Kegg Pathway: Biotin metabolism

Proteomics. 2008 Jul 9;
Tian R, Li L, Tang W, Liu H, Ye M, Zhao ZK, Zou H

A chemical proteomic approach was developed for profiling the noncovalent interactome of isoprenoid chain in the yeast proteome. A chemical probe that harbors a Biotin moiety and a photoreactive benzophenone group linked to the terminal of geranyl group was synthesized. Photoaffinity labeling was performed by incubating the Saccharomyces cerevisiae proteome and the probe under 365 nm UV light. Thirty proteins were identified by immobilized NeutraAvidin enrichment, on-bead digestion, online 2-D nano-LC/MS/MS identification and semi-quantitative proteomic analysis. As noted by Gene Ontology annotation, the identified proteins demonstrate a wide range of catalytic activity in several biological processes, especially in metabolism and biosynthesis. Further data analysis shows that hydrophobic binding of the synthetic probe is potentially the major interaction force leading to covalent labeling. These results argue that intracellular allosteric interactions conferred by the isoprenoid chain of the corresponding chemical structures may be widespread at an interactomic level.

Biochem J. 2008 Aug 1; 413(3): 369-87
Jitrapakdee S, St Maurice M, Rayment I, Cleland WW, Wallace JC, Attwood PV

PC (pyruvate carboxylase) is a Biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the Biotin carboxylase, carboxyltransferase and Biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the Biotin moiety transfers the carboxy group between the Biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

Arch Ophthalmol. 2008 Jun; 126(6): 816-21
Murano N, Ishizaki M, Sato S, Fukuda Y, Takahashi H

OBJECTIVE: To determine whether ultrasound oscillations in the anterior chamber cause corneal endothelial injury by free radicals. METHODS: A phacoemulsification probe was introduced into the anterior chamber of rabbits' eyes through a limbal incision, and ultrasound oscillation was performed without emulsifying the lens. Rabbits were assigned to 4 treatment groups: (1) no treatment (controls); (2) only irrigation with a salt solution; (3) ultrasound only; and (4) ultrasound oscillations with a salt solution of 0.001M ascorbic acid. The corneas were immunohistochemically examined for oxidative stress using 8-hydroxy-2-deoxyguanosine (8-OHdG), apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-Biotin nick-end labeling (TUNEL) staining, and ultrastructural changes by electron microscopy. The lipid peroxide levels in the aqueous humor were also measured. RESULTS: In the ultrasound-only group, 8-OHdG-positive cells and TUNEL-positive cells were detected at 24 hours; necrotic cells were detected at 12 to 24 hours. Also, lipid peroxide levels were significantly increased at later times in the ultrasound group. Such changes were not observed in other groups. CONCLUSION: Free radicals induced by ultrasound oscillation can cause corneal endothelial damages. Clinical Relevance Clinicians should be aware that free radicals associated with ultrasound oscillation can injure the corneal endothelial cells.

Acta Cytol. 2008 May-Jun; 52(3): 304-8
Sioutopoulou DO, Kampas LI, Gerasimidou D, Valeri RM, Boukovinas I, Tsavdaridis D, Destouni CT

OBJECTIVE: To evaluate the application of ThinPrep liquid-based cytology (LBC) and present our experience using LBC in the diagnosis of metastatic tumors in cerebrospinal fluid (CSF) samples. STUDY DESIGN: We examined 38 cytologic specimens of CSF, processed by ThinPrep technique. Of these, 18 presented with a previously diagnosed primary malignancy. Various immunocytochemical markers were performed. RESULTS: ThinPrep technology provided preservation of cytomorphologic features, high cellularity per slide and clear background. Analysis revealed 8 breast carcinomas, 5 lung carcinomas, 4 lymphomas, 3 adenocarcinomas of the gastrointestinal tract, 1 squamous cell carcinoma of uterine cervix and 1 urinary bladder carcinoma. Fifteen samples were negative for malignancy. CONCLUSION: CSF cytology is the only examination that verifies the presence of malignancy. Thin monolayer technology is suggested as an appropriate diagnostic method for metastatic tumors in CSF in everyday routine and seems to be a valuable tool for further management and planning of treatment.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jun 1; 64(Pt 6): 524-7
Gupta V, Gupta RK, Khare G, Surolia A, Salunke DM, Tyagi AK

