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Wenqing Zhang - One of the best experts on this subject based on the ideXlab platform.
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sumo1 activating Enzyme Subunit 1 is essential for the survival of hematopoietic stem progenitor cells in zebrafish
Development, 2012Co-Authors: Xiuling Li, Jin Xu, Wenqing ZhangAbstract:SUMMARY In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango hkz5 , which shows defects of definitive hematopoiesis. In tango hkz5 mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango hkz5 encodes SUMO1-activating Enzyme Subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango hkz5 mutation produces a truncated Sae1 protein (Sae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.
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SUMO1-activating Enzyme Subunit 1 is essential for the survival of hematopoietic stem/progenitor cells in zebrafish
Development, 2012Co-Authors: Xiuling Li, Jin Xu, Wenqing ZhangAbstract:SUMMARY In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango hkz5 , which shows defects of definitive hematopoiesis. In tango hkz5 mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango hkz5 encodes SUMO1-activating Enzyme Subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango hkz5 mutation produces a truncated Sae1 protein (Sae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.
Xiuling Li - One of the best experts on this subject based on the ideXlab platform.
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sumo1 activating Enzyme Subunit 1 is essential for the survival of hematopoietic stem progenitor cells in zebrafish
Development, 2012Co-Authors: Xiuling Li, Jin Xu, Wenqing ZhangAbstract:SUMMARY In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango hkz5 , which shows defects of definitive hematopoiesis. In tango hkz5 mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango hkz5 encodes SUMO1-activating Enzyme Subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango hkz5 mutation produces a truncated Sae1 protein (Sae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.
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SUMO1-activating Enzyme Subunit 1 is essential for the survival of hematopoietic stem/progenitor cells in zebrafish
Development, 2012Co-Authors: Xiuling Li, Jin Xu, Wenqing ZhangAbstract:SUMMARY In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango hkz5 , which shows defects of definitive hematopoiesis. In tango hkz5 mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango hkz5 encodes SUMO1-activating Enzyme Subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango hkz5 mutation produces a truncated Sae1 protein (Sae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.
Roberta F. Colman - One of the best experts on this subject based on the ideXlab platform.
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8-(4-Bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate: a new affinity label for purine nucleotide sites in proteins.
Archives of Biochemistry and Biophysics, 1999Co-Authors: Andrea Gorrell, Herbert J. Fromm, Roberta F. ColmanAbstract:Abstract A new affinity label, 8-(4-bromo-2,3-dioxobutylthio)guanosine 5′-triphosphate (8-BDB-TGTP), has been synthesized by initial reaction of GTP to form 8-Br-GTP, followed by its conversion to 8-thio-GTP, and finally coupling with 1,4-dibromobutanedione to produce 8-BDB-TGTP. 8-BDB-TGTP and its synthetic intermediates were characterized by thin-layer chromatography, UV, 31 P NMR spectroscopy, as well as by bromide and phosphorus analysis. Escherichia coli adenylosuccinate synthetase is inactivated by 8-BDB-TGTP at pH 7.0 at 25°C. Pretreatment of the Enzyme with N -ethylmaleimide (NEM) blocks the exposed Cys 291 and leads to simple pseudo-first-order kinetics of inactivation. The inactivation exhibits a nonlinear relationship of initial inactivation rate versus 8-BDB-TGTP concentration, indicating the reversible association of 8-BDB-TGTP with the Enzyme prior to the formation of a covalent bond. The inactivation kinetics exhibit an apparent K I of 115 μM and a k max of 0.0262 min −1 . Reaction of the NEM-treated adenylosuccinate synthetase with 8-BDB-[ 3 H]TGTP results in 1 mol of reagent incorporated/mol of Enzyme Subunit. Adenylosuccinate or IMP plus GTP completely protects the Enzyme against 8-BDB-TGTP inactivation, whereas IMP or GTP alone provide partial protection against inactivation. AMP is much less effective in protection. The results of ligand protection studies suggest that E. coli adenylosuccinate synthetase may accommodate 8-BDB-TGTP as a GTP analog. The new affinity label may be useful for identifying catalytic amino acid residues of protein proximal to the guanosine ring.
