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Báo cáo Y học: Function and cellular localization of farnesoic acid O -methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum....


Báo cáo Y học: A critical motif for oligomerization and chaperone activity of bacterial a-heat shock proteins

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all a-crystallin type heat shock proteins (a-Hsp), but the molecular details of complex assembly are poorly understood. The a-Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of  24 subunits.


Báo cáo Y học: Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening

Two genes (CM-AAT1 and CM-AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl-transferases and show at most 21% identity to the only other fruit acyl-transferase characterized so far in strawberry. RT-PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening. Expression was severely reduced in ethylene-suppressed antisense ACC oxidase (AS) fruit and in wild-type (WT) fruit treated...


Báo cáo Y học: Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R....


Báo cáo Y học: Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cellto-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach.


Báo cáo Y học: Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. ...


Báo cáo Y học: Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene (Cyp19 )

Department of Medical Chemistry, Kochi Medical School, Nankoku, Japan; 2Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan; 3Department of Anatomy and Cell Biology, Kochi Medical School, Nankoku, Japan We previously generated mice lacking aromatase activity by targeted disruption of Cyp19 (ArKO mice), and reported phenotypes of the female mice, showing hemorrhage formation and follicular depletion in the ovary, diminution in uterine size, and bone loss...


Báo cáo Y học: Thiol-modifying inhibitors for understanding squalene cyclase function

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by sitedirected mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3-carboxy-4-nitrophenyl-dithio1,1¢,2-trisnorsqualene (CNDT-squalene) showed that the cysteine residue at the channel constriction was covalently modified by the...


Báo cáo Y học: The unorthodox histidine kinases BvgS and EvgS are responsive to the oxidation status of a quinone electron carrier

The purified soluble forms of the histidine kinases BvgS and EvgS of Bordetella pertussis and Escherichia coli, respectively, are shown to be responsive to oxidized ubiquinone-0 (Q-0) in vitro. The oxidized ubiquinone is a strong inhibitor of kinase activity of both enzymes with half maximal inhibition occurring at 11 lM (BvgS) and 4 lM (EvgS). Reduced Q-0 has no effect on the histidine kinases. Kinase activity can reversibly be switched off and on by changing the oxidation status of the quinone....


Báo cáo Y học: Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) · striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges....


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....


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. ...


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. ...


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. ...


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).


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....


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....


Báo cáo Y học: Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12...


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....


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.


Báo cáo Y học: The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23–33 and orexin B 18–28, with high affinity (119 ± 49 and 49 ± 23 nM) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand–receptor interaction. However, these C-terminal fragments of orexin did not displace the 125 I-labelled...


Báo cáo Y học: Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. ...


Báo cáo Y học: Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family

4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent condensation, reduction, and transfer reactions in the biosynthesis of plant phenylpropanoids. Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid biosyntheses. In contrast, divergent members of a 4CL gene family were identified in soybean (Glycine max L.). We isolated three structurally and functionally distinct 4CL cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene Gm4CL3. A fourth cDNA encoding 4CL4 had high similarity with 4CL3....


Báo cáo Y học: Identification of novel membrane proteins by searching for patterns in hydropathy profiles

A technique has been developed to search a proteome database for new members of a functional class of membrane protein. It takes advantage of the highly conserved secondary structure of functionally related membrane proteins. Such proteins typically have the same number of transmembrane domains located at similar relative positions in their polypeptide sequence. This gives rise to a characteristic pattern of peaks in their hydropathy profiles. To conduct a search, each member of a polypeptide database is converted to a hydropathy profile, peaks are automatically detected, and the pattern of peaks is compared with a template. A template was designed...


Báo cáo Y học: Inactivation of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus by reactive oxygen species

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was inactivated by reactive oxygen species. Highest Na+-NQR activity was observed in anaerobically prepared membranes that exhibited 1 : 1 coupling of NADH oxidation and Q reduction activities (1.6 UÆmg)1). Optical and EPR spectroscopy documented the presence of b-type cytochromes, a [2Fe)2S] cluster and an organic radical signal in anaerobically pre- pared membranes from V. alginolyticus.


Báo cáo Y học: Kinetic properties of bifunctional 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase from spinach leaves

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. ...


Báo cáo Y học: The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider

Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin consists in a wellresolved two-stranded antiparallel b sheet connected by a noncanonical b turn.


Báo cáo Y học: Tissue factor pathway inhibitor A possible mechanism of action

We have analyzed several mathematical models that describe inhibition of the factor VIIa–tissue factor complex (VIIa–TF) by tissue factor pathway inhibitor (TFPI). At the core of these models is a common mechanism of TFPI action suggesting that only the Xa–TFPI complex is the inhibitor of the extrinsic tenase activity. However, the model based on this hypothesis could not explain well all the available experimental data. Here, we show that a good quantitative description of all experimental data could be achieved in a model that contains two more assumptions....


Báo cáo Y học: Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-deficient strain of Saccharomyces cerevisiae and study of its complementation

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan; 2Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Japan; 3Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6 ) was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase....


Báo cáo Y học: A chimeric scorpion a-toxin displays de novo electrophysiological properties similar to those of a-like toxins

Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunisia; 2De´partement d’Inge´nierie et d’Etude des Prote´ines, CEA, Saclay, France; 3UMR 6560, Universite´ de la Me´diterrane´e, CNRS, Inge´nierie des Prote´ines, Laboratoire de Biochimie, IFR Jean-Roche, Faculte´ de Me´decine Nord, Marseille, France; 4Laboratoire de Neurophysiologie UPRES EA 2647, UFR Sciences, Angers, France BotXIV and LqhaIT are two structurally related long chain scorpion a-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhaIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect a-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities....


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