including adoption as a new reference method are also presented. PMID:24969019

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

    PubMed

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

    2016-02-01

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

  10. Specific detection of avidin-biotin binding using liquid crystal droplets.

    PubMed

    Khan, Mashooq; Park, Soo-Young

    2015-03-01

    Poly(acrylicacid-b-4-cynobiphenyl-4'-undecylacrylate) (PAA-b-LCP)-functionalized 4-cyano-4'-pentylbiphenyl (5CB) droplets were made by using microfluidic technique. The PAA chains on the 5CB droplets, were biotinylated, and used to specifically detect avidin-biotin binding at the 5CB/aqueous interface. The avidin-biotin binding was characterized by the configurational change (from radial to bipolar) of the 5CB droplets, as observed through a polarized optical microscope. The maximum biotinylation was obtained by injecting a >100 μg/mL biotin aqueous solution, which enabled a limit of detection of 0.5 μg/mL avidin. This droplet biosensor could specifically detect avidin against other proteins such as bovine serum albumin, lysozyme, hemoglobin, and chymotrypsinogen solutions. Avidin detection with 5CBPAA-biotin droplets having high sensitivity, specificity, and stability demonstrates new applications of the functionalized liquid crystal droplets that can detect specific proteins or other analytes through a ligand/receptor model.

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

    PubMed

    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.

  12. Application of avidin-biotin technology to improve cell adhesion on nanofibrous matrices.

    PubMed

    Pan, Jian-feng; Liu, Ning-hua; Shu, Lin-yuan; Sun, Hui

    2015-05-16

    Electrospinning is an easy and effective technique to produce submicron fibers possessing a range of attractive characteristics such as interconnected porous structures similar to natural ECM and good resilience to movement. Rapid and efficient cell attachment to nanofibrous matrices is a necessary prerequisite in tissue engineering. Thus, the aim of this study is to evaluate poly(ε-caprolactone-co-lactide)/Pluronic (PLCL/Pluronic) nanofibrous matrices with avidin-biotin technology for improving cell adhesion for the first time. PLCL/Pluronic nanofibers had relatively homogeneous fibers and interconnected porous structures. Pluronic significantly modified the hydrophilicity of nanofibrous matrices and PLCL/Pluronic nanofibrous matrices had better performance on maintaining cell proliferation. Avidin-biotin technology had no negative effect on the hydrophilic property, mechanical property and cell proliferation. Meanwhile, the attachment and spreading of adipose-derived stem cells (ADSCs) onto PLCL/Pluronic nanofibrous matrices with avidin-biotin technology was promoted obviously. PLCL/Pluronic nanofibrous matrices inheriting the excellent characteristics of both PLCL and Pluronic have the better cell adhesion ability through avidin-biotin technology, implying a promising application in skin care, tissue regeneration and other related area.

  13. Biotin-tagged platinum(iv) complexes as targeted cytostatic agents against breast cancer cells.

    PubMed

    Muhammad, Nafees; Sadia, Nasreen; Zhu, Chengcheng; Luo, Cheng; Guo, Zijian; Wang, Xiaoyong

    2017-09-05

    A biotin-guided platinum(IV) complex is highly cytotoxic against breast cancer cells but hypotoxic against mammary epithelial cells. The mono-biotinylated Pt(IV) complex is superior to the di-biotinylated one and hence a promising drug candidate for the targeted therapy of breast cancer.

  14. Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria.

    PubMed

    Finkenwirth, Friedrich; Neubauer, Olivia; Gunzenhäuser, Julia; Schoknecht, Janna; Scolari, Silvia; Stöckl, Martin; Korte, Thomas; Herrmann, Andreas; Eitinger, Thomas

    2010-11-01

    BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor-acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor-acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

  15. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    ERIC Educational Resources Information Center

    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…

  16. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    ERIC Educational Resources Information Center

    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…

  17. Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

    PubMed Central

    Jurchenko, Carol; Chang, Yuan; Narui, Yoshie; Zhang, Yun; Salaita, Khalid S.

    2014-01-01

    The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼106 - 108 times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods. PMID:24703305

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

    PubMed

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

    2017-04-08

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

  19. Biotin treatment causing erroneous immunoassay results: A word of caution for clinicians.

    PubMed

    Sulaiman, Raashda Ainuddin

    Biotin or vitamin B7 when ingested in high doses may cause immunoassay interference leading to false potentially misleading results. It is important that clinicians should always interpret laboratory results in the context of patient's clinical state as erroneous results may lead to misdiagnosis and injudicious treatment with adverse patient outcome.

  20. Clinical study on the effect of biotin on skin conditions in dogs.

    PubMed

    Frigg, M; Schulze, J; Völker, L

    1989-01-01

    In a collaborative study with small-animal veterinary surgeons, dogs with fur and skin conditions were treated with biotin (approximately 5 mg biotin/10 kg body weight/day) for 3 to 5 weeks. In total 119 cases could be treated which were reported to show symptoms such as dull coat, brittle hair, loss of hair, scaly skin, pruritus or dermatitis. Cases requiring other treatments with e.g. glucocorticoids, were excluded from the study. In 60% of the cases all symptoms were reported to be cured after the biotin treatment and in a further 31% an improvement was noted; in only 9% no effect was recorded. All breeds responded but to a variable extent: e.g. in Poodles the response was lower (no response in 6 out of 11 cases) than in Alsatians where all improved and 14 out of 29 were completely cured. The results confirm the favourable effect of biotin for treatment of fur and skin conditions in dogs.

