Langmuir, 2004 Aug 31, 20(18), 7736 - 46 Role of Cell Surface Lipopolysaccharides in Escherichia coli K12 adhesion and transport; Walker SL et al.; The influence of bacterial surface lipopolysaccharides (LPS) on cell transport and adhesion has been examined by use of three mutants of Escherichia coli K12 with well-characterized LPS of different lengths and molecular composition . Two experimental techniques, a packed-bed column and a radial stagnation point flow system, were employed to investigate bacterial adhesion kinetics onto quartz surfaces over a wide range of solution ionic strengths . Although the two systems capture distinct deposition (adhesion) mechanisms because of their different hydrodynamics, similar deposition kinetics trends were observed for each bacterial strain . Bacterial deposition rates were directly related to the electrostatic double layer interaction between the bacteria and quartz surfaces, in qualitative agreement with classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory . However, DLVO theory does not fully explain the deposition behavior for the bacterial strain with the lengthy, uncharged O-antigen portion of the LPS . Neither the length nor the charge characteristics of the LPS molecule directly correlated to deposition kinetics, suggesting a complex combination of cell surface charge heterogeneity and LPS composition controls the bacterial adhesive characteristics . It is further suggested that bacterial deposition behavior is determined by the combined influence of DLVO interactions, LPS-associated chemical interactions, and the hydrodynamics of the deposition system. Shi Yan Sheng Wu Xue Bao, 2004 Jun, 37(3), 227 - 31 {Effective expression of ribonuclease-onconase in Escherichia coli and assaying its cytotoxic potential}; Xu GF et al.; Onconase, a ribonuclease purified from Rana pipiens oocytes, has cytotoxic activity against several tumor cell lines in vitro . With the characteristic of containing four pairs of disulfide bonds internal and N terminal sequence attributing mostly to its biological functions, it is difficult to obtain the active Onconase from Escherichia coli expression system with normal strategy . Here, we fused the cDNA coding for Onconase in frame with the pelB signal sequence in pET22b(+) expression vector . Onconase can be effectively expressed in inclusion body without additional residues at N terminal under the proper inducing condition . After refolding and purifying process, we can get the recombined Onconase which has a similar ribonuclease activity as the one isolated from oocytes . The recombined Onconase has a pronounced effect on proliferation of Hut-78 cells (IC50=0.5 micromol/L). J Biol Regul Homeost Agents, 2004 Jan-Mar, 18(1), 1 - 8 Structural elements responsible for transglutaminase activity of protein disulphide isomerases and thioredoxins; Blasko B et al.; According to recent results both protein disulphide isomerase (PDI) and thioredoxin (Trx) enzymes have transglutaminase activity which can be linked to the thioredoxin box found in these proteins . Analysis of known protein disulphide isomerase and thioredoxin sequences has revealed the presence of conserved Cys, His and Asp residues required for transglutaminases to catalyze the incorporation of primary amines into protein-bound glutamine residues . The available 3D structures of PDIs and Trxs show that these residues are in close proximity to achieve transglutamylation of substrate proteins . The shared activities of the members of the large protein disulphide isomerase, thioredoxin and transglutaminase enzyme families reviewed here may have general biological significance in the regulation of cellular and tissue processes. Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1681 - 9 Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem; Shyu DJ et al.; A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem . This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence . Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay . The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column . Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively . The recombinant cystatins were found to be thermally stable up to 60 degrees C . Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing. Protein Sci, 2004 Sep, 13(9), 2458 - 69 A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge; Kaderbhai MA et al.; A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide-mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli . The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E . coli . With-in "sequence spaces" ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins . Three clusters of hyper, median, and hypo exporters were isolated . These had corresponding net charges of -1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states . The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E . coli . We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E . coli. Protein Sci, 2004 Sep, 13(9), 2416 - 28 The HU-DNA binding interaction probed with UV resonance Raman spectroscopy: structural elements of specificity; Wojtuszewski K et al.; The Escherichia coli protein HU functions as an architectural DNA-binding protein by facilitating DNA looping or bending to form multiprotein complexes . Although HU does not recognize a specific DNA sequence, site-specific binding to a number of discontinuous, looped, or bent DNA substrates has been observed . In this study UV resonance Raman (UVRR) spectroscopy is used to identify structural elements associated with low- and high-affinity binding by examining three different HU-DNA complexes . UVRR spectra obtained with an excitation wavelength of 210 nm, which preferentially enhances protein backbone amide vibrations, indicate that HU secondary structure content increases and the protein structure becomes more rigid upon binding to DNA . The increase in alpha-helical content is attributed to the C-terminal helix, which interacts with the DNA and may play a role in binding affinity and specificity . UVRR spectra obtained with a 215 nm excitation wavelength demonstrate that Pro mode intensity at 1455 cm(-1) decreases upon complex formation . This intensity decrease is attributed to the intercalation of Pro residues between DNA base pairs to induce a bend in the DNA, as has been observed previously in the IHF-DNA and HU-DNA cocrystal structures . DNA vibrational modes are also indicative of significant base unstacking and opening of the minor groove upon protein binding, consistent with bending and distortion of the DNA . In all three complexes, A-DNA conformational features are indicated by deoxyribose-phosphate backbone modes . These and other results suggest that protein-induced bending plays an important role in HU site-specific binding and supports a model of a mutually induced fit. Protein Sci, 2004 Sep, 13(9), 2398 - 405 Long-range allosteric transitions in carbamoyl phosphate synthetase; Thoden JB et al.; Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine . The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel . The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia . The large subunit binds the two required molecules of MgATP and is involved in assembling the final product . Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme . Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E . coli, where Cys 248 in the small subunit was changed to an aspartate . This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished . Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes . The manner in which UMP binds to carbamoyl phosphate synthetase is described. Protein Sci, 2004 Sep, 13(9), 2378 - 87 Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases; Kuhlewein A et al.; E . coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity . The structure of E . coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 phi/psi restraints derived from the Chemical Shift Index) . Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 A within the well-defined secondary structure elements . E . coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins . The secondary structure of E . coli Par10 consists of four helical regions and a four-stranded antiparallel beta-sheet . The N terminus forms a beta-strand, followed by a large stretch comprising three alpha-helices . A loop region containing a short beta-strand separates these helices from a fourth alpha-helix . The C terminus consists of two more beta-strands completing the four-stranded anti-parallel beta-sheet with strand order 2143 . Interestingly, the third beta-strand includes a Gly-Pro cis peptide bond . The curved beta-strand forms a hydrophobic binding pocket together with alpha-helix 4, which also contains a number of highly conserved residues . The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins. J Biol Chem, 2004 Oct 29, 279(44), 46057 - 64 Epub 2004 Aug 20. Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase; Ahmad Z et al.; In the catalytic mechanism of ATP synthase, phosphate (P(i)) binding and release steps are believed to be correlated to gamma-subunit rotation, and P(i) binding is proposed to be prerequisite for binding ADP in the face of high cellular {ATP}/{ADP} ratios . In x-ray structures, residue betaAsn-243 appears centrally located in the P(i)-binding subdomain of catalytic sites . Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp . Mutation betaN243A caused 30-fold impairment of F(1)-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P(i) protected from inhibition . ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory . betaN243D F(1)-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium . 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F(1)-ATPase, and P(i) did not affect activation . We conclude that residue betaAsn-243 is not involved in P(i) binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues . It is also probably involved in orientation of the "attacking water" and of an associated second water. J Biol Chem, 2004 Oct 29, 279(44), 45540 - 5 Epub 2004 Aug 19. Cloning and characterization of the acid lipase from castor beans; Eastmond PJ; Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane . In order to identify this enzyme, proteomic analysis was performed on purified oil bodies . A approximately 60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei . RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue . RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima approximately 4.5) . RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids . The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase . Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies . This is the first evidence for the molecular identity of an oil body-associated lipase from plants . Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis. J Biol Chem, 2004 Oct 29, 279(44), 45485 - 94 Epub 2004 Aug 18. Hydroxylamine assimilation by Rhodobacter capsulatus E1F1 . requirement of the hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene region for hydroxylamine reduction; Cabello P et al.; Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source . Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library . A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP) . Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene . A His(6)-HCP was overproduced in Escherichia coli and purified . HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both {2Fe-2S} and {4Fe-2S-2O} clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant . The apparent K(m) values for NH(2)OH and methyl viologen were 1 mM and 7 microM, respectively, at the pH and temperature optima (9.3 and 40 degrees C) . The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents . R . capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH(2)OH as nitrogen source, and up to 10 mM NH(2)OH was taken up by anaerobic resting cells . Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor . In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities . However, R . capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH(2)OH . Also, E . coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth . These results suggest that HCP is involved in assimilation of NH(2)OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step. J Biol Chem, 2004 Oct 29, 279(44), 45477 - 84 Epub 2004 Aug 17. Concomitant reconstitution of TraI-catalyzed DNA transesterase and DNA helicase activity in vitro; Csitkovits VC et al.; TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation . Regulation of the functional domains of the enzyme is poorly understood . TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic) . The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase . This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities . Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation . Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed . Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation . The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates . This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic. J Biol Chem, 2004 Nov 5, 279(45), 46794 - 801 Epub 2004 Aug 17. Crystal structural studies of changes in the native dinuclear iron center of ribonucleotide reductase protein R2 from mouse; Strand KR et al.; Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms . The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical . Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions . In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron . The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1 . There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies . Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study . Compared with E . coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible . Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2. J Biol Chem, 2004 Oct 22, 279(43), 44628 - 38 Epub 2004 Aug 18. TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking; Wang Y et al.; The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c'') relative to the integral subunit a . The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'' . Subunit c'' contains an additional buried acidic residue in TM4 that is not required for proton transport . The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation . We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al . (2003) J . Biol . Chem . 278, 41908-41913) . We have now analyzed interaction of subunits a and c'' using disulfide-mediated cross-linking . The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c'' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c' . By contrast, TM4 of subunit c'' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity . These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other. Infect Immun, 2004 Sep, 72(9), 5089 - 96 Dendritic cells stimulated with Actinobacillus actinomycetemcomitans elicit rapid gamma interferon responses by natural killer cells; Kikuchi T et al.; Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-gamma) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production . DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans . These results prompted the hypothesis that an interaction between mDCs and A . actinomycetemcomitans promotes IFN-gamma production, and IFN-gamma is known to promote both immunopathology and protective IgG2 . A . actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A . actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-gamma within just 24 h . In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent . A . actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-gamma responses . NK cells of the innate immune system were the primary source of this early IFN-gamma, although CD8 T cells also contributed some . The NK cell-derived IFN-gamma was IL-12 dependent, and A . actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent . A . actinomycetemcomitans and A . actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E . coli LPS in the ability to induce DC IL-12 and IFN-gamma . The ability of A . actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-gamma in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1 . This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A . actinomycetemcomitans. Am J Physiol Lung Cell Mol Physiol, 2004 Dec, 287(6), L1178 - 85 Epub 2004 Aug 20. Vascular changes after intra-amniotic endotoxin in preterm lamb lungs; Kallapur SG et al.; Chorioamnionitis is associated with preterm delivery and bronchopulmonary dysplasia (BPD), characterized by impaired alveolar and pulmonary vascular development and vascular dysfunction . To study the vascular effects in a model of chorioamnionitis, preterm lambs were exposed to 20 mg of intra-amniotic endotoxin or saline for 1, 2, 4, or 7 days and delivered at 122 days gestational age (term = 150 days) . This intra-amniotic endotoxin dose was previously shown to induce lung maturation . The effect of intra-amniotic endotoxin on expression of endothelial proteins was evaluated . Muscularization of the media and collagen deposition in adventitia of small pulmonary arteries was used to assess vascular remodeling . Compared with controls, bronchoalveolar lavage fluid protein content was increased 2 days after intra-amniotic endotoxin exposure . Vascular endothelial growth factor (VEGF) 165 isoform mRNA decreased 2-4 days after intra-amniotic endotoxin . VEGF, VEGF receptor-2, endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, and Tie-2 protein expression in the lung coordinately decreased 1-7 days after intra-amniotic endotoxin . Intra-amniotic endotoxin appeared to selectively decrease eNOS expression in small pulmonary vessels compared with large vessels . Medial smooth muscle hypertrophy and increased adventitial fibrosis were observed 4 and 7 days after intra-amniotic endotoxin . These results demonstrate that, in the preterm lamb lung, antenatal inflammation inhibits endothelial cell protein expression followed by vascular remodeling changes in small pulmonary arteries . Exposure to antenatal inflammation may cause vascular remodeling and contribute to the development of BPD. Thyroid, 2004 Aug, 14(8), 560 - 70 Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function; Sanders J et al.; The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described . Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation . Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation . Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22 . These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity . Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation . The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain . The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction. J Biol Chem, 2004 Oct 22, 279(43), 44966 - 75 Epub 2004 Aug 19. Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli; Jia W et al.; The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments . The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide . PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix . The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane . We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet . We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A . Although expression of the E . coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis . EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane . A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo . Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro . These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet. Am J Physiol Heart Circ Physiol, 2004 Dec, 287(6), H2545 - 54 Epub 2004 Dec. Differing effects of epinephrine, norepinephrine, and vasopressin on survival in a canine model of septic shock; Minneci PC et al.; During sepsis, limited data on the survival effects of vasopressors are available to guide therapy . Therefore, we compared the effects of three vasopressors on survival in a canine septic shock model . Seventy-eight awake dogs infected with differing doses of intraperitoneal Escherichia coli to produce increasing mortality were randomized to receive epinephrine (0.2, 0.8, or 2.0 microg.kg(-1).min(-1)), norepinephrine (0.2, 1.0, or 2.0 microg.kg(-1).min(-1)), vasopressin (0.01 or 0.04 U/min), or placebo in addition to antibiotics and fluids . Serial hemodynamic and biochemical variables were measured . Increasing doses of bacteria caused progressively greater decreases in survival (P <0.06), mean arterial pressure (MAP) (P <0.05), cardiac index (CI) (P <0.02), and ejection fraction (EF) (P=0.02) . The effects of epinephrine on survival were significantly different from those of norepinephrine and vasopressin (P=0.03) . Epinephrine had a harmful effect on survival that was significantly related to drug dose (P=0.02) but not bacterial dose . Norepinephrine and vasopressin had beneficial effects on survival that were similar at all drug and bacteria doses . Compared with concurrent infected controls, epinephrine caused greater decreases in CI, EF, and pH, and greater increases in systemic vascular resistance and serum creatinine than norepinephrine and vasopressin . These epinephrine-induced changes were significantly related to the dose of epinephrine administered . In this study, the effects of vasopressors were independent of severity of infection but dependent on the type and dose of vasopressor used . Epinephrine adversely affected organ function, systemic perfusion, and survival compared with norepinephrine and vasopressin . In the ranges studied, norepinephrine and vasopressin have more favorable risk-benefit profiles than epinephrine during sepsis. Hybrid Hybridomics, 2004 Aug, 23(4), 244 - 9 Development of single-domain recombinant antibodies to reverse transcriptase domain of human hTERT; Huang M et al.; This paper describes the development of single-domain recombinant antibodies against human telomerase core protein . A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E . coli and used to immunize BALB/c mice . The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library . All VHs were cloned into phagemid vectors and displayed to generate 4 x 10(10) phage libraries . The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot . Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay . DNA sequencing proved their mouse VH origin . The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3 . The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition. Lik Sprava, 2004 Apr-May, (3-4), 54 - 8 {Effect of Escherichia coli endotoxin on thymus-dependent activation of T-immunity system and expression of lymphocyte endotoxin receptors in adolescents with reccurent bronchitis}; Slobozhan LI; Endotoxin-dependent direct inhibition of functional activity of T-cell immunity and biological activity of thymus factors was revealed in patients with recurrent chronic bronchitis and with systemic endoxinemia . The findings are considered by authors as an important pathogenetic mechanism inhibiting stable regression of inflammation in bronchopulmonary system in juvenile patients with recurrent chronic bronchitis . The correction of systemic endoxinemia in such patients is pathophysiologicaly substantiated. J Anim Sci, 2004 Aug, 82(8), 2364 - 74 Effect of diet composition on postweaning colibacillosis in piglets; Montagne L et al.; The weaning of piglets is often associated with digestive disorders, particularly diarrhea--postweaning colibacillosis (PWC)--which is caused by infection with enterotoxigenic strains of Escherichia coli . It has been shown previously that a diet for newly weaned pigs based on cooked white rice and animal protein decreases the occurrence of PWC, whereas the addition of carboxymethylcellulose (CMC) to this diet enhances PWC . The aims of the current work were to 1) determine whether substitution of animal protein with plant proteins in the cooked-white-rice diet influenced its protective effects on PWC and 2) confirm that an increase in viscosity of the digesta by adding CMC to the diet favors the development of PWC--with (Exp . 1) or without (Exp . 2) experimental infection of piglets with E . coli . The diets were 1) cooked white rice and animal protein sources (RAP), 2) RAP + CMC added at 40 g of CMC/kg (air-dry basis) of diet, 3) cooked white rice and plant protein sources (RPP), and 4) wheat and plant protein sources (WPP) . Experiments 1 and 2 were conducted using 32 and 24 piglets (eight and six per treatment), respectively . Piglets were weaned at 21 d (d 1), and fed ad libitum until slaughter on d 9 . In Exp . 1, piglets were orally infected with enterotoxigenic E . coli on d 4, 5, 6, and 7 . On d 8 of Exp . 1, the E . coli scores in feces of pigs fed RAP + CMC were higher than with RAP (P < 0.01) . On d 9 after weaning, feces from pigs fed diet RAP were normal or moist, whereas feces from pigs fed RAP + CMC were wet to diarrheic . On d 7 of Exp . 2, pigs fed diets RAP + CMC and WPP had wetter feces than pigs fed diets RAP or RPP (P < 0.05) . On d 8, the E . coli scores in feces were higher (P < 0.01) with pigs fed RAP + CMC than with all other diets . The E . coli scores in the digesta were also higher with pigs fed RAP + CMC, and to a lesser extent with diet WPP, than with pigs fed RAP or RPP (P < 0.01) . The large intestine was heavier in pigs fed diets RPP and WPP, and the digesta were more acidic (P < 0.05) . This study confirmed that diet RAP was protective against PWC, and that substitution of animal proteins with plant protein in a rice-based diet did not diminish its protective effects . The addition of CMC to cooked white rice increased digesta viscosity and enhanced PWC . Consequently, this diet represents a useful model for studying this condition. Water Sci Technol, 2004, 50(1), 33 - 8 Repressive effects of yeast extract on photoreactivation of Escherichia coli; Oguma J et al.; Photoreactivation of Escherichia coli K12 (IFO 3301), in the presence or absence of yeast extract (YE), was investigated after inactivation by low-pressure UV lamp . An endonuclease sensitive site (ESS) assay was used to determine the UV-induced pyrimidine dimers in the genome of E . coli, while a colony-forming ability (CFA) test was also used to examine the survival ratio of E . coli . The YE solution reduced the CFA recovery at a final concentration of 125 mg/L . A dialysis of the YE solution indicated that the YE fraction (with nominal molecular weight >1,000 and <3,500) was effective at repressing the CFA recovery . Interestingly, the repair of ESS was equivalent regardless of the presence of the YE dialysate, while the CFA recovery was significantly repressed in the presence of YE . It was, therefore, suggested that YE components, probably with molecular weights of 1,000-3,500, were effective at repressing the CFA recovery of E . coli without affecting the ESS repair during photoreactivation. EMBO J, 2004 Sep 1, 23(17), 3570 - 82 Epub 2004 Aug 19. Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1; Matsuzawa T et al.; Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease . Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria . Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro . A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation . Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor . These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity . This report reveals for the first time that a pathogen can exploit the host factor GEF-H1. RNA, 2004 Sep, 10(9), 1359 - 65 A stop codon-dependent internal secondary translation initiation region in Escherichia coli rpoS; Subbarayan PR et al.; Sigma S (sigmaS) encoded by rpoS is a stationary phase-specific sigma subunit of the Escherichia coli RNA polymerase holoenzyme . In many E . coli strains, rpoS has an amber stop as codon 33 (rpoSAm), resulting in a 32-amino-acid-long peptide . Nevertheless, suppressor-free rpoSAm strains have functional sigmaS . This led us to hypothesize the presence of an intracistronic secondary translational initiation region (STIR) in the E . coli rpoS gene . Here, we demonstrate that the STIR is functional and is controlled by the upstream amber stop codon 33 . Removal of the primary translational initiation region did not abolish translation from STIR, ruling out translational coupling . Importantly, the functional STIR conferred survival advantage . Taken together, our results reveal a hitherto unknown physiologically significant post-transcriptional process in E . coli rpoSAm strains. Nucleic Acids Res, 2004 Aug 18, 32(15), 4469 - 79 Print 2004. Transcription influences the types of deletion and expansion products in an orientation-dependent manner from GAC*GTC repeats; Mochmann LH et al.; The genetic instability of (GAC*GTC)n (where n = 6-74) was investigated in an Escherichia coli-based plasmid system . Prior work implicated the instability of a (GAC*GTC)5 tract in the cartilage oligomeric matrix protein (COMP) gene to the 4, 6 or 7mers in the etiology of pseudoachondroplasia and multiple epiphyseal dysplasia . The effects of triplet repeat length and orientation were studied after multiple replication cycles in vivo . A transcribed plasmid containing (GAC*GTC)49 repeats led to large deletions (>3 repeats) after propagation in E.coli; however, if transcription was silenced by the LacI(Q) repressor, small expansions and deletions (<3 repeats) predominated the mutation spectra . In contrast, propagation of similar length but opposing orientation (GTC*GAC)53 containing plasmid led to small instabilities that were unaffected by the repression of transcription . Thus, by inhibiting transcription, the genetic instability of (GAC*GTC)49 repeats did not significantly differ from the opposing orientation, (GTC*GAC)53 . We postulate that small instabilities of GAC*GTC repeats are achieved through replicative slippage, whereas large deletion events are found when GAC*GTC repeats are transcribed . Herein, we report the first genetic study on GAC*GTC repeat instability describing two types of mutational patterns that can be partitioned by transcription modulation . Along with prior biophysical data, these results lay the initial groundwork for understanding the genetic processes responsible for triplet repeat mutations in the COMP gene. Nucleic Acids Res, 2004 Aug 18, 32(15), 4462 - 8 Print 2004. Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons; Cruz-Vera LR et al.; Minigenes encoding the peptide Met-Arg-Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria . The codon sequences of the 'mini-ORFs' employed were initiator, combinations of AGA and CGA, and terminator . Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA(Arg4), CGA is recognized by the abundant tRNA(Arg2) . Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNA(Arg4) and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet . The minigenes carrying CGA in the third position were not toxic . Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNA(Arg4) but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome . Consistent with these findings, peptidyl-tRNA(Arg4) was identified to be mainly associated with ribosomes in a stand-by complex . These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome. Nucleic Acids Res, 2004 Aug 18, 32(15), 4429 - 38 Print 2004. The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity; Takaku H et al.; Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA . There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300-400 amino acids, and the other is a long form (tRNase ZL) that contains 800-900 amino acids . Here we investigated whether the short and long forms have different preferences for various RNA substrates . We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA(Arg) and the RNase 65 activity . All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA . We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA . These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL . Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the K(m) values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs. J Bacteriol, 2004 Sep, 186(17), 5968 - 71 Overexpression of gnsA, a multicopy suppressor of the secG null mutation, increases acidic phospholipid contents by inhibiting phosphatidylethanolamine synthesis at low temperatures; Sugai R et al.; GnsA overproduction was previously found to suppress both the secG null mutation and the fabA6 mutation in Escherichia coli by increasing the unsaturated fatty acid contents . We report here that it also increased the acidic phospholipid contents at 20 degrees C but not at 37 degrees C . GnsA overproduction at 20 degrees C specifically inhibited phosphatidylethanolamine synthesis and therefore caused the increase in the proportion of acidic phospholipids. J Bacteriol, 2004 Sep, 186(17), 5899 - 905 The Escherichia coli argU10(Ts) phenotype is caused by a reduction in the cellular level of the argU tRNA for the rare codons AGA and AGG; Sakamoto K et al.; The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C . In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation . An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature . The arginine acceptance is also impaired significantly but to similar extents at the two temperatures . An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated . Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA . At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes . We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase. J Bacteriol, 2004 Sep, 186(17), 5826 - 33 Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli; Grass G et al.; The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli . Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I) . In this report we provide evidence that CueO protects E . coli cells by oxidizing enterobactin, the catechol iron siderophore of E . coli, in the presence of copper . In vitro, a mixture of enterobactin and copper was toxic for E . coli cells, but the addition of purified CueO led to their survival . Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin . In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone . The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate. J Bacteriol, 2004 Sep, 186(17), 5762 - 74 The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp . strain CIB; Lopez Barragan MJ et al.; We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp . strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator . The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway . The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases . The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp . strain CIB extracts . The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica . Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp . strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell. J Bacteriol, 2004 Sep, 186(17), 5730 - 40 Mutations altering the N-terminal receiver domain of NRI (NtrC) That prevent dephosphorylation by the NRII-PII complex in Escherichia coli; Pioszak AA et al.; The phosphorylated form of NRI is the transcriptional activator of nitrogen-regulated genes in Escherichia coli . NRI approximately P displays a slow autophosphatase activity and is rapidly dephosphorylated by the complex of the NRII and PII signal transduction proteins . Here we describe the isolation of two mutations, causing the alterations DeltaD10 and K104Q in the receiver domain of NRI, that were selected as conferring resistance to dephosphorylation by the NRII-PII complex . The mutations, which alter highly conserved residues near the D54 site of phosphorylation in the NRI receiver domain, resulted in elevated expression of nitrogen-regulated genes under nitrogen-rich conditions . The altered NRI receiver domains were phosphorylated by NRII in vitro but were defective in dephosphorylation . The DeltaD10 receiver domain retained normal autophosphatase activity but was resistant to dephosphorylation by the NRII-PII complex . The K104Q receiver domain lacked both the autophosphatase activity and the ability to be dephosphorylated by the NRII-PII complex . The properties of these altered proteins are consistent with the hypothesis that the NRII-PII complex is not a true phosphatase but rather collaborates with NRI approximately P to bring about its dephosphorylation. J Bacteriol, 2004 Sep, 186(17), 5621 - 8 Slr1293 in Synechocystis sp . strain PCC 6803 Is the C-3',4' desaturase (CrtD) involved in myxoxanthophyll biosynthesis; Mohamed HE et al.; When grown at high light intensity, more than a quarter of the total carotenoids in the unicellular cyanobacterium Synechocystis consists of myxoxanthophyll, a polar carotenoid glycoside . The biosynthetic pathway of myxoxanthophyll is unknown but is presumed to involve a number of enzymes, including a C-3',4' desaturase required to add one double bond to generate 11 conjugated double bonds in the monocyclic myxoxanthophyll . A candidate for this desaturase is Slr1293, which was identified by genome similarity searching . To determine whether Slr1293 is a desaturase recognizing neurosporene and lycopene, slr1293 was expressed in Escherichia coli strains accumulating neurosporene or lycopene . Confirming such a desaturase function for Slr1293, these E . coli strains accumulated 3',4'-didehydroneurosporene and 3',4'-didehydrolycopene, respectively . Indeed, deletion of slr1293 in Synechocystis provides further evidence that Slr1293 is a desaturase recognizing neurosporene: In the slr1293 deletion mutant, neurosporene was found to accumulate and was further processed to produce neurosporene glycoside . Neurosporene hereby becomes a primary candidate to be the branch point molecule between carotene and myxoxanthophyll biosynthesis in this cyanobacterium . The slr1293 gene was concluded to encode a C-3',4' desaturase that is essential for myxoxanthophyll biosynthesis, and thus it was designated as crtD . Furthermore, as Slr1293 appears to recognize neurosporene and to catalyze the first committed step on the myxoxanthophyll biosynthesis pathway, Slr1293 plays a pivotal role in directing a portion of the precursor pool for carotenoid biosynthesis toward myxoxanthophyll biosynthesis in Synechocystis sp . strain PCC 6803. Eur J Biochem, 2004 Sep, 271(17), 3539 - 46 Comparison of native and recombinant chlorite dismutase from Ideonella dechloratans; Danielsson Thorell H et al.; A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme . Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis . Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein . Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein . Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low pK(a) . The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination . These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions. Eur J Biochem, 2004 Sep, 271(17), 3512 - 22 Structural characterization of the human Nogo-A functional domains . Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration; Li M et al.; The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury . Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration . So far, no structural information is available for Nogo-A or any of its structural domains . We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli . CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble . Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration . Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment . The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol . The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands . Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration. Eur J Biochem, 2004 Sep, 271(17), 3488 - 502 An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum . Cloning and biochemical characterization of the enzyme; Tripathi AK et al.; Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites . The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli . The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate . Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD . The enzyme exhibited strict substrate and cofactor specificity . The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts . A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets . The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs . Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also . Treatment of a synchronized culture of P . falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged . Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites . Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs. J Med Chem, 2004 Aug 26, 47(18), 4373 - 90 Functionality maps of the ATP binding site of DNA gyrase B: generation of a consensus model of ligand binding; Schechner M et al.; The Multiple Copy Simultaneous Search method (MCSS) was used to construct consensus functionality maps for functional group binding in the ATP binding site of DNA gyrase B . To account for the conformational flexibility of the protein active site, which involves small side chain fluctuations as well as large-scale loop motions, the calculations were done for three different conformations of the 24 kDa subdomain of DNA gyrase B . A postprocessing procedure that employs a continuum dielectric model to include solvent effects was used to calculate the binding free energy for every functional group . These results were ranked according to their affinity for DNA gyrase B and clustered using a new procedure based on van der Waals contacts that is better adapted for cases where multiple conformations are being considered . A total of 23 different functional groups were tested . The results gave consensus maps that indicate those functional group binding sites that are insensitive to the specific protein conformation . The maps also demonstrate that functional groups other than those found in the known ligands may bind competitively in the binding sites of known ligands . This suggests numerous scaffolds that can be used in the development of new ligands for the ATP and coumarinic binding sites in DNA gyrase B . Finally, the calculations show the existence of alternative binding sites near the known binding sites that could be targeted in the rational design for new inhibitors. J Neurol, 2004 Aug, 251(8), 1006 - 11 The significance of titin antibodies in myasthenia gravis--correlation with thymoma and severity of myasthenia gravis; Chen XJ et al.; Myasthenia gravis (MG) is caused by autoantibodies to acetylcholine receptors (AChR) . Non-AChR muscle autoantibodies, such as titin antibodies, are present in sera of many MG patients . To study the correlation between titin antibodies and the features of MG, the cDNA segment encoding MGT-30 was amplified and sequenced . The cloned MGT-30 cDNA was expressed in vector pET-30a, and then transfected into E.coli . BL21 . We examined titin antibodies in sera of 265 normal subjects, 154 MG patients with different thymic pathology and 48 patients with other neurological diseases . Titin antibodies occurred more frequently in MGT, especially in MG with epithelial predominant-thymoma, and were correlated with the severity of disease . The levels of titin antibodies were reduced 6 months after thymectomy . The specificity of titin antibodies for the detection of thymoma was higher than that of CT examination in MG with thymoma . These results suggest that titin antibodies could be useful in both diagnosis and follow-up of MG patients with thymoma. Shock, 2004 Sep, 22(3), 248 - 53 Age and caloric restriction diets are confounding factors that modify the response to lipopolysaccharide by peritoneal macrophages in C57BL/6 mice; Vega VL et al.; Aging is the result of several detrimental changes that lead to a decrease in homeostasis, an increase in the incidence of degenerative diseases, and death . A caloric-restricted diet (CR), which consists of a significant reduction in calorie intake (40%) without malnutrition, has been shown to delay the onset of age-related diseases and pathologies and to extend life span . The aims of this study were to assess the effects of aging and CR on lipopolysaccharide (LPS)-dependant cytokine production by peritoneal macrophages (PMphis) . Resident naive PMphis were isolated from 2- to 24-month-old male C57BL/6 mice and were stimulated with Escherichia coli LPS (100 ng/mL) for 1 to 5 h in culture conditions . A linear decrease in the production of LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 10 was observed with age . LPS-induced IL-6 and IL-1beta levels were also reduced with age, but in a nonlinear fashion . Expression of CD14, the major receptor for LPS, on the PMphi surface was also observed to decline with age . Moreover, TNF-alpha production by PMphis was reduced in mice undergoing the two different CR diets of limited daily feeding and intermittent fasting, as compared with ad libitum-fed mice . The results of this study add the new variables age and diet to the paradigm proposing that the response to LPS is modulated by multiple components, including genetic background and sex. J Biol Chem, 2004 Oct 22, 279(43), 45004 - 12 Epub 2004 Aug 16. Calmodulin mediates Ca2+ sensitivity of sodium channels; Kim J et al.; Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus . Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood . Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels . In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini . Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2 . In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex . Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone . Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels . The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex . In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death . Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function. J Biol Chem, 2004 Nov 5, 279(45), 47192 - 200 Epub 2004 Aug 16. Structural basis for the inhibitory role of tomosyn in exocytosis; Pobbati AV et al.; Upon Ca2+ influx synaptic vesicles fuse with the plasma membrane and release their neurotransmitter cargo into the synaptic cleft . Key players during this process are the Q-SNAREs syntaxin 1a and SNAP-25 and the R-SNARE synaptobrevin 2 . It is thought that these membrane proteins gradually assemble into a tight trans-SNARE complex between vesicular and plasma membrane, ultimately leading to membrane fusion . Tomosyn is a soluble protein of 130 kDa that contains a COOH-terminal R-SNARE motif but lacks a transmembrane anchor . Its R-SNARE motif forms a stable core SNARE complex with syntaxin 1a and SNAP-25 . Here we present the crystal structure of this core tomosyn SNARE complex at 2.0-A resolution . It consists of a four-helical bundle very similar to that of the SNARE complex containing synaptobrevin . Most differences are found on the surface, where they prevented tight binding of complexin . Both complexes form with similar rates as assessed by CD spectroscopy . In addition, synaptobrevin cannot displace the tomosyn helix from the tight complex and vice versa, indicating that both SNARE complexes represent end products . Moreover, data bank searches revealed that the R-SNARE motif of tomosyn is highly conserved throughout all eukaryotic kingdoms . This suggests that the formation of a tight SNARE complex is important for the function of tomosyn. J Biol Chem, 2004 Nov 5, 279(45), 47264 - 71 Epub 2004 Aug 16. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres; Ye JZ et al.; Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2 . The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis . The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response . A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres . Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes . Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex . Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction . Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2 . Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins . This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends . The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection. Biophys J, 2004 Nov, 87(5), 2942 - 53 Epub 2004 Aug 17. OmpT: molecular dynamics simulations of an outer membrane enzyme; Baaden M et al.; Five molecular dynamics simulations (total duration >25 ns) have been performed on the Escherichia coli outer membrane protease OmpT embedded in a dimyristoylphosphatidylcholine lipid bilayer . Globally the protein is conformationally stable . Some degree of tilt of the beta-barrel is observed relative to the bilayer plane . The greatest degree of conformational flexibility is seen in the extracellular loops . A complex network of fluctuating H-bonds is formed between the active site residues, such that the Asp210-His212 interaction is maintained throughout, whereas His212 and Asp83 are often bridged by a water molecule . This supports a catalytic mechanism whereby Asp83 and His212 bind a water molecule that attacks the peptide carbonyl . A configuration yielded by docking calculations of OmpT simulation snapshots and a model substrate peptide Ala-Arg-Arg-Ala was used as the starting point for an extended Huckel calculation on the docked peptide . These placed the lowest unoccupied molecular orbital mainly on the carbon atom of the central C=O in the scissile peptide bond, thus favoring attack on the central peptide by the water held by residues Asp83 and His212 . The trajectories of water molecules reveal exchange of waters between the intracellular face of the membrane and the interior of the barrel but no exchange at the extracellular mouth . This suggests that the pore-like region in the center of OmpT may enable access of water to the active site from below . The simulations appear to reveal the presence of specific lipid interaction sites on the surface of the OmpT barrel . This reveals the ability of extended MD simulations to provide meaningful information on protein-lipid interactions. Vaccine, 2004 Sep 9, 22(27-28), 3751 - 61 Non-toxic Stx derivatives from Escherichia coli possess adjuvant activity for mucosal immunity; Ohmura-Hoshino M et al.; Both B subunit of Shiga toxin 1 (Stx1-B), which mediates the binding of toxin to the membrane, and mutant Stx1 (mStx1), which is a non-toxic double-mutated Stx1 harboring double amino acid substitutions in the A subunit, possess potent mucosal adjuvant activity . Nasal immunization of mice with ovalbumin (OVA) plus the Stx1-B or mStx1 induced OVA-specific serum IgG and mucosal IgA responses . IgG subclass analysis revealed that mStx1 and