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Báo cáo Y học: A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function
The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the a subunit of cytosolic casein kinase (CK2) that contained an N-terminal extension for a putative plastid transit peptide. Using in organello chloroplast import studies, immunodetection and MS, we found that the corresponding protein, termed cpCK2a, is targeted to the chloroplast and is associated with the plastid RNA polymerase PEP-A. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues
Aralar1 and citrin are members of the subfamily of calciumbinding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown....
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Báo cáo Y học: A new conceptual framework for enzyme catalysis Hydrogen tunneling coupled to enzyme dynamics in flavoprotein and quinoprotein enzymes
Recent years have witnessed high levels of activity in identifying enzyme systems that catalyse H-transfer by quantum tunneling. Rather than being restricted to a small number of specific enzymes as perceived initially, it has now become an accepted mechanism for H-transfer in a growing number of enzymes. Furthermore, H-tunneling is driven by the thermally induced dynamics of the enzyme. In some of those enzymes that break stable C–H bonds the reaction proceeds purely by quantum tunneling, without the need to partially ascend the barrier. Enzymes studied that fall into this category include the flavoprotein and quinoprotein amine dehydrogenases, which have...
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Báo cáo Y học: Barrier passage and protein dynamics in enzymatically catalyzed reactions
This review describes studies of particular enzymatically catalyzed reactions to investigate the possibility that catalysis is mediated by protein dynamics. That is, evolution has crafted the protein backbone of the enzyme to direct vibrations in such a fashion to speed reaction. The review presents the theoretical approach we have used to investigate this problem, but it is designed for the nonspecialist.
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Báo cáo Y học: Environmentally coupled hydrogen tunneling Linking catalysis to dynamics
Many biological C-H activation reactions exhibit nonclassical kinetic isotope effects (KIEs). These nonclassical KIEs are too large (kH/kD 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH/AD) fall outside of the semiclassical range near unity. The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model. Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy-atom coordinate, from which the hydrogen transfers via nuclear tunneling. Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates...
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Báo cáo Y học: Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB) 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites
2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 lM, respectively, measured at pH 7.2)....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda b-glycosidase
The design of b-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the b-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzymetransition state complex. In an attempt to fill this gap, sitedirected mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive b-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbgly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes....
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Báo cáo Y học: Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn’s disease
Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy
We report here the direct evidence for the formation of alternative DNA structures in a plasmid DNA, termed pTIR10, containing a 0.23-kb pyrimidine/purine-biased (Pyr/Pur) stretch isolated from the rat genome. Long Pyr/Pur sequences are abundant in eukaryotic genomes, and they may modulate the biological activity of genes and genomes via formation of various types of triplex-related structures. The plasmid DNA in sodium acetate buffer (pH 4.35) was deposited on APS-modified mica, and after drying it was imaged with an atomic force microscope in air. ...
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Báo cáo Y học: GNA33 from Neisseria meningitidis serogroup B encodes a membrane-bound lytic transglycosylase (MltA)
In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48-kDa lipoprotein that is 34.5% identical with membrane-bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma
Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol Comparison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains
The human immunodeficiency virus type 1, HIV-1, genome encodes a highly conserved regulatory gene product, Vpr (96 amino acids), which is incorporated into virions in quantities equivalent to those of the viral Gag protein. In infected cells, Vpr is believed to function during the early stages of HIV-1 replication (such as transcription of the proviral genome and migration of preintegration nuclear complex), blocks cells in G2 phase and triggers apoptosis. Vpr also plays a critical role in long-term AIDS disease by inducing viral infection in nondividing cells such as monocytes and macrophages. To gain deeper insight of the structure–function relationship...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes
Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin-diabetic rats. Glutamine increased cell volume and induced an anabolic response characterized by an activation of acetyl-CoA carboxylase (ACC), glycogen synthase (GS) and p70 ribosomal S6 kinase (p70S6K), the key enzymes in fatty acid, glycogen and protein synthesis, respectively. The effects of glutamine were independent of...
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Báo cáo Y học: Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils
The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0–10 lM did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 2.5 lM).