  1. Biotin kinetics in serum of cattle after intravenous and oral dosing.

    PubMed

    Frigg, M; Hartmann, D; Straub, O C

    1994-01-01

    Single oral (p.o.) or intravenous (i.v.) doses of biotin were given to four cattle (400-450 kg body weight) in two consecutive tests two weeks apart. Dosages were p.o. 20, 40, 80 or 160 and i.v. 5, 10, 20, 40 mg biotin per 300 kg body weight. A three-compartment model was used to describe the course of serum concentrations with time. After i.v. administration, terminal half-lives of about 8 h were found. Areas under the curves were linearly related to both the p.o. and the i.v. doses. The systemically available fraction of the p.o. dose was 50 to 60%. On the basis of kinetic parameters, the biotin uptake via the feed was estimated to be 2.5 mg/day, which was about half of that estimated to be in the hay consumed. The data suggest that there was no relevant ruminal synthesis of biotin.

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

    PubMed Central

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

    2015-01-01

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

  3. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms

    PubMed Central

    Srinivasan, Padmanabhan; Kapadia, Rubina; Biswas, Arundhati

    2014-01-01

    Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5′-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na+ dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms. PMID:25214397

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

    PubMed

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

    2015-03-01

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

  5. Contributions of the Peroxisome and β-Oxidation Cycle to Biotin Synthesis in Fungi*

    PubMed Central

    Magliano, Pasqualina; Flipphi, Michel; Arpat, Bulak A.; Delessert, Syndie; Poirier, Yves

    2011-01-01

    The first step in the synthesis of the bicyclic rings of d-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi. PMID:21998305

  6. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms.

    PubMed

    Srinivasan, Padmanabhan; Kapadia, Rubina; Biswas, Arundhati; Said, Hamid M

    2014-11-01

    Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms. Copyright © 2014 the American Physiological Society.

  7. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  8. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  9. Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

    PubMed Central

    Gitlin, G; Bayer, E A; Wilchek, M

    1990-01-01

    The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin. PMID:2386489

  10. Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

    PubMed

    Gitlin, G; Bayer, E A; Wilchek, M

    1990-07-15

    The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

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

    PubMed

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

    2015-07-09

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. Low noise, tunable Ho:fiber soliton oscillator for Ho:YLF amplifier seeding

    NASA Astrophysics Data System (ADS)

    Li, Peng; Ruehl, Axel; Bransley, Colleen; Hartl, Ingmar

    2016-06-01

    We present a passively mode-locked, tunable soliton Ho:fiber ring oscillator, optimized for seeding of holmium-doped yttrium lithium flouride (Ho:YLF) amplifiers. The oscillator is independently tunable in central wavelength and spectral width from 2040 to 2070 nm and from 5 to 10 nm, respectively. At all settings the pulse energy within the soliton is around 800 pJ. The soliton oscillator was optimized to fully meet the spectral requirements for seeding Ho:YLF amplifiers. Its Kelly sidebands are located outside the amplifier gain spectrum, resulting in a train of about 1 ps long pedestal-free pulses with relative intensity noise of only 0.13% RMS when integrated from 1 Hz to Nyquist frequency.

  15. High Energy Directly Pumped Ho:YLF Laser

    NASA Technical Reports Server (NTRS)

    Petros, Mulugeta; Yu, Ji-Rong; Singh, Upendra N.; Barnes, Norman P.

    2000-01-01

    The most commonly used crystal architecture to produce 2 micrometer laser is co-doping Ho and Tm into a single host crystal. In this method, the stored energy transfer from the Tm (3)F4 to the Ho (5)I7 manifold is not fast enough to warrant high efficiency for short pulse applications. By separating the Ho and the Tm ions and doping the Tm in YALO3 and the Ho in YLF, we were able to directly pump the Ho (5)I7 manifold with 1.94 micrometers. The Ho:YLF laser has produced 33 mJ at 2.062 micrometers with a quantum efficiency of 0.88. The performance of each laser will be presented.

  16. Hydration and mobility of HO-(aq)

    PubMed Central

    Asthagiri, D.; Pratt, Lawrence R.; Kress, J. D.; Gomez, Maria A.

    2004-01-01

    The hydroxide anion plays an essential role in many chemical and biochemical reactions. But a molecular-scale description of its hydration state, and hence also its transport, in water is currently controversial. The statistical mechanical quasichemical theory of solutions suggests that \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{HO}}{\\cdot}[{\\mathrm{H}}_{2}{\\mathrm{O}}]_{3}^{-}\\end{equation*}\\end{document} is the predominant species in the aqueous phase under standard conditions. This result agrees with recent spectroscopic studies on hydroxide water clusters and with the available thermodynamic hydration free energies. In contrast, a recent ab initio molecular dynamics simulation has suggested that \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{HO}}{\\cdot}[{\\mathrm{H}}_{2}{\\mathrm{O}}]_{4}^{-}\\end{equation*}\\end{document} is the only dominant aqueous solution species. We apply adiabatic ab initio molecular dynamics simulations and find good agreement with both the quasichemical theoretical predictions and experimental results. The present results suggest a picture that is simpler, more traditional, but with additional subtlety. These coordination structures are labile but the tricoordinate species is the prominent case. This conclusion is unaltered with changes in the electronic density functional. No evidence is found for rate-determining activated interconversion of a \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength

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

    PubMed

    van Gog, F B; Visser, G W; Gowrising, R W; Snow, G B; van Dongen, G A

    1998-10-01

    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.

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

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

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

  19. Spectroscopic and lasing properties of Ho:Tm:LuAG

    NASA Technical Reports Server (NTRS)

    Barnes, Norman P.; Filer, Elizabeth D.; Naranjo, Felipe L.; Rodriguez, Waldo J.; Kokta, Milan R.