The gene encoding Biotin acetyl-CoA carboxylase ligase (BirA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli with a C-terminal Strep-tag. PEG 4000 as well as PEG 8000 were used as precipitants at pH 7.5 to crystallize the protein using the vapour-diffusion technique. X-ray characterization of crystals at room temperature indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.7, b = 62.8, c = 105.8 A. Assuming the presence of two BirA molecules in the asymmetric unit, the solvent content of the crystals was 44% (V(M) = 2.2 A(3) Da(-1)). When transferred to a cryoprotectant, crystals grown in the same drop exhibited a difference in one unit-cell parameter, with a = 60.1, b = 64.0, c = 103.6 A, but belonged to the same P2(1)2(1)2(1) space group. These crystals, with two molecules of BirA present per asymmetric unit, appeared to have a very low solvent content of 28% (V(M) = 1.7 A(3) Da(-1)).

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jun 1; 64(Pt 6): 520-3
Pendini NR, Polyak SW, Booker GW, Wallace JC, Wilce MC

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

PLoS ONE. 2008; 3(6): e2328
Shen W, Hao J, Tian C, Ren J, Yang L, Li X, Luo C, Cotma CW, Liu J

BACKGROUND: Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto-Kakizaki rats. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-alpha-lipoic acid, acetyl-L-carnitine, nicotinamide, and Biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-alpha (Ppar alpha), peroxisome proliferator-activated receptor-delta (Ppar delta), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain. CONCLUSIONS/SIGNIFICANCE: These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.

J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jun 15; 869(1-2): 93-100
Hayakawa K, Katsumata N, Hirano M, Yoshikawa K, Ogata T, Tanaka T, Nagamine T

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 0.7M 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 1.39pmol with a detection limit of 69.5fmol. Biotin was measured after hydrolysis in 2.25M 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.

Anal Chim Acta. 2008 Jun 23; 618(2): 218-26
Bouças RI, Trindade ES, Tersariol IL, Dietrich CP, Nader HB

Sulfated glycosaminoglycans were labeled with Biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using Biotin-hydrazide, under different conditions. The structural characteristics of the Biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and Biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of Biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the Biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the Biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of Biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

Nat Methods. 2008 Jun; 5(6): 507-16
Roy R, Hohng S, Ha T

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

J Mol Biol. 2008 Jun 27; 380(1): 223-36
Zhao H, Beckett D

Proteins can perform completely distinct functions in response to the particular partners that they bind to. Consequently, determination of the mechanism of functional regulation in such systems requires elucidation of the mechanism switching between binding partners. The central protein of the Escherichia coli Biotin regulatory system, BirA, switches between its function as a metabolic enzyme or a transcriptional repressor in response to binding either the Biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase or a second BirA monomer. These two protein-protein interactions are structurally mutually exclusive. The results of earlier studies suggest that the system is regulated by kinetic partitioning between the two protein-protein interactions. In this work, sedimentation velocity was employed to monitor the partitioning directly. The results indicate similar equilibrium parameters governing formation of the two protein-protein interactions. Kinetic analysis of the sedimentation velocity data indicated that holoBirA dimerization is governed by very slow forward and reverse rate constants. The slow kinetics of holoBirA dimerization combined with fluctuations in the intracellular apoBCCP pool are critical determinants in partitioning BirA between its distinct biological functions.

