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Báo cáo Y học: Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL
Many groups of proteins play important roles in the cell’s response to various stresses. The molecular chaperone GroEL of Escherichia coli represents one such highly conserved family of stress proteins. We have observed that isolated GroEL complexes from stationary cultures contain various polypeptides that can be released from the chaperonin by GroES and/or ATP, and identified two such polypeptides as the proteins GatY and UP12. Whereas GatY had been isolated previously, as an in vivo substrate of GroEL, the isolation of UP12 in a complex with GroEL was intriguing, because based on sequence similarity it was suggested that UP12 might...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase Electron entry and kinetic phases of the reaction
Introducing site-directed mutations in surface-exposed residues of subunit II of the heme aa3 cytochrome c oxidase of Paracoccus denitrificans, we analyze the kinetic parameters of electron transfer from reduced horse heart cytochrome c. Specifically we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101) A high-pressure stopped-flow study
The high-pressure stopped-flow technique is applied to study the CO binding in cytochrome P450cam (P450cam) bound with homologous substrates (1R-camphor, camphane, norcamphor and norbornane) and in the substrate-free protein. The activation volume DV # of the CO on-rate is positive for P450cam bound with substrates that do not contain methyl groups. The kon rate constant for these substrate complexes is in the order of 3 · 106 M)1Æs)1. In contrast, P450cam complexed with substrates carrying methyl groups show a negative activation volume and a low kon rate constant of % 3 · 104 M)1Æs)1. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Interaction of bovine coagulation factor X and its glutamic-acid-containing fragments with phospholipid membranes A surface plasmon resonance study
The interaction of blood coagulation factor X and its Gla-containing fragments with negatively charged phospholipid membranes composed of 25 mol% phosphatidylserine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho) was studied by surface plasmon resonance. The binding to 100 mol% PtdCho membranes was negligible. The calcium dependence in the membrane binding was evaluated for intact bovine factor X (factor X) and the fragment containing the Gla-domain and the N-terminal EGF (epidermal growth factor)-like domain, Gla–EGFN, from factor X....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Determinants of the inhibition of a Taiwan habu venom metalloproteinase by its endogenous inhibitors revealed by X-ray crystallography and synthetic inhibitor analogues
Venoms from crotalid and viperid snakes contain several peptide inhibitors which regulate the proteolytic activities of their snake-venom metalloproteinases (SVMPs) in a reversible manner under physiological conditions. In this report, we describe the high-resolution crystal structures of a SVMP, TM-3, from Taiwan habu (Trimeresurus mucrosquamatus) cocrystallized with the endogenous inhibitors pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) or pyroGlu-Lys-Trp (pEKW).
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Báo cáo Y học: Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase b subunit in the energy coupling mechanism of the Na+ pump
Mikrobiologisches Institut der Eidgeno¨ssischen Technischen Hochschule, ETH-Zentrum, Zu¨rich, Switzerland The membrane-bound b subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the b subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum
The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases
Business Unit Cell Cybernetics, Plant Research International, Wageningen, the Netherlands; 2University of Wu¨rzburg, Chair of Food Chemistry, Germany; 3Laboratory of Plant Physiology, Wageningen University, the Netherlands Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated.
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Molecular mechanisms underlying SHP-1 gene expression
SHP-1, a protein-tyrosine phosphatase with two srchomology 2 domains, is expressed predominantly in hematopoietic and epithelial cells and has been implicated in numerous signaling pathways as a negative regulator. Two promoters direct the expression of human and murine SHP-1, and two types of transcripts (I) and (II) SHP-1, are initiated from each of these promoters. The cDNA sequences of (I)SHP-1 and (II)SHP-1 are identical except in the 5¢ untranslated region and in the first few coding nucleotides. In this report, we show that promoter usage is similar in mouse and human hematopoietic cells, but different in epithelial cells. In human...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4
We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124–126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of 52 kb. The first intron is 16 kb in length and is followed by an unusually large exon ( 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase
The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1
Sensitization to cockroach allergens is associated with the development of asthma. Bla g 1 is a German cockroach allergen that shows allergenic cross-reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino-acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1. By molecular mass, purified natural Bla g 1 would contain approximately two duplexes.