    1993-01-01

    Ho:Tm:LuAG has been grown, examined spectroscopically, and lased at 2.1 microns. Ho:Tm:LuAG was selected for this experimental investigation when quantum-mechanical modeling predicted that it would be a good laser material for Ho laser operation on one of the 5I7 to 5I8 transitions. Lasing was achieved at 2.100 microns, one of the three wavelengths predicted to be most probable for laser action.

  20. Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

    PubMed Central

    Norman, E; De Smet, K A; Stoker, N G; Ratledge, C; Wheeler, P R; Dale, J W

    1994-01-01

    The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins. Images PMID:7909542

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

    PubMed

    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.

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

    PubMed

    Brylinski, Michal; Waldrop, Grover L

    2014-04-02

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

  3. Role of the Recombination Channel in the Reaction between the HO and HO2 Radicals.

    PubMed

    Badenes, María P; Tucceri, María E; Cobos, Carlos J

    2017-01-19

    The kinetics of the gas phase recombination reaction HO + HO2 + He → HOOOH + He has been studied between 200 and 600 K by using the SACM/CT model and the unimolecular rate theory. The molecular properties of HOOOH were derived at the CCSD(T)/aug-cc-pVTZ ab initio level of theory, while relevant potential energy features of the reaction were calculated at the CCSD(T)/aug-cc-pVTZ//CCSD(T)/aug-cc-pVDZ level. The resulting high and low pressure limit rate coefficients are k∞ = 3.55 × 10(-12) (T/300)(0.20) cm(3) molecule(-1) s(-1) and k0 = [He] 1.55 × 10(-31) (T/300)(-3.2) cm(3) molecule(-1) s(-1). The rate coefficients calculated over the 6 × 10(-4) - 400 bar range are smaller at least in a factor of about 60 than the consensus value determined for the main reaction channel HO + HO2 → H2O + O2, indicating that the recombination pathway is irrelevant.

  4. Determination of biotin in Antarctic krill (Euphausia superba) by high-performance TLC with different post-chromatographic derivatizations.

    PubMed

    Teo, Peishan; Liu, Daicheng

    2013-08-01

    A new efficient method was developed to detect biotin in Antarctic krill by Vis-absorbance detection. DMF was used after chloroform pretreatment to extract biotin and two chromogenic methods were developed. The development system consisted of dichloromethane/dimethylcarbinol/methanol/glacial acetic acid (3:3:2:0.015, v/v/v/v). Samples were separated on precoated silica gel GF254 high-performance TLC plates. Densitometric analysis of biotin was carried out in the absorbance mode at 400 and 530 nm. The biotin content was determined to be 1.0948 ± 0.0097 and 1.1212 ± 0.0155 mg/g in Antarctic krill with the two chromogenic methods, which had no significant difference. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Comparison of avidin and biotin levels in eggs from turkey hens with high and low hatchability records.

    PubMed

    Robel, E J

    1987-01-01

    Levels of biotin and avidin were assayed in eggs from Small White turkey hens having 12-15 week average hatchability of fertile egg records (HF) of 13 and 99%. The median level and concentration of avidin was higher in eggs of the 13% HF (2.72 mg and 42.5 micrograms/ml, respectively) as compared with the 99% HF (1.39 mg and 23.8 micrograms/ml, respectively). Calculations for total biotin to avidin binding in the eggs revealed that all eggs in the 13% HF and residual free avidin levels after total biotin was complexed to avidin. This complex formation is expected to have limited the availability of biotin for embryonic development.

  6. Role of HO-1 in protective effect of electro-acupuncture against endotoxin shock-induced acute lung injury in rabbits.

    PubMed

    Yu, Jian-Bo; Jianbo, Yu; Dong, Shu-An; Shuan, Dong; Luo, Xiao-Qing; Xiaoqing, Luo; Gong, Li-Rong; Lirong, Gong; Zhang, Yuan; Yuan, Zhang; Wang, Man; Man, Wang; Cao, Xin-Shun; Xinshun, Cao; Liu, Da-Quan; Daquan, Liu

    2013-06-01

    Heme oxygenase (HO)-1 has been reported to play a great role in attenuating lung injury during endotoxic shock in our previous research. Although electro-acupuncture has been explored to reduce oxidative stress and decrease inflammatory reaction in animals with endotoxic shock, the mechanism of this effect is still unclear. The aim of this study was to determine whether HO-1 is involved in the effect of electro-acupuncture on the injured lung during endotoxic shock in rabbits. Sixty New England white rabbits were randomly divided into groups C, Z, ES, EA, AP, and EAZ. Before inducing endotoxic shock, group ES received no electro-acupuncture, while group EA received electro-acupuncture at ST36 (zusanli) and BL13 (feishu) acupoints on both sides for five days and group AP received electro-acupuncture (EA) stimulation at a non-acupoint. Groups ES, AP, EA, and EAZ received LPS to replicate the experimental model of injured lung induced by endotoxic shock, and electro-acupuncture was performed throughout the procedure with the same parameter. Groups EAZ and Z received the HO-1 inhibitor, ZnPP-IX, intraperitoneally. The animals were sacrificed by blood-letting at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, detection of alveolaer epithelial cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay), determination of wet to dry ratio, measurement of Evans blue (EB) contents, and determination of HO-1protein and mRNA expression. According to the results, EA at ST36 and BL13 could increase the expression of HO-1. At the same time, index of quantitative assessment (IQA) score and the number of TUNEL-positive cells decreased, while electro-acupuncture at the other points did not exert this effect, and pretreatment with ZnPP-IX in group EAZ suppressed the efficacy of electro-acupuncture preconditioning. In summary, electro