Nat Biotechnol. 2008 Jun; 26(6): 685-94
Schilling O, Overall CM

We introduce human proteome-derived, database-searchable peptide libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used peptides in such libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P') sides of the scissile bond. Prime-side cleavage products were tagged with Biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6'. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.

Neuron. 2008 May 22; 58(4): 613-24
Yan Z, Tan J, Qin C, Lu Y, Ding C, Luo M

Olfactory sensory neurons expressing a common receptor gene converge onto one or a few glomeruli with stereotyped positions within the mouse main olfactory bulb (MOB), producing a map of approximately 1800 olfactory columns representing approximately 1000 odorant receptors. Here, we report that this precise olfactory map is maintained over several synapses that ultimately cross MOB hemispheres to link bilateral isofunctional olfactory columns. Focal injection of tracer into genetically identified glomeruli revealed an exquisite topography that involves a bilateral connection via the anterior olfactory nucleus pars externa (AONpE) that links isofunctional olfactory columns in the contralateral MOB. Physiological and behavioral assays revealed an important role for the AONpE in bilateral exchange of odorant-specific information. These results indicate that the interbulbar link through the AONpE integrates bilateral olfactory sensory maps and exchanges olfactory information, positioning it as a unique model system for studying interhemispheric connections.

Histol Histopathol. 2008 Aug; 23(8): 945-52
Nemeth G, Felszeghy S, Kenyeres A, Szentmary N, Berta A, Suveges I, Modis L

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-Biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.

Acta Ophthalmol. 2008 May; 86(3): 302-6
Donati G, Kapetanios A, Dubois-Dauphin M, Pournaras CJ

PURPOSE: Acute brain ischaemia (stroke) causes a central area of coagulation necrosis. Peripheral to it and after a few hours, apoptosis causes neurons throughout the entire area to die progressively. However, this sequence of events is related to the reperfusion of regenerated capillaries or collateral circulation, and is considered to be potentially salvageable. Similar findings have been reported in the retina after ischaemia-reperfusion injury in rats. In the present study, we intended to investigate whether delayed cell death is involved in neuronal injuries to the inner retina during chronic retinal ischaemia. METHODS: Experimental branch retinal vein occlusion (BRVO) was induced in miniature pigs using indirect argon laser. The eyes were prelevated at 4, 24 and 48 hours and at 1 and 3 weeks following BRVO. The caspase inhibitor Z-VAD was injected intravitreally 24 hours after BRVO. Affected retinas were examined 24 hours later for any protective effect from apoptotic cell death. Histological examination with cresyl violet staining and TUNEL (TdT-mediated dUTP-Biotin nick-end labelling) was performed on the samples. RESULTS: A progressive oedema of the nerve fibre, ganglion cell and inner plexiform layers, related to a widely diffused cell necrosis, was observed in the affected territory within 4-24 hours after BRVO. This was followed by a wave of apoptosis localized at the periphery of the affected territory, which peaked approximately 48 hours after BRVO and was associated with a diffuse oedema of the inner nuclear layer. A progressive atrophy of the inner retina was observed 1-3 weeks after BRVO. Injection of the caspase inhibitor Z-VAD (24 hours after BRVO) decreased the amount of apoptotic cell bodies 48 hours after BRVO. CONCLUSIONS: This study shows that although necrosis is the predominant form of neuronal death in the early phase, massive delayed neuronal cell death caused by apoptosis occurs on a widespread basis as a result of chronic ischaemia after BRVO in the retina. Further studies are needed to evaluate the possibility of rescuing retinal neurons from death by neuroprotective treatments.