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Báo cáo Y học: The solution structure and activation of visual arrestin studied by small-angle X-ray scattering
Visual arrestin is converted from a ÔbasalÕ state to an ÔactivatedÕ state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 lM. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Granule-bound starch synthase I A major enzyme involved in the biogenesis of B-crystallites in starch granules
Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity
An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+-dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short-chain alcohols. We have characterized the cytosolic and membrane-bound forms of the yeast PtdEtn-PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions. Both forms of PtdEtn-PLD activity were similarly activated by Ca2+ ions in a biphasic manner. Likewise, other divalent cations affected both cytosolic and membrane-bound forms to the same extent....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes
Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity
In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11b-hydroxylase [Bulow, H.E., Mobius, ¨ ¨ K., Bahr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. ¨ Commun. 221, 304–312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability...
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Báo cáo Y học: Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors
The transcription factor serum response factor (SRF) regulates expression of growth factor-dependent genes and muscle-specific genes in vertebrates. Homologous factors regulate differentiation of some ectodermic tissues in invertebrates. To explore the molecular basis of these different physiological functions, the functionality of human, Drosophila melanogaster and Artemia franciscana SRFs in mammalian cells has been compared in this article. D. melanogaster and, to a lesser extend, A. franciscana SRF co-expression represses the activity of strong SRF-dependent promoters, such as those of the mouse c-fos and A. ...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Presence and regulation of the endocannabinoid system in human dendritic cells
Cannabinoid receptors and their endogenous ligands, the endocannabinoids, have been detected in several blood immune cells, including monocytes/macrophages, basophils and lymphocytes. However, their presence in dendritic cells, which play a key role in the initiation and development of the immune response, has never been investigated. Here we have analyzed human dendritic cells for the presence of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), the cannabinoid CB1 and CB2 receptors, and one of the enzymes mostly responsible for endocannabinoid hydrolysis, the fatty acid amide hydrolase (FAAH)....
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Báo cáo Y học: Dissecting the effect of trifluoroethanol on ribonuclease A Subtle structural changes detected by nonspecific proteases
With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At 30% (v/v) trifluoroethanol (pH 8.0; 25 °C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity....
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Báo cáo Y học: Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe
Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose6-P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies
Porcine heart cytoplasmic malate dehydrogenase (s-MDH) is a dimeric protein (2 · 35 kDa). We have studied equilibrium unfolding and refolding of s-MDH using activity assay, fluorescence, far-UV and near-UV circular dichroism (CD) spectroscopy, hydrophobic probe-1-anilino8-napthalene sulfonic acid binding, dynamic light scattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second one is an expanded monomer (ME), which is little less compact than the native monomer and has most of the characteristic features...
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Báo cáo Y học: The proximal cis-acting elements Sp1, Sp3 and E2F regulate mouse mer gene transcription in Sertoli cells
Mer belongs to the Tyro 3 family of receptor tyrosine kinases (RTKs). Together with Axl and Rse, the three RTKs are believed to play important functional roles in the male gonads because gene knockout male mice lacking all of these receptors are infertile. In the present study, postnatal expression of Axl and Rse in mouse testes decreased during maturation while expression of Mer increased age-dependently during testicular development.
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Báo cáo Y học: The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea
Shikimate kinase was chosen as a convenient representative example of the subclass of a/b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 M urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJÆmol)1. Approximately 95% of the enzyme activity could...
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Coordination structures of Ca21 and Mg21 in Akazara scallop troponin C in solution FTIR spectroscopy of side-chain COO – groups
FTIR spectroscopy has been applied to study the coordination structures of Mg2þ and Ca2þ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2þ binding site. The region of the COO – antisymmetric stretch provides information about the coordination modes of COO – groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm21 in the apo state, whereas additional bands were observed at 1543 and 1601 cm21 in the Ca2þ-bound and Mg2þ-bound states, respectively. The intensity of the band at 1567 cm21 in the Mg2þ-bound state was identical...
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Báo cáo Y học: Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M-type homopolymer
Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is composed of a single subunit that contains both the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleation site, typical of mammalian L chains. Comparison of the amino-acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M-type homopolymer. Interestingly, the T. bernacchii ferritin chain shows 85.7% identity with a coldinducible ferritin chain of the rainbow trout Salmo gairdneri....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans
Dystrobrevin is one of the intracellular components of the transmembrane dystrophin–glycoprotein complex (DGC). The functional role of this complex in normal and pathological situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affecting other members of the DGC complex. These findings therefore suggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases....
8/30/2018 3:05:01 AM +00:00
Báo cáo Y học: Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC Fluorescence resonance energy transfer and crystallographic study
The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/ sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300–380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380–500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cisparinaric acid interactions occurring in the presence of sodium taurodeoxycholate....
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