  7. Biotin-mediated epigenetic modifications: Potential defense against the carcinogenicity of benzo[a]pyrene.

    PubMed

    Xia, Bo; Pang, Li; Zhuang, Zhi-xiong; Liu, Jian-jun

    2016-01-22

    Environmental pollution and an unhealthy lifestyle result in direct exposure to dangerous chemicals that can modify endogenous pathways and induce malignant transformation of human cells. Although the molecular mechanisms of tumorigenesis are still not well understood, epigenetic alteration may be associated with exogenous chemical-induced carcinogenicity. Given the association between nutrition and cancer, nutrient supplementation may reduce aberrant epigenetic modifications induced by chemicals, thus decreasing carcinogenesis. This paper provides an overview of the epigenetic events caused by benzo[a]pyrene, a procarcinogenic and environmental pollutant, and biotin, an essential water-soluble vitamin, and investigates potential connections between them. This paper also discusses the potential inhibitory effect of biotin-related epigenetic modifications on the carcinogenicity of benzo[a]pyrene. The effect of nutritional supplementation on tumorigenesis involving epigenetic modifications is also discussed.

  8. Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

    PubMed

    Gruber, H J; Kada, G; Marek, M; Kaiser, K

    1998-07-23

    A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes. Copyright 1998 Elsevier Science B.V. All rights reserved.

  9. Thiol- and Biotin-Labeled Probes for Oligonucleotide Quartz Crystal Microbalance Biosensors of Microalga Alexandrium Minutum

    PubMed Central

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-01-01

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency. PMID:25585927

  10. Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae.

    PubMed

    Batsios, Petros; Meyer, Irene; Gräf, Ralph

    2016-01-01

    The identification of a bona fide lamin-like protein in Dictyostelium made this lower eukaryote an attractive model organism to study evolutionarily conserved nuclear envelope (NE) proteins important for nuclear organization and human laminopathies. Proximity-dependent biotin identification (BioID), reported by Roux and colleagues, is a powerful discovery tool for lamin-associated proteins. In this method, living cells express a bait protein (e.g., lamin) fused to an R118G-mutated version of BirA, an Escherichia coli biotinylase. In the presence of biotin, BirA-R118G biotinylates target proteins in close proximity in vivo, which are purified using streptavidin and identified by immunoblotting or mass spectrometry. We adapted the BioID method for use in Dictyostelium amoebae. The protocols described here successfully revealed Dictyostelium lamin-like protein NE81 proximity to Sun1, a conserved inner nuclear membrane protein.

  11. Thiol- and biotin-labeled probes for oligonucleotide quartz crystal microbalance biosensors of microalga alexandrium minutum.

    PubMed

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-07-04

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency.

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

    PubMed Central

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

    2008-01-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 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. PMID:18540065

  13. Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay.

    PubMed

    Indyk, H E; Evans, E A; Bostrom Caselunghe, M C; Persson, B S; Finglas, P M; Woollard, D C; Filonzi, E L

    2000-01-01

    Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.

  14. Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures.

    PubMed

    Gown, A M; Garcia, R; Ferguson, M; Yamanaka, E; Tippens, D

    1986-03-01

    We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.

  15. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

    NASA Astrophysics Data System (ADS)

    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'.

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

    PubMed Central

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

    2007-01-01

    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 V M value of 2.45 Å3 Da−1 and a solvent content of 50%. PMID:17401210

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

    PubMed

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

    2007-04-01

    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 (BCCPDeltaN76), 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*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 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*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%.

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

    PubMed

    Ghosal, Abhisek; Sekar, Thillai V; Said, Hamid M

    2014-08-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. Copyright © 2014 the American Physiological Society.

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

    PubMed Central

    Ghosal, Abhisek; Sekar, Thillai V.

    2014-01-01

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

  20. Urinary Excretion of 3-Hydroxyisovaleryl Carnitine Is an Early and Sensitive Indicator of Marginal Biotin Deficiency in Humans12

    PubMed Central

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

    2011-01-01

    Mounting evidence indicates that marginal biotin deficiency is not rare, contrary to previous assumptions. Accordingly, robust indicators of biotin status would be useful. In a study of 10 healthy adults, we recently provided evidence that abnormally increased plasma concentration of 3-hydroxyisovaleryl carnitine (3HIA-carnitine) is a sensitive indicator of marginal biotin deficiency. We sought to determine whether urinary excretion of 3HIA-carnitine (expressed as the ratio to urinary creatinine) significantly increases in marginal biotin deficiency. Marginal, asymptomatic biotin deficiency was induced experimentally in the same 10 healthy adults (8 women) by feeding undenatured egg white with meals for 28 d. Biotin status was repleted by a mixed general diet plus biotin supplementation. Urinary excretion of 3HIA-carnitine was determined by liquid chromatography-tandem MS on d 0, 14, and 28 (depletion) and on d 35 and 50 (repletion). Mean urinary 3HIA-carnitine concentration increased with depletion (P < 0.0001; d 0 vs. 28) and decreased with repletion (P = 0.0002; d 28 vs. 50). Urinary 3HIA-carnitine excretion was greater than the upper limit of normal in 9 of 10 participants by d 14 and decreased to within normal limits by d 50 in all participants. This study provides evidence that urinary excretion of 3HIA-carnitine is an early and sensitive indicator of marginal biotin deficiency. The ease of collection of untimed urine samples and application of a new analytical method with simplified sample preparation suggest that urinary 3HIA-carnitine is likely to be a useful indicator for large population studies. PMID:21248194

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

    PubMed Central

    Uram, Łukasz; Szuster, Magdalena; Gargasz, Krzysztof; Filipowicz, Aleksandra; Wałajtys-Rode, Elżbieta; Wołowiec, Stanisław

    2013-01-01

    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 significantly decreases that effect, confirming that PAMAM G3 is a useful candidate as a carrier for active biocompound delivery. PMID:24376351

  2. Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin densities

    PubMed Central

    Mock, Donald M.; Matthews, Nell I.; Zhu, Shan; Strauss, Ronald G.; Schmidt, Robert L.; Nalbant, Demet; Cress, Gretchen A.; Widness, John A.