J Am Chem Soc. 2008 Jun 11; 130(23): 7276-85
Limoges B, Marchal D, Mavré F, Savéant JM

The two articles in this series are dedicated to bioaffinity electrodes with in situ detection of the product of the enzyme label after recognition by its conjugate immobilized on the electrode. Part 1 was devoted to direct electrochemical detection, whereas the present contribution deals with homogeneous chemical and enzymatic amplification of the primary electrochemical signal. The theoretical relationships that are established for these modes of amplification are applied to the avidin-Biotin recognition in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichloro-phenyl phosphate as substrate, generating 2,6-dichloro-4-aminophenol as electrochemically active product. Chemical amplification then results from the addition of NADH, which reduces the 2,6-dichloro-quinonimine resulting from the electrochemical oxidation of 2,6-dichloro-4-aminophenol. An increased amplification is obtained when the reduction of 2,6-dichloro-quinonimine involves diaphorase in solution with NADH as substrate. The excellent agreement between theoretical predictions and experimental data required a detailed theoretical analysis and the independent determination of the key kinetic parameters of the system. The theoretical analysis was extended to monolayer and multilayered films of auxiliary enzyme as well as to electrochemical amplification by means of closely spaced dual electrodes so as to offer a rational comparative panorama of the amplification capabilities of the various possible strategies. Confinement of the profile of the product, and/or its oxidized form, in the vicinity the electrode surface appears as a key parameter of amplification.

Biochemistry. 2008 Jun 17; 47(24): 6361-9
Wang P, Shim E, Cravatt B, Jacobsen R, Schoeniger J, Kim AC, Paetzel M, Dalbey RE

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-Biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-Biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.

Histopathology. 2008 Jun; 52(7): 806-15
McLean MH, Murray GI, Fyfe N, Hold GL, Mowat NA, El-Omar EM

AIMS: To assess cyclooxygenase-2 (COX-2) expression in sporadic colonic adenomas and to explore the association of COX-2 positivity with adenoma characteristics linked to increased risk of malignant transformation. METHODS AND RESULTS: COX-2 expression and localization were assessed in 64 colorectal adenomas and 35 paired adjacent normal colonic mucosal biopsy specimens. The number of adenoma specimens was then extended to include polyps exhibiting an increasing degree of epithelial dysplasia. Forty colonic hyperplastic polyps were also identified from the pathology diagnostic database and included in the analysis. Immunohistochemistry was performed with the Envision+ peroxidase-linked Biotin-free system incorporating a signal amplification step. There was a statistically significant increase in COX-2 expression in colonic polyps compared with paired adjacent normal mucosa, chi(2) = 40.1, P = 0.001. The probability of COX-2 expression increased along with increasing adenoma size and increasing degree of epithelial dysplasia. Fifty-five per cent of the hyperplastic polyp specimens expressed COX-2. CONCLUSIONS: This study associates COX-2 epithelial expression with a number of adenoma characteristics that convey an increased risk of malignant transformation. This is in keeping with a positive role for COX-2 in early colorectal carcinogenesis.

Anal Chem. 2008 Jun 1; 80(11): 4007-13
Linman MJ, Taylor JD, Yu H, Chen X, Cheng Q

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of Biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using Biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the Biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the Biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Biosci Biotechnol Biochem. 2008 May; 72(5): 1290-8
Sugita Y, Shirakawa H, Sugimoto R, Furukawa Y, Komai M

Biotin functions as a coenzyme for four carboxylases involved in energy metabolism in mammals. Besides these classical functions, Biotin has novel functions in the cellular processes via the modulation of gene expression. In this study, we examined the alteration of gene expression by Biotin administration in the liver of streptozotocin (STZ)-induced diabetic rats. In comparison with the control, the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were significantly reduced and glucokinase mRNA was increased 3 h after the administration of Biotin or insulin. The expression of hepatocyte nuclear factor 4alpha, one of the transcription factors responsible for gluconeogenic gene expression, was decreased by Biotin at both mRNA and protein levels. In addition, forkhead box O1 and sterol regulatory element-binding protein 1c mRNA expression that was enhanced by the insulin treatment was inversely decreased by Biotin. These results indicate that Biotin repressed the gluconeogenic genes and their transcription factors via a pathway independent of insulin-signaling and could improve the diabetic condition.