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Identification of the Nonsubstrate Steroid Binding Site of Rat Liver GlutathioneS-Transferase, Isozyme 1-1, by the Steroid Affinity Label, 3β-(Iodoacetoxy)dehydroisoandrosterone
Archives of Biochemistry and Biophysics, 1997Co-Authors: Joseph J. Barycki, Roberta F. ColmanAbstract:Abstract 3β-(Iodoacetoxy)dehydroisoandrosterone (3β-IDA), an analogue of the electrophilic substrate, Δ 5 -androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S -transferase, was tested as an affinity label of rat liver glutathione S -transferase, isozyme 1-1. A time-dependent loss of Enzyme activity is observed upon incubation of 3β-IDA with the Enzyme. The rate of Enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K i of 21 μ m . Upon complete inactivation of the Enzyme, a reagent incorporation of approximately 1 mol/mol of Enzyme Subunit or 2 mol/mol of Enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17β-estradiol-3,17-disulfate but, surprisingly, not by Δ 5 -androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the Enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the Enzyme. Two cysteine residues, Cys 17 and Cys 111 , are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol Enzyme Subunit. Isolation of Enzyme Subunits indicates the presence of unmodified, singly labeled, and doubly labeled Subunits, consistent with mutually exclusive modification of cysteine residues across Enzyme Subunits; i.e., modification of Cys 111 on Subunit A prevents modification of Cys 111 on Subunit B and similarly for Cys 17 . Molecular modeling analysis suggests that Cys 17 and Cys 111 are located in the nonsubstrate steroid binding site, within the cleft between the Subunits of the dimeric Enzyme.
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identification of his141 in the active site of bacillus subtilis adenylosuccinate lyase by affinity labeling with 6 4 bromo2 3 dioxobutyl thioadenosine 5 monophosphate
Journal of Biological Chemistry, 1997Co-Authors: Carolyn A Worby, Jack E Dixon, Roberta F. ColmanAbstract:Abstract Adenylosuccinate lyase of Bacillus subtilis is inactivated by 25-400 μM 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5′-monophosphate (6-BDB-TAMP) at pH 7.0 and 25°C. The initial inactivation rate constant exhibits nonlinear dependence on the concentration of 6-BDB-TAMP, implying there is reversible formation of Enzyme-reagent complex (KI = 30 ± 4 μM) prior to irreversible modification (kmax = 0.139 ± 0.005 min−1). The tetrameric Enzyme incorporates about 1 mol of 6-BDB-[32P]TAMP per mol of Enzyme Subunit concomitant with complete inactivation. Protection against inactivation and incorporation of [32P]reagent is provided by adenylosuccinate or a combination of AMP and fumarate, whereas either AMP or fumarate alone is much less effective. These observations suggest that 6-BDB-TAMP targets the adenylosuccinate-binding site. Hydrolyzed 6-BDB-TAMP is a competitive inhibitor with respect to adenylosuccinate in the catalytic reaction and also decreases the rate of inactivation by 6-BDB-TAMP. These results account for the decrease in the inactivation rate as the reaction of 6-BDB-TAMP with the Enzyme proceeds. Purification by chromatography on dihydroxyboryl-agarose and high performance liquid chromatography of the tryptic digest of inactivated Enzyme yields a single radioactive peptide, Thr140-Phe150, as determined by gas-phase sequencing. Modified His141 is the reaction product of 6-BDB-TAMP and adenylosuccinate lyase. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analog in modifying His141 in the substrate-binding site of adenylosuccinate lyase, where it may serve as a general base accepting a proton from the succinyl group during catalysis.
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activation of bovine liver glutamate dehydrogenase by covalent reaction of adenosine 5 o s 4 bromo 2 3 dioxobutyl thiophosphate with arginine 459 at an adp regulatory site
Biochemistry, 1994Co-Authors: Kazimierz O Wrzeszczynski, Roberta F. ColmanAbstract:: Bovine liver glutamate dehydrogenase is an allosteric Enzyme which is activated by ADP. The affinity label adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPSBDB), a new ADP analog featuring a reactive group at a position equivalent to that of the pyrophosphate, reacts with this glutamate dehydrogenase to yield Enzyme containing about 0.9 mol/mol of Enzyme Subunit. The reaction results in a time-dependent irreversible activation of the Enzyme. Glutamate dehydrogenase (8.9 microM Subunit) modified with 10-60 microM AMPSBDB is about 3.2-fold more active than native Enzyme. The modified Enzyme is still inhibited by GTP and by high concentrations of NADH, but is no longer activated by ADP. The addition to the reaction mixture of (a) NADH or alpha-ketoglutarate; (b) GTP + NADH; or (c) alpha-ketoglutarate + NADH has little effect on the functional changes produced by AMPSBDB; whereas, the reaction is prevented by ADP. Purification of labeled peptide from proteolytic and chemical digests of [2-3H]AMPSBDB-modified Enzyme leads to identification of Arg459 as the target amino acid. We conclude that AMPSBDB functions as an ADP mimic covalently bound to Arg459 within the ADP activator site of the allosteric bovine liver glutamate dehydrogenase.