    2010-01-01

    BACKGROUND Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 (51Cr) -labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs). STUDY DESIGN AND METHODS Aliquots of autologous RBCs from eight healthy adult subjects were labeled separately at four discrete biotin densities, mixed, and infused. The proportion of each population of BioRBCs circulating was determined serially by flow cytometry over 20 weeks. For each population, RCS was assessed by the following: 1) post-transfusion BioRBC recovery at 24 hour (PTR24); 2) time to decrease to 50% of the enrichment at 24 hours (T50); and 3) mean potential lifespan (MPL). RESULTS Among the four BioRBC densities, no significant differences in PTR24 were observed. T50 and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T50 and MPL. CONCLUSION RBCs labeled at four biotin densities can be used to independently and accurately measure PTR24 and two lowest biotin densities can accurately quantitate long-term RCS. This method provides a tool for investigating anemia in infants, fetuses, and pregnant women with the following advantages over the standard 51Cr method: 1) study subjects are not exposed to radiation; 2) small blood volumes (e.g., 20 μL) are required; and 3) multiple independent RCS measurements can be made simultaneously in the same individual. PMID:21062290

  3. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    PubMed Central

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-01-01

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or “flip-flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC. PMID:18725455

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

    SciTech Connect

    Nan, Alexandrina; Turcu, Rodica; Liebscher, Jürgen

    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.

  5. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    SciTech Connect

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-10-24

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or 'flip-flop' their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF{sub 2}P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.

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

    SciTech Connect

    Chou, Chi-Yuan; Tong, Liang

    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.

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

    SciTech Connect

    C Chou; L Tong

    2011-12-31

    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.

  8. Revisiting the streptavidin-biotin binding by using an aptamer and displacement isothermal calorimetry titration.

    PubMed

    Kuo, Tai-Chih; Tsai, Ching-Wei; Lee, Peng-Chen; Chen, Wen-Yih

    2015-03-01

    The association constant of a well-known streptavidin-biotin binding has only been inferred from separately measured kinetic parameters. In a single experiment, we obtained Ka 1 × 10(12)  M(-1) by using a streptavidin-binding aptamer and ligand-displacement isothermal titration calorimetry. This study explores the challenges of determining thermodynamic parameters and the derived equilibrium binding affinity of tight ligand-receptor binding.

  9. EGR-1 regulates Ho-1 expression induced by cigarette smoke

    SciTech Connect

    Chen, Huaqun; Wang, Lijuan; Gong, Tao; Yu, Yang; Zhu, Chunhua; Li, Fen; Wang, Li; Li, Chaojun

    2010-05-28

    As an anti-oxidant molecule, heme oxygenase-1 (HO-1) has been implicated in the protection of lung injury by cigarette smoke (CS). The mechanisms regulating its expression have not been defined. In this report, the role of early growth response 1 (EGR-1) in the regulation of Ho-1 expression was investigated. In C57BL/6 mice with CS exposure, HO-1 was greatly increased in bronchial epithelial cells and alveolar inflammatory cells. In primary cultured mouse lung fibroblasts and RAW264.7 cells exposed to cigarette smoke water extract (CSE), an increase in HO-1 protein level was detected. In addition, CSE induced HO-1 expression was decreased in Egr-1 deficient mouse embryo fibroblasts (Egr-1{sup -/-} MEFs). Nuclear localization of EGR-1 was examined in mouse lung fibroblasts after exposure to CSE. Luciferase reporter activity assays showed that the enhancer region of the Ho-1 gene containing a proposed EGR-1 binding site was responsible for the induction of HO-1. A higher increase of alveolar mean linear intercept (Lm) was observed in lung tissues, and a larger increase in the number of total cells and monocytes/macrophages from bronchial alveolar lavage fluid was found in CS-exposed mice by loss of function of EGR-1 treatment. In summary, the present data demonstrate that EGR-1 plays a critical role in HO-1 production induced by CS.

  10. Structural Characterization of the Mycobacterium tuberculosis Biotin Biosynthesis Enzymes 7,8-Diaminopelargonic Acid Synthase and Dethiobiotin Synthetase†,‡

    PubMed Central

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

    2010-01-01

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 Å and 1.85 Å. Superimposition of the DAPAS structures bound either to the SAM analog sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 Å, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb. PMID:20565114