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids
The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 lM for ADP, and 105 lM or 140 lM, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors
We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( 30 min) caused a profound inhibition of protein synthesis. In each case this was accompanied by dephosphorylation of the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and increased binding of this repressor protein to eIF4E. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter
An immuno-homologue of maize Ca2+/calmodulin (CaM)dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperaturemediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Transglutaminase-mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity
Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional characterization of three VIP polyaminated adducts (VIPDap, VIPSpd, and VIPSpm), obtained by a transglutaminasecatalysed reaction between the VIP Gln16 residue and 1,3-diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes
The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the 3JH-C coupling...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Role of histidine 42 in ascorbate peroxidase Kinetic analysis of the H42A and H42E variants
To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, l ¼ 0.10 M, 25.0 °C) revealed wavelength maxima [kmax (nm): 397, 509, % 540sh, 644 (H42A); 404, 516, % 538sh, 639 (H42E)] consistent with a predominantly fiveco-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 ± 0.3 UÆmg)1) was % 30-fold lower than that of the recombinant wild-type...
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Báo cáo Y học: Balanced expression of single subunits in a multisubunit protein, achieved by cell fusion of individual transfectants
To establish stable cell lines that produce recombinant multisubunit proteins, it is usually necessary to cotransfect cells with several independent gene constructs. Here, we show that a stepwise fusion of individually transfected cells, results in a fused cell-line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a defined expression ratio between each protein subunit. The cell-fusion technology described allows a predefined expression level of each subunit. Using SIgA as a model protein we demonstrate that the majority of the fused cells inherit the molar expression ratio of the parental transfected cells. These results indicate that...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle
The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils
In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2– generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac ` (Doussiere J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun. 285, 1317–1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A–Sepharose bound to antip67phox antibodies, the...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPcNH2 and GTPcF
Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPcS are widely used to stabilize rapidly hydrolyzing protein-nucleotide complexes and to investigate biochemical reaction pathways. Here we describe the chemical synthesis of guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH2) and a new synthesis of guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on 31P, 19F and 1H NMR resonances are tabulated. For GTPcNH2 the enthalpy of magnesium coordination is DH° ¼ 3.9 kcalÆmol)1 and the association constant Ka is 0.82 mM)1....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: The evolution of monomeric and oligomeric bc-type crystallins
The case of homologous monomeric c-type and oligomeric b-type crystallins has been described and analyzed in evolutionary terms. Data and hypotheses from molecular genetics and structural investigations converge and suggest a novel three-phase model for the evolutionary history of crystallin-type proteins. In the divergent cascades of monomeric and oligomeric crystallins, a pivotal role was played by alterations in the gene segments encoding the C-terminal extensions and the intermotif or interdomain linker peptides. These were genomic hot spots where evolution experimented to produce the modern variety of bc-crystallin-type quaternary structures. Keywords: crystallins; evolution; quaternary structure; introns late; introns early. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Structure of human immunodeficiency virus type 1 Vpr(34–51) peptide in micelle containing aqueous solution
Human immunodeficiency virus type 1 protein R (HIV-1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G2 cell cycle arrest and is involved in cellular differentiation and cell death. Vpr subcellular localization is as variable as its functions. It is known, that consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr is clearly dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 34–51 of HIV-1 Vpr. This...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Pyruvate decarboxylase from Kluyveromyces lactis An enzyme with an extraordinary substrate activation behaviour
Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells
This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP-76, Gads, Vav and ZAP70. We show that both SLP-76 and ZAP70 co-immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co-immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP-76, Gads and Vav associate independently of each other to different domains or regions, of Shb.
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Báo cáo Y học: The structure of the O-chain of the lipopolysaccharide of a prototypal diarrheagenic strain of Hafnia alvei that has characteristics of a new species under the genus Escherichia
The structure of the O-polysaccharide of the lipopolysaccharide from a diarrheal strain isolated in Bangladesh was studied with sugar, and methylation analysis, NMR spectroscopy, mass spectrometry and partial acid hydrolysis. The strain was first designated as Hafnia alvei, but later found to be a possible new species in the genus Escherichia. Two different polysaccharides were detected, a major and a minor one. The structure of the major polysaccharide is given below, while the structure of the minor one was not investigated. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration
Department of Medical Biochemistry and Biology, University of Bari, Italy; 2Department of Zoology, Laboratory of Histology and Comparative Anatomy, University of Bari, Italy; 3Center for the Study of Mitochondria and Energy Metabolism (CNR) Bari, Italy Mitochondrial bioenergetic impairment has been found in the organelles isolated from rat liver during the prereplicative phase of liver regeneration. To gain insight into the mechanism underlying this impairment, we investigated mitochondrial ultrastructure and membrane permeability properties in the course of liver regeneration after partial hepatectomy, with special interest to the role played by Ca2+ in this process. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK
PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a b turn. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: The cold-active lipase of Pseudomonas fragi Heterologous expression, biochemical characterization and molecular modeling
A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%).
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