Stx1-B as mucosal adjuvants supported Ag-specific IgG1 followed by IgG2b Abs . The co-administration of either mStx1 or Stx1-B with OVA enhanced the production of IL-4, IL-5, IL-6 and IL-10 with low IFN-gamma, by OVA-specific CD4+ T cells . To better elucidate the mechanisms underlying mStx1's and Stx1-B's adjuvant activity, we next sought to examine whether or not dendritic cells (DC) residing in the nasopharyngeal-associated lymphoreticular tissue (NALT) were activated by nasal administration of Stx1-B or mStx1 . We found that mice nasally administered with Stx1-B or mStx1 showed an up-regulation in the expression of CD80, CD86 and especially CD40 on NALT DCs . Taken together, these results suggest that non-toxic Stx derivatives could be effective mucosal adjuvants for the induction of Th2-type, CD4+ T cell mediated, antigen-specific mucosal IgA and systemic IgG Ab responses, and that they likely owe their adjuvant activity to the up-regulation of co-stimulatory molecules including CD80, CD86 and CD40 on NALT DCs. Vaccine, 2004 Sep 9, 22(27-28), 3727 - 37 Evaluation of immune responses elicited in mice against a recombinant malaria vaccine based on Plasmodium vivax Duffy binding protein; Yazdani SS et al.; Plasmodium vivax Duffy binding protein (PvDBP) binds the Duffy blood group antigen as the obligate receptor for erythrocyte invasion . We have tested in mice the immunogenicity of recombinant P . vivax region II (PvRII), the receptor-binding domain of PvDBP, formulated with five adjuvants, namely, Montanide ISA720, AS02A, alum, QS21 and MF59 . All the formulations elicited high titer antibodies, with Montanide ISA720 and AS02A yielding the highest titers followed by MF59, QS21 and alum . Sera raised against PvRII formulated with AS02A and Montanide ISA720 followed by alum were most effective at blocking PvRII binding to erythrocytes in a functional assay . Analysis of cellular immune responses indicated that all adjuvant groups induced significant interferon-gamma, with alum being the highest interferon-gamma inducer . These results suggest that recombinant PvRII formulated with human compatible adjuvants is immunogenic in small animal models and that Montanide ISA720, AS02A and alum perform better than MF59 and QS21 in terms of their ability to elicit high titer binding inhibitory antibodies. Vaccine, 2004 Sep 9, 22(27-28), 3713 - 21 A recombinant luteinising-hormone-releasing-hormone immunogen bioeffective in causing prostatic atrophy; Talwar GP et al.; Previous studies with a semi-synthetic vaccine indicated the utility of immunization against luteinising-hormone-releasing-hormone (LHRH) in prostate cancers . To overcome the limitations of the previous vaccine, which caused carrier induced suppression of antibody response on repeated immunizations and was costly to synthesize, two recombinant vaccines were designed, in which diptheria or tetanus toxoid used as carriers were replaced by 4-5 T non B peptides . The paper reports the immunogenecity, efficacy and safety of these multimer vaccines in rats, a homologous experimental animal . All animals generated anti-LHRH antibodies, which caused the decline of testosterone to castration levels at and above 0.15 OD units of antibody titres . The prostate was significantly atrophied in all animals immunized with these vaccines. Vaccine, 2004 Sep 9, 22(27-28), 3595 - 602 Mucosal immunisation of murine neonates using whole cell and acellular Pertussis vaccines; Hale C et al.; Groups of neonatal mice were immunised with different mucosal vaccines based on acellular (Pertactin antigen) or whole cell (inactivated Bordetella pertussis with Diphtheria and Tetanus toxoid) Pertussis vaccines, using Escherichia coli heat-labile enterotoxin (LT) as a mucosal adjuvant . Neonatal mice tolerated mucosal vaccination well and a significant cellular infiltrate was detected in the lungs of mice receiving mucosal vaccines compared to PBS controls . This infiltrate included B lymphocytes, gammadelta T cells and interferon-gamma producing T cells . Neonatal mice, in contrast to adult mice, responded poorly in terms of the production of serum antibody to Pertussis antigens delivered mucosally, although they were able to mount an anti-Tetanus response to those vaccines harbouring Tetanus toxoid and whole cell Pertussis antigen . Neonatal mice immunised with Pertactin or whole cell Pertussis antigen together with LT were protected against virulent B . pertussis challenge. Cell, 2004 Aug 20, 118(4), 465 - 75 Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor; Spahn CM et al.; Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs . An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA . Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome . The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites . Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome . CrPV-like IRES elements seem to act as RNA-based translation factors. Acta Anaesthesiol Scand, 2004 Sep, 48(8), 935 - 43 Dobutamine compensates deleterious hemodynamic and metabolic effects of vasopressin in the splanchnic region in endotoxin shock; Martikainen TJ et al.; BACKGROUND: Vasopressin is a potent vasopressor in septic shock, but it may impair splanchnic perfusion . We compared the effects of vasopressin alone and in combination with dobutamine on systemic and splanchnic circulation and metabolism in porcine endotoxin shock . METHODS: Twelve pigs were randomized to receive either vasopressin (VASO, n = 6) or vasopressin in combination with dobutamine (DOBU, n = 6) during endotoxin shock (E . coli endotoxin infusion) . Endotoxin infusion rate was increased to induce hypotension after which vasoactive drugs were started . We aimed to keep systemic mean arterial pressure (MAP) >70 mmHg by vasopressin; the goal of dobutamine infusion was to prevent decrease in cardiac output often associated with vasopressin infusion . Regional blood flows, oxygen delivery and consumption, arterial and regional lactate concentrations were measured . RESULTS: Mean arterial pressure >70 mmHg was achieved in both the VASO and DOBU groups . After the primary decrease of cardiac output by vasopressin, systemic blood flow remained stable in vasopressin-treated animals . However, vasopressin as a monotherapy decreased portal venous blood flow . This was prevented by dobutamine . Vasopressin also induced splanchnic lactate release and arterial hyperlactatemia, which were not observed when dobutamine was combined with vasopressin . CONCLUSION: Dobutamine prevents adverse hemodynamic and metabolic effects of vasopressin in septic shock. J Am Chem Soc, 2004 Aug 25, 126(33), 10296 - 305 Oxyanion selectivity in sulfate and molybdate transport proteins: an ab initio/CDM study; Dudev T et al.; A striking feature of sulfate (SO(4)(2-)) and molybdate (MoO(4)(2-)) transport proteins, such as SBP and ModA, which specifically bind SO(4)(2-) and MoO(4)(2-), respectively, is their ability to discriminate very similar anions with the same net charge, geometry, and hydrogen-bonding properties . Here, we determine to what extent (1) oxyanion-solvent interactions, (2) oxyanion-amino acid interactions, and (3) the anion-binding pocket sizes of the cognate protein contribute to the anion selectivity process in SO(4)(2-) and MoO(4)(2-) transport proteins by computing the free energies for replacing SO(4)(2-) with MoO(4)(2)(-)/WO(4)(2-) in model SO(4)(2-)-binding sites of varying degrees of solvent exposure using a combined quantum mechanical/continuum dielectric approach . The calculations reveal that MoO(4)(2-) transport proteins, such as ModA, specifically bind MoO(4)(2-)/WO(4)(2-) but not SO(4)(2-), mainly because the desolvation penalty of MoO(4)(2-)/WO(4)(2-) is significantly less than that of SO(4)(2-) and, to a lesser extent, because the large and rigid cavity in these proteins attenuates ligand interactions with SO(4)(2-), as compared to MoO(4)(2-) . On the other hand, SO(4)(2-) transport proteins prefer SO(4)(2-) to MoO(4)(2-)/WO(4)(2-) because the small anion-binding pocket characteristic of these proteins inhibits binding of the larger MoO(4)(2-) and WO(4)(2-) anions . The calculations also help to explain the absence of positively charged Lys/Arg side chains in the anion-binding sites of SBP and ModA . During evolution, these transport proteins may have excluded cationic ligands from their binding sites because, on one hand, Lys/Arg do not contribute to the selectivity of the binding pocket and, on the other, they substantially stabilize the complex between the oxyanion and protein ligands, which in turn would prohibit the rapid release of the bound oxyanion at a certain stage during the transport process. J Mol Biol, 2004 Sep 3, 342(1), 57 - 71 Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease; Reuven NB et al.; The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins . We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction . Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles . Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end . ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases . Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end . In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8 . This study further illustrates the complexity of the multi-functional ICP8 protein. J Mol Biol, 2004 Sep 3, 342(1), 1 - 7 A role for the interdomain linker region of the Escherichia coli CytR regulator in repression complex formation; Kallipolitis BH et al.; Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli . The formation of the complexes is mediated by protein-DNA interactions and protein-protein interactions between the two regulators . Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR . When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp . However, in the presence of CRP, CytR recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp . A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation . These observations suggest that CRP alters the DNA-binding mode of CytR . Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis . Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation . Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated . The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors. Methods Enzymol, 2004, 389, 320 - 38 Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1; Willard FS et al.; The model organism Arabidopsis thaliana contains a restricted set of heterotrimeric G-protein subunits, with only one canonical Galpha subunit (AtGPA1), one Gbeta subunit (AtAGB1), and two Ggamma subunits (AtAGG1 and AtGG2) identified . We have identified a novel additional component of heterotrimeric G-protein signaling in the A . thaliana genome, regulator of G-protein signaling-1 (AtRGS1) . This protein has the predicted topology and structure of a G-protein-coupled receptor in that it contains seven transmembrane domains, but AtRGS1 also contains a unique C-terminal extension, namely a regulator of G-protein signaling domain (RGS box) . This article describes methods for the purification and in vitro functional analysis of the RGS box of AtRGS1. Comp Biochem Physiol C Toxicol Pharmacol, 2004 May, 138(1), 23 - 32 The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases; Garcia LT et al.; Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms . Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries . Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively . Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro . This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA) . The heterologous proteins were produced in E . coli, an in vivo expression system that does not make post-translational modifications . The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH . However, they preserved their proteolytic activity on fibrinogen and fibronectin . It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications. Res Microbiol, 2004 Sep, 155(7), 553 - 8 Replication of Mu prophages lacking the central strong gyrase site; Pato ML; Replication of Mu prophages lacking the central strong gyrase site (SGS) is severely slowed . To study details of the replication of these prophages, an assay was developed for determining the rate and extent of introduction of nicks at the 3'-ends of a Mu prophage, an early step in Mu replicative transposition . The maximal level of end-nicking of a prophage with the SGS, about 70-90% depending upon the host strain, was achieved within about 15 min after induction, whereas at that time less than 5% nicking was observed with a prophage lacking the SGS . The amount of nicking at the end of the SGS(-) prophage increased with time, and approx . 30% nicking of the SGS(-) prophage was achieved by 60 min post-induction . Nicking kinetics were identical at each end of the prophages, and no nicking was observed at the 5'-ends of the prophages, verifying in vivo the earlier results with in vitro systems . To determine if prophage location affects the kinetics of replication, we examined prophages at numerous chromosomal locations . SGS(+) prophages at different chromosomal locations showed essentially identical replication kinetics . SGS(-) prophages showed a range of delays in replication and host lysis . A gradient of delays was apparent, with prophages further from the chromosomal origin of replication, oriC, showing longer delays than ones nearer to oriC . However, there were also exceptions to this overall gradient . Possible explanations for the differences in the delays observed with SGS(-) prophages are discussed. Curr Opin Struct Biol, 2004 Aug, 14(4), 405 - 12 The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily; Lemieux MJ et al.; The major facilitator superfamily represents the largest group of secondary active membrane transporters in the cell . The 3.3A resolution structure of a member of this protein superfamily, the glycerol-3-phosphate transporter from the Escherichia coli inner membrane, reveals two domains connected by a long central loop . These N- and C-terminal domains, each containing a six-helix bundle, are related by pseudo-twofold symmetry . A substrate translocation pore is located between the two domains and is open to the cytoplasm . Two arginines at the closed end of the pore comprise the substrate-binding site . Biochemical experiments show that, upon substrate binding, the protein adopts a more compact conformation . The crystal structure suggests that the transporter operates through a single binding site, alternating access mechanism via a rocker-switch type of movement of the N- and C-terminal domains . The structure and mechanism of the glycerol-3-phosphate transporter form a paradigm for other members of the major facilitator superfamily. Arch Biochem Biophys, 2004 Sep 15, 429(2), 224 - 30 Catalytic properties of the PepQ prolidase from Escherichia coli; Park MS et al.; The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus . The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity . The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively . The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters . A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ . The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1) . The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1) . The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters . The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX. Biochem Biophys Res Commun, 2004 Sep 10, 322(1), 126 - 32 Role of H164 in a unique dye-decolorizing heme peroxidase DyP; Sugano Y et al.; The expression system of a unique dye-decolorizing peroxidase DyP in Escherichia coli has been constructed . The molecular mass of the expressed DyP (eDyP) is 47kDa, indicating no any modification with saccharides . The characteristics of eDyP were almost the same as those of native DyP from a fungus Thanatephorus cucumeris Dec 1 and recombinant DyP with Aspergillus oryzae except thermostability . As H164 was suggested to be the proximal histidine based on the preliminary X-ray crystallographic analysis of DyP, the site-directed mutations H164A and H166A (residue near H164) were introduced into the gene encoding DyP . The specific activity and RZ value of the purified H164A were 1.52U/mg and 0.11, respectively, which were 99.8% and 95% lower than those of eDyP, respectively . On the contrary, those of H166A were not different from those of eDyP . Therefore, H164 was confirmed to be the proximal histidine. Biochem Biophys Res Commun, 2004 Sep 10, 322(1), 1 - 8 Mouse spermine oxidase: a model of the catalytic cycle and its inhibition by N,N1-bis(2,3-butadienyl)-1,4-butanediamine; Bellelli A et al.; Spermine oxidase (SMO) is a recently described flavoenzyme belonging to the class of polyamine oxidases (PAOs) and participating in the polyamine metabolism in animal cells . In this paper we describe the expression, purification, and characterization of the catalytic properties of a recombinant mouse SMO (mSMO) . The purified enzyme has absorbance peaks at 457nm (epsilon=11mM(-1)cm(-1)) and 378nm, shows a molecular mass of approximately 63kDa, and has K(m) and k(cat) values of 170microM and 4.8s(-1), using spermine as substrate; it is unable to oxidize other free or acetylated polyamines . The mechanism-based PAO inhibitor N,N(1)-bis(2,3-butadienyl)-1,4-butanediamine (MDL72,527) acts as a competitive inhibitor of mSMO, with an apparent dissociation constant K(i)=63microM . If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100microM MDL72,527 . The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme. Int J Parasitol, 2004 Aug, 34(9), 1037 - 45 Ac-SAA-1, an immunodominant 16 kDa surface-associated antigen of infective larvae and adults of Ancylostoma caninum; Zhan B et al.; A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera . The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A . caninum L(3) . Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids . Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum . A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages . In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms . Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant . Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro . Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen. J Mol Biol, 2004 Jul 30, 341(1), 241 - 53 Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry; Rojsajjakul T et al.; The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively . The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form . To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS . The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry . Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection . Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured . The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate . A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS. J Mol Biol, 2004 Jul 30, 341(1), 227 - 39 Structural analyses of peptide release factor 1 from Thermotoga maritima reveal domain flexibility required for its interaction with the ribosome; Shin DH et al.; We have determined the crystal structure of peptide chain release factor 1 (RF1) from Thermotoga maritima (gi 4981173) at 2.65 Angstrom resolution by selenomethionine single-wavelength anomalous dispersion (SAD) techniques . RF1 is a protein that recognizes stop codons and promotes the release of a nascent polypeptide from tRNA on the ribosome . Selenomethionine-labeled RF1 crystallized in space group P2(1) with three monomers per asymmetric unit . It has approximate dimensions of 75 Angstrom x 70 Angstrom x 45 Angstrom and is composed of four domains . The overall fold of each RF1 domain shows almost the same topology with Escherichia coli RF2, except that the RF1 N-terminal domain is shorter and the C-terminal domain is longer than that of RF2 . The N-terminal domain of RF1 indicates a rigid-body movement relative to that of RF2 with an angle of approximately 90 degrees . Including these features, RF1 has a tripeptide anticodon PVT motif instead of the SPF motif of RF2, which confers the specificity towards the stop codons . The analyses of three molecules in the asymmetric unit and comparison with RF2 revealed the presence of dynamic movement of domains I and