  11. Effect of dietary palm kernel oil and biotin on the fatty liver and kidney syndrome in broiler chicken.

    PubMed

    Oloyo, R A; Ogunmodede, B K

    1989-01-01

    The effect of feeding biotin and palm kernel oil to broiler chicks on the appearance of Fatty Liver and Kidney Syndrome (FLKS) was investigated. A total of 480 broiler chicks was divided into two equal batches each of which was divided into 6 groups of 40 chicks per group. Each group was further subdivided into equal units of 20 chicks. Six dietary levels of biotin (40, 80, 120, 160, 200, and 240 mcg/kg feed) were given to the first batch of chicks, while the second batch had 2% palm kernel oil added to the six dietarybiotin levels. These two basic rations were supplemented with biotin in order to obtain six levels of the vitamin in the rations. The results showed that the 2% palm kernel oil forage affected FLKS mortality and the minimum biotin requirement. FLKS mortality was significantly reduced in case of palm kernel oil supplement. A lower amount of biotin (120 mcg/kg feed) was needed in case of palm kernel oil supplement-as compared with the necessary biotin (160 mcg/kg feed)-in order to prevent FLKS mortality when palm kernel oil was not contained in the rations. The biochemical analysis of the liver and kidney syndrome-coupled with the correlation and regression analysis of the data collected-showed that a minimum of 120 mcg/kg feed was needed by broiler chicks for the prevention of FLKS.

  12. A small-molecule-linked DNA-graphene oxide-based fluorescence-sensing system for detection of biotin.

    PubMed

    Zhang, Hao; Li, Yan; Su, Xingguang

    2013-11-15

    In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin-DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3' to the 5' terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5-20nmol/L. The detection limit for biotin is 0.44nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein-small molecule ligand binding.

  13. Recognition of oxidatively modified bases within the biotin-binding site of avidin.

    PubMed

    Conners, Rebecca; Hooley, Elizabeth; Clarke, Anthony R; Thomas, Siân; Brady, R Leo

    2006-03-17

    Oxidative damage of DNA results in the formation of many products, including 8-oxodeoxyguanosine, which has been used as a marker to quantify DNA damage. Earlier studies have demonstrated that avidin, a protein prevalent in egg-white and which has high affinity for the vitamin biotin, binds to 8-oxodeoxyguanosine and related bases. In this study, we have determined crystal structures of avidin in complex with 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. In each case, the base is observed to bind within the biotin-binding site of avidin. However, the mode of association between the bases and the protein varies and, unlike in the avidin:biotin complex, complete ordering of the protein in this region does not accompany binding. Fluorescence studies indicate that in solution the individual bases, and a range of oligonucleotides, bind to avidin with micromolar affinity. Only one of the modes of binding observed is consistent with recognition of oxidised purines when incorporated within a DNA oligomer, and from this structure a model is proposed for the selective binding of avidin to DNA containing oxidatively damaged deoxyguanosine. These studies illustrate the molecular basis by which avidin might act as a marker of DNA damage, although the low levels of binding observed are inconsistent with the recognition of oxidised purines forming a major physiological role for avidin.

  14. Three-dimensional structures of avidin and the avidin-biotin complex.

    PubMed Central

    Livnah, O; Bayer, E A; Wilchek, M; Sussman, J L

    1993-01-01

    The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis. Images Fig. 3 Fig. 4 PMID:8506353

  15. Biotin synthase: insights into radical-mediated carbon-sulfur bond formation.

    PubMed

    Fugate, Corey J; Jarrett, Joseph T

    2012-11-01

    The enzyme cofactor and essential vitamin biotin is biosynthesized in bacteria, fungi, and plants through a pathway that culminates with the addition of a sulfur atom to generate the five-membered thiophane ring. The immediate precursor, dethiobiotin, has methylene and methyl groups at the C6 and C9 positions, respectively, and formation of a thioether bridging these carbon atoms requires cleavage of unactivated CH bonds. Biotin synthase is an S-adenosyl-l-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals, high-energy carbon radicals that can directly abstract hydrogen atoms from dethiobiotin. The available experimental and structural data suggest that a [2Fe-2S](2+) cluster bound deep within biotin synthase provides a sulfur atom that is added to dethiobiotin in a stepwise reaction, first at the C9 position to generate 9-mercaptodethiobiotin, and then at the C6 position to close the thiophane ring. The formation of sulfur-containing biomolecules through a radical reaction involving an iron-sulfur cluster is an unprecedented reaction in biochemistry; however, recent enzyme discoveries suggest that radical sulfur insertion reactions may be a distinct subgroup within the burgeoning Radical SAM superfamily. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

  16. Structure guided design of biotin protein ligase inhibitors for antibiotic discovery.

    PubMed

    Paparella, Ashleigh S; Soares da Costa, Tatiana P; Yap, Min Y; Tieu, William; Wilce, Matthew C J; Booker, Grant W; Abell, Andrew D; Polyak, Steven W

    2014-01-01

    Biotin protein ligase (BPL) represents a promising target for the discovery of new antibacterial chemotherapeutics. Here we review the central role of BPL for the survival and virulence of clinically important Staphylococcus aureus in support of this claim. X-ray crystallography structures of BPLs in complex with ligands and small molecule inhibitors provide new insights into the mechanism of protein biotinylation, and a template for structure guided approaches to the design of inhibitors for antibacterial discovery. Most BPLs employ an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5´-AMP from substrates biotin and ATP. Recent studies reporting chemical analogs of biotin and biotinyl-5´-AMP as BPL inhibitors that represent new classes of anti-S. aureus agents are reviewed. We highlight strategies to selectively inhibit bacterial BPL over the mammalian equivalent using a 1,2,3-triazole isostere to replace the labile phosphoanhydride naturally present in biotinyl-5´-AMP. A novel in situ approach to improve the detection of triazole-based inhibitors is also presented that could potentially be widely applied to other protein targets.

  17. Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach.

    PubMed

    Kwon, Sun Jae; Choi, Eun Young; Seo, Jong Bok; Park, Ohkmae K

    2007-10-31

    Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.