Kazunori Ikebukuro - One of the best experts on this subject based on the ideXlab platform.
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Construction of target molecule sensing system using aptameric Enzyme Subunit based on PQQGDH activity.
Nucleic acids symposium series (2004), 2020Co-Authors: Kazunori Ikebukuro, Wataru Yoshida, Yo Morita, Koji SodeAbstract:We previously reported a novel target sensing element named aptameric Enzyme Subunit (AES). AES is composed of an aptamer binding to the target molecule and an aptamer affecting the enzymatic activity. In our previous work, we have succeeded in adenosine or DNA sensing by measuring the thrombin activity change resulting from adenosine or DNA binding to the AES. In this work, we have screened the pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) aptamers which affected the PQQGDH activity and have constructed an AES using the PQQGDH aptamer. As a model system, we connected the PQQGDH aptamer and an adenosine aptamer to make AES, and detected adenosine by measuring the PQQGDH activity change resulting from adenosine binding to the AES.
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An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin
Analytical Letters, 2013Co-Authors: Wataru Yoshida, H. Yamamoto, Kazunori IkebukuroAbstract:The aptameric Enzyme Subunit (AES) is an artificial Enzyme Subunit that can allosterically control partner Enzyme activity. By means of an AES, target molecules can be detected by measurement of enzymatic activity in a homogeneous solution. We have developed a thrombin aptamer-based AES that can detect several targets by measuring clotting time in a fibrinogen solution. Measurement of the clotting activity in the fibrinogen solution is not suitable for a convenient biosensor. However, kinetics measurement of clotting activity is suitable for convenient detection of thrombin activity. The interference-enhanced reflection (IER) method is a simple, real-time technique to detect the thickness and/or refractive index of a thin film formed on a glass substrate surface. We demonstrated that clotting activity on a glass substrate with immobilized thrombin can be monitored in real time by IER. By means of the IER method, we were able to detect the target molecules of an AES. IER-based sensors have already been com...
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selection of dna aptamers against insulin and construction of an aptameric Enzyme Subunit for insulin sensing
Biosensors and Bioelectronics, 2009Co-Authors: Wataru Yoshida, Koji Sode, Yo Morita, Eriko Mochizuki, Madoka Takase, Hijiri Hasegawa, Hiroki Yamazaki, Kazunori IkebukuroAbstract:We selected DNA aptamers against insulin and developed an aptameric Enzyme Subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a “natural” insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation.
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label free homogeneous detection of immunoglobulin e by an aptameric Enzyme Subunit
Biotechnology Letters, 2008Co-Authors: Wataru Yoshida, Koji Sode, Kazunori IkebukuroAbstract:We have developed an aptameric Enzyme Subunit (AES) for immunoglobulin E (IgE) sensing. AES is an artificial Enzyme Subunit constructed from two different aptamers and does not require any modification. Using the AES, the target molecule can be detected by measuring enzymatic activity in homogeneous solution. We connected IgE-binding aptamer and its complementary strand to split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin activity and it decreased in the presence of IgE. We were able to detect IgE by using this AES in homogeneous solution with a detection limit of 50 pmol.
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aptameric Enzyme Subunit for homogeneous dna sensing
Biotechnology Letters, 2008Co-Authors: Kazunori Ikebukuro, Wataru Yoshida, Koji SodeAbstract:We have developed an aptameric Enzyme Subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial Enzyme Subunit composed of an Enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA. Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution.
A. Martínez - One of the best experts on this subject based on the ideXlab platform.
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Evidence for a functionally important histidine residue in human tyrosine hydroxylase
Amino Acids, 1995Co-Authors: A. MartínezAbstract:Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M^−1 min^−1 at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native Enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per Enzyme Subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoEnzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.