III, which are anchored to the central domain by hinge loops . The crystal structure of RF1 elucidates the intrinsic property of this family of having large domain movements for proper function with the ribosome. J Mol Biol, 2004 Jul 30, 341(1), 185 - 98 The structure and biochemical properties of the human spliceosomal protein U1C; Muto Y et al.; The spliceosomal U1C protein is critical to the initiation and regulation of precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear ribonucleoprotein particle (snRNP) . We have produced full-length and 61 residue constructs of human U1C in soluble form in Escherichia coli . Atomic absorption spectroscopy and mass spectrometry show that both constructs contain one Zn atom and are monomeric . Gelmobility-shift assays showed that one molecule of recombinant U1C, either full-length or 61 residue construct, can be incorporated into the U1 snRNP core domain in the presence of U1 70k . This result is in perfect agreement with the previous experiment with U1C isolated from the HeLa U1 snRNP showing that the recombinant U1C is functionally active . We have determined the solution structure of the N-terminal 61 residue construct of U1C by NMR . A Cys(2)His(2)-type zinc finger, distinct from the TFIIIA-type, is extended at its C terminus by two additional helices . The two Zn-coordinating histidine residues are separated by a five residue loop . The conserved basic residues in the first two helices and the intervening loop may be involved in RNA binding . The opposite beta-sheet face with two surface-exposed Tyr residues may be involved in protein contacts . Both the full-length and 61 residue constructs of human U1C fail to bind RNA containing the 5' splice site sequence, in contrast to what has been reported for the Saccharomyces cerevisiae orthologue. J Mol Biol, 2004 Jul 30, 341(1), 37 - 54 Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces; Schubert M et al.; S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G . Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy . The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core . Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing . Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range . The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E . Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains . This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain . Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al . in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme. J Microsc, 2004 Sep, 215(Pt 3), 297 - 301 Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica; Ellis JS et al.; Multivalent ions in solution are known to mediate attraction between two like-charged molecules . Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid . Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate . Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions . Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions . Our data depict E . coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods . These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions . We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule. Biochemistry, 2004 Aug 24, 43(33), 10800 - 8 6-S-cysteinyl flavin mononucleotide-containing histamine dehydrogenase from Nocardioides simplex: molecular cloning, sequencing, overexpression, and characterization of redox centers of enzyme; Fujieda N et al.; Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine . The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli . The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands . The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer . The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur . These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole . The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase. J Biol Inorg Chem, 2004 Oct, 9(7), 818 - 27 Epub 2004 Aug 11. Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants; implications for the O2 activation mechanism; Lu S et al.; The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O(2) to produce a tyrosine radical (Y122.) . In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent {Fe(III)-Fe(IV)} state called compound X, but in related diiron proteins such as methane monooxygenase, Delta(9)-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized . Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate (mu-1,2-peroxo)diiron species to accumulate . To investigate the possible involvement of a bridging peroxo species within the O(2) activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/D84E by using vibrational spectroscopy . Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled {(FeNO)(7)}(2) center . In the wt and variant proteins, we also observe that both irons bind one NO to form a (FeNO)(7) dimer where both Fe-N-O units share a common vibrational signature . In the wt protein, nu(Fe-NO), delta(Fe-N-O), and nu(N-O) bands are observed at 445, 434 and 1742 cm(-1), respectively, while in the variant proteins the nu(Fe-NO) and delta(Fe-N-O) bands are observed approximately 10 cm(-1) higher and the nu(N-O) approximately 10 cm(-1) lower at 1735 cm(-1) . These results demonstrate that all three proteins accommodate fully symmetric {(FeNO)(7)}(2) species with two identical Fe-N-O units . The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O(2) activation process in R2-wt. Nat Struct Mol Biol, 2004 Sep, 11(9), 830 - 7 Epub 2004 Aug 15. An unstructured initiation site is required for efficient proteasome-mediated degradation; Prakash S et al.; The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus . Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains . The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins . We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate . The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially . These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently . The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region. Nucleic Acids Res, 2004 Aug 13, 32(14), 4332 - 9 Print 2004. N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites; Ma H et al.; Repair of most modified and mispaired bases in the genome is initiated by DNA glycosylases, which bind to their respective targets and cleave the N-glycosyl bond to initiate base excision repair (BER) . The mammalian homolog of the Escherichia coli MutY DNA glycosylase (MYH) cleaves adenine residues paired with either oxidized or non-modified guanines . MYH is crucial for the avoidance of mutations resulting from oxidative DNA damage . Multiple N-terminal splice variants of MYH exist in mammalian cells and it is likely that different variants result in the production of enzymes with altered properties . To investigate whether modifications in the N-terminus are consequential to MYH function, we overexpressed intact and N-terminal-deletion rat MYH proteins and examined their activities . We found that deletion of 75 amino acids, which perturbs the catalytic core that is conserved with E.coli MutY, abolished excision activity . In contrast, deletions limited to the extended mammalian N-terminal domain, differentially influenced steady-state excision rates . Notably, deletion of 50 amino acids resulted in an enzyme with a significantly lower K(m) favoring formation of excision products with 3'-OH termini . Our findings suggest that MYH isoforms divergent in the N-terminus influence excision rates and processing of abasic sites. Plant Physiol, 2004 Aug, 135(4), 1976 - 83 Epub 2004 Aug 13. Herbivore-induced defense response in a model legume . Two-spotted spider mites induce emission of (E)-beta-ocimene and transcript accumulation of (E)-beta-ocimene synthase in Lotus japonicus; Arimura G et al.; Indirect defense of plants against herbivores often involves the induced emission of volatile infochemicals including terpenoids that attract natural enemies of the herbivores . We report the isolation and characterization of a terpene synthase cDNA (LjEbetaOS) from a model legume, Lotus japonicus . Recombinant LjEbetaOS enzyme produced (E)-beta-ocimene (98%) and its Z-isomer (2%) . Transcripts of LjEbetaOS were induced in L . japonicus plants infested with two-spotted spider mites (Tetranychus urticae), coinciding with increasing emissions of (E)-beta-ocimene as well as other volatiles, (Z)-3-hexenyl acetate and (E)-4,8-dimethyl-1,3,7-nonatriene, by the infested plants . We suggest that LjEbetaOS is involved in the herbivore-induced indirect defense response of spider mite-infested L . japonicus via de novo formation and emission (E)-beta-ocimene . Mechanical wounding of the leaves or application of alamethicin (ALA), a potent fungal elicitor of plant volatile emission, also induced transiently increased levels of LjEbetaOS transcripts in L . japonicus . However, wounding or ALA did not result in elevated release of (E)-beta-ocimene . Differences in volatile emissions after herbivory, mechanical wounding, or treatment with ALA suggest that neither a single mechanical wounding event nor ALA simulate the effect of herbivore activity and indicate that herbivore-induced emission of (E)-beta-ocimene in L . japonicus involves control mechanisms in addition to up-regulation of LjEbetaOS transcripts. J Biol Chem, 2004 Oct 22, 279(43), 44613 - 20 Epub 2004 Aug 13. Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis; Fujii I et al.; The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases . We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus . To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene . The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid . Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity . To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined . The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p . These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p . From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate . These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain . Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis . An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been report