  18. Effects of age and biotin status on postnatal development of plasma biotinidase activity in rats.

    PubMed

    Heard, G S; Tanner, R W; Blevins, T L; Evans, J S; Redmond, J B; Roth, K S; Wolf, B

    1991-02-01

    Biotinidase activity was measured in plasmas of 1-, 7-, 14-, and 21-day-old rats from control dams and dams that had been fed a biotin-depleting diet from Day 15 of gestation. Biotinidase activity increased significantly in the plasma of rats from control and depleted mothers until Postnatal Day 14, after which there was a small but significant decline at Day 21. Differences between the mean activities of the two groups of pups on each sampling day were not significant and there were no significant differences in activity levels attributable to sex. Plasma albumin concentrations increased from birth until Day 21, and plasma biotinidase activity and albumin concentration were significantly correlated (r = +/- 0.43). We suggested that these two proteins may be controlled by a common mechanism in the early postnatal period, and that biotin deficiency does not affect the development of biotinidase activity. Because biotin-depleted neonatal pups show developmental changes in biotinidase activity similar to those of human newborns, and they can be produced reliably by depleting dams from Day 15 of gestation, they may be useful models for studying the developmental abnormalities associated with human biotinidase deficiency.

  19. Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney.

    PubMed

    Salto, R; Girón, M D; del Mar Sola, M; Vargas, A M

    1999-10-01

    The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period.

  20. Targeting quantum dots to surface proteins in living cells with biotin ligase

    NASA Astrophysics Data System (ADS)

    Howarth, Mark; Takao, Keizo; Hayashi, Yasunori; Ting, Alice Y.

    2005-05-01

    Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling. BirA | labeling | streptavidin | glutamate receptor | single molecule

  1. Biotin-Avidin Based Universal Cell-Matrix Interaction for Promoting Three-Dimensional Cell Adhesion.

    PubMed

    Dou, Xiao-Qiu; Zhang, Jia; Feng, Chuanliang

    2015-09-23

    To promote cell adhesion in three-dimensional (3D) extracellular matrix (ECM) is crucial for avoiding cell anoikis, which is one of the most important issues for fundamental cell biology. Herein, a biotin-avidin based universal cell-matrix interaction for different types of cells is developed in order to achieve the promoted adhesion in 3D ECM. For the purpose, biotinylated nanofibrous hydrogels are constructed by coassembling 1,4-benzyldicarboxamide (C2) based non-biotinylated and biotinylated supramolecular gelators. The used cells are modified by avidin (AV-cells) through biotinylating cells and then interacting with avidin. After in situ encapsulating AV-cells in the hydrogels, the adhered amount can be increased by tens of percent even with adding several percentages of the biotinylated C2 gelators in the coassembly due to the specific biotin-avidin interaction. Reverse transcription polymerase chain reaction (RT-PCR) confirms that AV-cells can proliferate without varying gene expression and denaturation. Compared with the interaction between RGD and cells, this avidin-biotin interaction should be much more universal and it is feasible to be employed to promote cell adhesion for most types of cells in 3D matrix.

  2. Magnetic molecularly imprinted polymer for the isolation and detection of biotin and biotinylated biomolecules.

    PubMed

    Ben Aissa, A; Herrera-Chacon, A; Pupin, R R; Sotomayor, M D P T; Pividori, M I

    2017-02-15

    Magnetic separation based on biologically-modified magnetic particles is a preconcentration procedure commonly integrated in magneto actuated platforms for the detection of a huge range of targets. However, the main drawback of this material is the low stability and high cost. In this work, a novel hybrid molecularly-imprinted polymer with magnetic properties is presented with affinity towards biotin and biotinylated biomolecules. During the synthesis of the magneto core-shell particles, biotin was used as a template. The characterization of this material by microscopy techniques including SEM, TEM and confocal microscopy is presented. The application of the magnetic-MIPs for the detection of biotin and biotinylated DNA in magneto-actuated platforms is also described for the first time. The magnetic-MIP showed a significant immobilization capacity of biotinylated molecules, giving rise to a cheaper and a robust method (it is not required to be stored at 4°C) with high binding capacity for the separation and purification under magnetic actuation of a wide range of biotinylated molecules, and their downstream application including determination of their specific targets.

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

    PubMed

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

    2015-11-01

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

  4. Antibody immobilization using pneumatic spray: comparison with the avidin-biotin bridge immobilization method.

    PubMed

    Figueroa, Jhon; Magaña, Sonia; Lim, Daniel V; Schlaf, Rudy

    2012-12-14

    The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin-biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin-biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10(5)-10(7)cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24h for the avidin-biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices. Copyright © 2012. Published by Elsevier B.V.

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

    PubMed

    Malý, J; Lampová, H; Semerádtová, A; Stofik, M; Kovácik, L

    2009-09-23

    This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH(4) silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

    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.

  7. [Interaction of triiodothyronine-biotin conjugate with binding proteins in immunoassay systems].

    PubMed

    Navakouskiĭ, M J; Vashkevich, I I; Sviridov, O V

    2012-01-01

    A new construction of a test-system for free thyroid hormone 3,3',5-triiodothyronine determination (free T3) in human blood serum in ELISA and IRMA variants was described. For this purpose a low-molecular weight bifunctional conjugate containing T3 and vitamin H (biotin, Bt) residues was synthesized. The conjugate (T3-Bt) can be bound via its biotin function to biotin-binding proteins on a solid phase whereas its T3-portion can interact with monoclonal antibody against T3 (anti-T3-MAb) labeled with horseradish peroxidase or iodine-125 in an enzyme immunoassay or an immunoradiometric assay system (ELISA or IRMA), respectively. The immunochemical interaction is hindered if the T3 residue takes part as a component of a preformed solid phase complex between T3-Bt and avidin. Computer modeling shows that in this complex glycan component of avidin shields iodothyronine residue. The binding of T3-Bt both with T3-free sites on anti-T3-MAb and the solid phase avidin achieves with help of a delayed T3-Bt addition into avidin-covered containers with preliminary co-incubated labeled anti-T3-MAb and an assayed T3 sample.

