oalib
Search Results: 1 - 10 of 100 matches for " "
All listed articles are free for downloading (OA Articles)
Page 1 /100
Display every page Item
Uptake of Biotin by Chlamydia Spp. through the Use of a Bacterial Transporter (BioY) and a Host-Cell Transporter (SMVT)  [PDF]
Derek J. Fisher, Reinaldo E. Fernández, Nancy E. Adams, Anthony T. Maurelli
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0046052
Abstract: Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB) and/or transport (bioY). Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613) from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF). Type II ECFs are typically composed of a transport specific component (S) and a chromosomally unlinked energy module (AT). Intriguingly, Chlamydia lack recognizable AT modules. Using 3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module) and capacity (apparent K(m) of 3.35 nM and V(max) of 55.1 pmol×min?1×mg?1). Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT), which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s) would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin from the host cell into the bacterial cytoplasm.
Optimal use of tandem biotin and V5 tags in ChIP assays
Katarzyna E Kolodziej, Farzin Pourfarzad, Ernie de Boer, Sanja Krpic, Frank Grosveld, John Strouboulis
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-6
Abstract: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking.The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.Affinity tags have been widely used for the study of protein interactions and the isolation of protein complexes. Such tags are also increasingly used in ChIP assays in detecting the in vivo binding of transcription factors and associated co-factors to their target genes in chromatin. In searching for the optimal affinity tag for ChIP applications, three criteria are important: (a) tags must have high binding affinity; (b) tags should be preferably small and not strongly charged so as to minimize possible interference with transcription factor function (c) tags should be fairly insensitive to formaldehyde fixation. The latter is true for most tags that contain no or few lysine, arginine or histidine residues [1-3].The biotin/(strept)avidin affinity system fulfils the above criteria due to its unique characteristics [4], which include: (a) the very tight and specific binding of biotin by avidin (or streptavidin) which, with a Kd of 1015 L*mol -1, is one of the highest non covalent interactions known in nature, close to almost 103 – 106times greater than the interaction of epitopes with their specific antibodies. Once formed, the biotin-streptavidin complex is not disturbed by chang
Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter  [PDF]
Rugmani Padmanabhan Iyer, Sumin Gu, Bruce J. Nicholson, Jean X. Jiang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0056792
Abstract: SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.
Crosslinking Of Polysaccharides: Methods And Applications  [cached]
Sachin Patil,D. R. Jadge
Pharmaceutical Reviews , 2008,
Abstract: Polysaccharides are polymers of simple sugar building blocks. Crosslinked polysaccharides are having many applications in pharmaceuticals.Different methods are available for crosslinking of polysaccharides. Different chemical and physical methods are summarized and discussed. Chemical crosslinking of polysaccharide is highly versatile method with good mechanical stability. However, the crosslinking agents used are not only affects the integrity of substances but are often toxic compounds, which have been extracted from gel before they can be applied. Such adverse effects are avoided with the use of physically crosslinked gels.
Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri  [PDF]
Irati Martinez-Malaxetxebarria, Rudy Muts, Linda van Dijk, Craig T. Parker, William G. Miller, Steven Huynh, Wim Gaastra, Jos P. M. van Putten, Aurora Fernandez-Astorga, Marc M. S. M. W?sten
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0044796
Abstract: The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintenance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor, indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.
Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum
Jens Schneider, Petra Peters-Wendisch, K Corinna Stansen, Susanne G?tker, Stanislav Maximow, Reinhard Kr?mer, Volker F Wendisch
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-6
Abstract: 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.Biotin is a vitamin in humans (vitamin H or B7). Biotin deficiency is rarely observed in humans, e.g. after prolonged consumption of raw egg whites that contains biotin-binding avidin [1], as the normal microflora of the large intestine is considered to provide sufficient supply of biotin. If biotin is lacking, multiple carboxylase deficiencies arise [1] because biotin is a cofactor of the biotin-dependent carboxylases, which occur in all domains of life [2]. Many bacteria can synthesize biotin, but biotin auxotrophic bacteria such as Corynebacterium glutamicum require uptake of biotin from the habitat.Biotin synthesis can be subdivided into synthesis of pimelic acid followed by the biotin ring assembly [3]. Biotin ring ass
Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σAntA  [PDF]
Ryan F. Seipke,Elaine Patrick,Matthew I. Hutchings
PeerJ , 2015, DOI: 10.7717/peerj.253
Abstract: Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF) RNA polymerase sigma factor named σAntA which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σAntA controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σAntA is highly conserved in antimycin producing strains and the 10 and 35 elements at the σAntA regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σAntA proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.
Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σAntA  [PDF]
Ryan F Seipke,Elaine Patrick,Matt I Hutchings
PeerJ , 2015, DOI: 10.7287/peerj.preprints.203v1
Abstract: Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF) RNA polymerase sigma factor named σAntA which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σAntA controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σAntA is highly conserved in antimycin producing strains and the -10 and -35 elements at the σAntA regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σAntA proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.
Determination of biotin content in beet molasses by Lactobacillus plantarum
Lon?ar Eva S.,Do?enovi? Irena S.,Markov Sini?a L.,Malba?a Radomir V.
Acta Periodica Technologica , 2005, DOI: 10.2298/apt0536215l
Abstract: D-biotin content in beet molasses was determined by microbiological method using Lactobacillus plantarum, based on the comparison of the growth of this microorganism in molasses solutions with those in standard solutions of biotin. Incubation of the microorganism was performed on original Vitamin Biotin Testbouillon and laboratory prepared liquid culture medias. The amount of "real" biotin in molasses is low. The results depend upon the sample and volume of molasses solutions. Biotin contents obtained on both liquid media are close.
Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli
Teegan A Delli-Bovi, Maroya D Spalding, Sean T Prigge
BMC Biotechnology , 2010, DOI: 10.1186/1472-6750-10-73
Abstract: In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.Biotin, or vitamin H, was first identified as a yeast growth factor over 100 years ago[1] and was subsequently isolated from egg yolk[2] and liver[3]. It is an essential cofactor for a small family of enzymes that catalyze carboxylation and decarboxylation reactions, in which biotin serves as a covalent attachment site for CO2[4]. The number of biotinylated proteins varies from one to five in different organisms[5]. Biotin-dependent enzymes include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase[6]. These enzymes participate in central metabolic processes such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. The most widespread biotin-dependent enzyme is acetyl-CoA carboxylase (ACC)[7], which catalyzes the ATP-dependent transfer of a carboxyl group from carbonate to acetyl-CoA to form malonyl-CoA, in the first committed step of fatty acid biosynthesis[8].ACC is the o
Page 1 /100
Display every page Item


Home
Copyright © 2008-2017 Open Access Library. All rights reserved.