  8. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

    PubMed

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  9. Improved surface stability and biotin binding properties of streptavidin coating on polystyrene.

    PubMed

    Ylikotila, Johanna; Välimaa, Lasse; Takalo, Harri; Pettersson, Kim

    2009-05-01

    The ultimate nature of streptavidin to bind biotin tightly is widely utilized in many solid-phase based applications to provide a universal binding surface for biotinylated molecules. However, the preparation of the streptavidin coatings by passive adsorption may heavily alter the binding properties of native streptavidin and may not result in the best possible capture surface for demanding solid-phase assays. By introducing sulphydryl groups through primary amines in the protein, we have activated and conjugated native streptavidin into larger protein polymers resulting in high local binding density when coated on polystyrene. This thiolated streptavidin formed through chemical modification has improved adsorption properties and biotin binding capability, compared to the native streptavidin. When this thiolated streptavidin is coated on polystyrene, a dense surface is formed, which provides up to 3-fold increase in the biotin binding efficiency and improves the surface stability by minimizing the desorption of the adsorbed protein from the surface during incubation. Furthermore, this high-capacity surface is resistant to harsh chemical treatments, such as denaturing conditions or mild reducing conditions. The improved adsorption properties of the thiolated streptavidin allow the coating process to be performed with shorter incubation times (15min), still providing enhanced solid-phase properties, compared to a reference streptavidin surface.

  10. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies.

    PubMed

    Hsu, S M; Raine, L; Fanger, H

    1981-05-01

    A highly sensitive immunoenzymatic technic is presented. The method involves three sequential steps: (1) primary antibody, (2) biotin-labeled secondary antibody, and (3) avidin-biotin-peroxidase complex. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin. Many moieties of biotin can be coupled to the peroxidase molecule. Thus, since a relatively large amount of avidin is incubated with biotin-labeled peroxidase, avidin serves as a link between biotin-peroxidase molecules; in turn, biotin-peroxidase serves as a link between avidin molecules. Consequently, this large lattice-like complex with biotin-binding capability can be attracted to the sites of biotin-labeled antibody, producing a superior staining sensitivity. Several commercially available radioimmunoassay antibodies (e.g., antiglucagon, prolactin, gastrin, growth hormone, and thyroid-stimulating hormone antibodies) were tested for immunohistochemical staining. The unlabeled antibody peroxidase-antiperoxidase method fails to stain gastrin or thyroid-stimulating secretory cells when using these antibodies, and a relatively high antibody concentration is required to produce a positive reaction for glucagon, prolactin, and growth hormone. In contrast, the avidin-biotin-peroxidase complex method successfully demonstrates polypeptide hormones even when antibodies are diluted 20 to 40 times.

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

    PubMed Central

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

    2008-01-01

    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 biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (KD = 26 nM) and a slow dissociation rate (koff = 5.4 × 10-4 s-1) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LC-biotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patch-like surface distribution of BK channel protein. The results support the feasibility of using IbTx-LC-biotin and similar biotin-tagged K+ channel toxins for diverse applications in cellular neurobiology. PMID:16704206

  12. Monitoring DNA recombination initiated by HO endonuclease.

    PubMed

    Sugawara, Neal; Haber, James E

    2012-01-01

    DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.

  13. Towards Performance Portability with GungHo

    NASA Astrophysics Data System (ADS)

    Ford, Rupert; Glover, Matthew; Ham, David; Hobson, Mike; Maynard, Chris; Mitchell, Lawrence; Mullerworth, Steve; Pickles, Stephen; Rezny, Mike; Riley, Graham; Wood, Nigel; Ashworth, Mike

    2014-05-01

    The Met Office's numerical weather prediction and climate model code, the Unified Model (UM), is almost 25 years old. Up to the present day the UM has been able to be run efficiently on many of the worlds most powerful computers, helping to keep the Met Office at the forefront of climate prediction and weather forecasting. However, with performance increases from each new generation of computers now being primarily provided by an increase in the amount of parallelism rather than an increase in the clock-speed of the processors themselves, running higher resolutions of the UM now faces the double challenge of code scalability and numerical accuracy. The UM's atmospheric dynamical core makes use of a finite-difference scheme on a regular latitude-longitude grid. The regular latitude-longitude mesh results in an increasingly disparate grid resolution as the mesh resolution increases due to lines of longitude converging at the poles. For example, a 10km resolution at mid-latitudes would result in a 12m resolution at the poles. The difference in resolution leads to increased communication at the poles and load balance issues which are known to impair scalability; it also leads to issues with numerical accuracy and smaller time-steps due to the difference in scale. To address this problem the Met Office, NERC and STFC initiated the GungHo project. The primary aim of this project is to deliver a scalable, numerically accurate dynamical core. This dynamical core is scheduled to become operational around the year 2022. The project is currently investigating the use of quasi-uniform meshes, such as triangular, icosahedral and cubed-sphere meshes, using finite element methods. The associated GungHo software infrastructure is being developed to support multiple meshes and element types thus allowing for future model development. GungHo is also proposing a novel separation of concerns for the software implementation of the dynamical core. This approach distinguishes between