J Biol Chem, 2004 Jan 30, 279(5), 3169 - 79 Epub 2003 Nov 12. Properties of recombinant human cytosolic sialidase HsNEU2 . The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules; Tringali C et al.; Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied . HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3 . alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant . The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range . HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase . HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations . With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively . Remarkably, GM1 and GM2 were recognized only as monomers . HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion . These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides . The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions. Bioorg Chem, 2003 Dec, 31(6), 453 - 63 The effect of three-dimensional structure on the solid state isotope exchange of hydrogen in polypeptides with spillover hydrogen; Zolotarev YA et al.; The effect of the three-dimensional structure of polypeptides and proteins on their ability to undergo isotopic exchange under the action of spillover hydrogen (SH) in the high temperature solid state catalytic isotope exchange reaction (HSCIE) was theoretically and experimentally studied . The HSCIE reaction in the beta-galactosidase protein from Thermoanaerobacter ethanolicus (83kDa) was studied . The influence of the beta-galactosidase structure on isotopic exchange as peptide fragments with spillover tritium was studied . The most accessible peptide fragment, which does not contribute to alpha-helix and beta-strand formations (KEMQKE215-220), had the largest relative reactivity . The one located in the contact area between the subunits (YLRDSE417-422) showed the smallest relative reactivity . The relative reactivities of these peptides differ more than 150 times . Data collected during a study devoted to the HSCIE reaction of the beta-galactosidase protein indicate that the HSCIE reaction might be employed for acquiring information about their three-dimensional structure and protein-protein interactions . The results of ab initio calculations have shown that alpha-helix formation in polypeptides decreases the reactivity in HSCIE . Hydrogen exchange in the alpha-helical fragment Trp1-Leu8 of zervamycin IIB was also analyzed using theoretical methods . It was shown by ab initio quantum-chemical calculations that the high degree of substitution of C(alpha)H for tritium in Gln3 might be associated with the participation of electron donor O and N atoms in transition state stabilization in the HSCIE reaction. J Biomed Mater Res A, 2003 Dec 1, 67(3), 868 - 75 Metal ions alter lipopolysaccharide-induced NF kappa B binding in monocytes; Lewis JB et al.; Metals are components of a variety of biomaterials used in orthopedic and dental appliances; however, their biocompatibility with the surrounding tissues is not completely understood . Monocytes are important immune cells that respond to inflammatory stimuli by rapidly producing a variety of inflammatory proteins . Regulation of this response often involves activation of the transcription factor NF kappa B . The current study was designed to determine whether monocyte activation of NF kappa B in response to bacterial lipopolysaccharide (LPS) is affected by pretreatment with metal ions . Concentrations of metal ions that affected cell number after 24 h of exposure were first determined . Then THP-1 human monocytes were cultured for 2 h in media containing metal ions at concentrations below levels that altered cell growth . Parallel cultures were treated with 10 microg/mL Escherichia coli LPS, and all samples were cultured an additional 2 h . Nuclear proteins were extracted and normalized amounts were incubated with {(32)P}-end-labeled NF kappa B consensus oligonucleotide . NF kappa B-DNA complexes were identified and quantified by electrophoretic mobility shift analysis . The extent of NF kappa B-DNA complex formation after metal ion pretreatment with or without LPS induction was compared to no treatment or LPS-only treated controls . Finally, LPS-induced IL1 beta secretion was measured from palladium-treated and control cells . Concentrations were identified for each metal ion (Ag(+), Co(2+), Cu(2+), Hg(2+), Ni(2+), and Pd(2+)) that did not reduce cell number after 24 h of exposure (ranging from 5 microM for Ag(+) and Hg(2+) to 200 microM for Ni(2+)) . Exposures of 2 h at these concentrations did not alter cell morphology, staining with trypan blue, or cell number . LPS exposure had no effect on cell number with or without metal ions after 2 h . When metal treatment alone was assessed, none of the metal ions had a significant effect on NF kappa B-DNA binding . However, pretreatment with Co(2+), Ni(2+), Ag(1+), Hg(2+), and Pd(2+) significantly decreased NF kappa B-DNA binding by 40-70% versus LPS alone . Only Cu(2+) had no effect on LPS-induced NF kappa B-DNA complex formation . Pd(2+) lowered, but did not abolish, IL1 beta secretion at concentrations comparable to those that altered NF kappa B-DNA binding . These results suggest that many commonly used metals alter monocyte function at concentrations that are not overtly toxic, and that protein levels controlled in part by NF kappa B also may be altered . Electrophoresis, 2003 Nov, 24(21), 3620 - 32 Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis; Zhang S et al.; We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection . The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source . This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip . This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles . The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time . The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover . The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts . The identification of 50 spots from E . coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software . In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed . Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented. Chembiochem, 2003 Nov 7, 4(11), 1206 - 15 Chemical and enzymatic synthesis of fluorinated-dehydroalanine-containing peptides; Zhou H et al.; Michael acceptors have long been recognized as reactive functionalities that may link a biologically active molecule to its cellular target . 1,2-Dehydro amino acids are potential Michael acceptors present in a large number of natural products, but their reactivity is modulated by the deactivating nature of the alpha-amino group engaged in an amide bond . We describe here the preparation of 3-fluoro-1,2-dehydroalanine moieties within peptides that significantly enhance the reactivity of the Michael acceptor . Two different routes were designed to access these compounds, one relying on chemical means to introduce the desired functionality and the second taking advantage of a peptide epimerase . In the chemical approach, the fluoro-Pummerer reaction of cysteine derivatives afforded 3-fluorocysteine residues that were oxidized to the corresponding sulfoxides, followed by thermolytic elimination to provide the desired 3-fluorodehydroalanines . The mechanism of the fluoro-Pummerer reaction was investigated and several possible pathways were ruled out . The enzymatic approach utilized the dipeptide epimerase YcjG from Escherichia coli . Difluorinated alanine was incorporated at the C terminus of a dipeptide by chemical means . The resulting peptide proved to be a substrate for YcjG, which catalyzed fluoride elimination to provide the 3-fluorodehydroalanine-containing peptide . Mechanistic investigations showed that fluoride elimination occurred faster than epimerization and at a rate close to that of epimerization of Ala-Ala. Mar Biotechnol (NY), 2004 Jan-Feb, 6(1), 53 - 9 Epub 2003 Nov 14. Expression in Escherchia coli and purification of sea bass (Dicentrarchus labrax) interleukin 1beta, a possible immunoadjuvant in aquaculture; Buonocore F et al.; Interleukin 1beta (IL-1beta) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses . Our group has recently cloned IL-1beta from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species . The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1 . As for trout and carp IL-1beta precursor sequence, no candidate cut site for ICE (IL-1beta converting enzyme) enzyme was apparent in the alignments of sea bass IL-1beta with other mammalian IL-1betas . Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids . The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector . The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified . Finally, we demonstrated that this purified recombinant IL-1beta was able to induce IL-1beta gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments. Curr Opin Allergy Clin Immunol, 2003 Dec, 3(6), 421 - 5 Hyper-immunoglobulin-M syndromes caused by an intrinsic B cell defect; Durandy A et al.; PURPOSE OF REVIEW: Elucidation of the molecular basis of hyper-immunoglobulin-M syndromes has provided considerable insight into the molecular events involved in antibody maturation, including immunoglobulin class switch recombination and the generation of somatic hypermutation . RECENT FINDINGS: The identification of activation-induced cytidine deaminase deficiency (hyper-immunoglobulin-M syndrome 2) has revealed the key role played by this inducible B cell-specific molecule in both class switch recombination and somatic hypermutation . Data from Escherichia coli and in-vitro assays have strongly suggested that activation-induced cytidine deaminase acts as a DNA-editing enzyme in these processes . The recent description of a new hyper-immunoglobulin-M syndrome caused by mutations in the gene encoding the uracil-N glycosylase provided further evidence that activation-induced cytidine deaminase acts on deoxycytidine in the switch and variable regions . Indeed, uracil-N glycosylase is required to remove the uracil residues integrated into DNA following deoxycytidine deamination by activation-induced cytidine deaminase . Another hyper-immunoglobulin-M condition has recently been described (hyper-immunoglobulin-M syndrome 4) . Its molecular basis is unknown, but it appears to be a homogeneous entity characterized by an intrinsic B cell defective class switch recombination but normal generation of somatic hypermutation . It is probably caused by a class switch recombination-specific DNA repair defect because class switch recombination-induced DNA breaks in S regions are normally detected in patients with this condition . SUMMARY: The heterogeneity in hyper-immunoglobulin-M syndromes will continue to shed light on the molecular mechanisms of class switch recombination and somatic hypermutation . The description of hyper-immunoglobulin-M syndromes may therefore lead to improvements in the care of these patients. J Vet Sci, 2000 Jun, 1(1), 27 - 31 Activation domain in P67phox regulates the steady state reduction of FAD in gp91phox; Han CH et al.; An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system {10} To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored . Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect . A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction . The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558) . The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction . Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD. J Vet Sci, 2000 Jun, 1(1), 19 - 26 Expression and characterization of the flavoprotein domain of gp91phox; Han CH et al.; Truncated forms of gp91(phox) were expressed in E . coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX) . TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive . Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme . Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI) . In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect . Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity . Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox). J Biol Chem, 2004 Jan 30, 279(5), 3340 - 7 Epub 2003 Nov 11. Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli . Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and Met-95 mutants; Taguchi S et al.; The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis . The rate of O(2) association (k(on)) with full-length Ec DOS is extremely slow at 0.0019 microM(-1) s(-1), compared with >9.5 microM(-1) s(-1) for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method . This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain . Dissociation constants (K(d)) calculated from the kinetic parameters are 340 and 20 microm for the full-length wild-type enzyme and its isolated PAS domain, respectively . Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the k(on) value by more than 30-fold, and consequently, a decrease in the K(d) value to less than 1 microM . The k(on) value for CO binding to the full-length wild-type enzyme is also very low (0.00081 microM(-1) s(-1)) . The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O(2) . However, the K(d) values for CO are considerably lower than those for O(2). J Biol Chem, 2004 Feb 13, 279(7), 5177 - 83 Epub 2003 Nov 11. Pygopus residues required for its binding to Legless are critical for transcription and development; Townsley FM et al.; Pygopus and Legless/Bcl-9 are recently discovered core components of the Wnt signaling pathway that are required for the transcriptional activity of Armadillo/beta-catenin and T cell factors . It has been proposed that they are part of a tri-partite adaptor chain (Armadillo>Legless>Pygopus) that recruits transcriptional co-activator complexes to DNA-bound T cell factor . Here, we identify four conserved residues at the putative PHD domain surface of Drosophila and mouse Pygopus that are required for their binding to Legless in vitro and in vivo . The same residues are also critical for the transactivation potential of DNA-tethered Pygopus in transfected mammalian cells and for rescue activity of pygopus mutant embryos . These residues at the Legless>Pygopus interface thus define a specific molecular target for blocking Wnt signaling during development and cancer. J Biol Chem, 2004 Feb 13, 279(7), 5588 - 96 Epub 2003 Nov 11. Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli; Schiefner A et al.; Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water . They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure . Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity . To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine . This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX . The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding. J Biol Chem, 2004 Jan 23, 279(4), 3078 - 83 Epub 2003 Nov 11. Occurrence of a bound ubiquinone and its function in Escherichia coli membrane-bound quinoprotein glucose dehydrogenase; Elias MD et al.; The membrane-bound pyrroloquinoline quinone (PQQ)-containing quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli functions by catalyzing glucose oxidation in the periplasm and by transferring electrons directly to ubiquinone (UQ) in the respiratory chain . To clarify the intramolecular electron transfer of mGDH, quantitation and identification of UQ were performed, indicating that purified mGDH contains a tightly bound UQ(8) in its molecule . A significant increase in the EPR signal was observed following glucose addition in mGDH reconstituted with PQQ and Mg(2+), suggesting that bound UQ(8) accepts a single electron from PQQH(2) to generate semiquinone radicals . No such increase in the EPR signal was observed in UQ(8)-free mGDH under the same conditions . Moreover, a UQ(2) reductase assay with a UQ-related inhibitor (C49) revealed different inhibition kinetics between the wild-type mGDH and UQ(8)-free mGDH . From these findings, we propose that the native mGDH bears two ubiquinone-binding sites, one (Q(I)) for bound UQ(8) in its molecule and the other (Q(II)) for UQ(8) in the ubiquinone pool, and that the bound UQ(8) in the Q(I) site acts as a single electron mediator in the intramolecular electron transfer in mGDH. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 151 - 7 Mutations in deoB and deoC alter an extracellular signaling pathway required for activation of the gab operon in Escherichia coli; Joloba ML et al.; In Escherichia coli, a lacZ fusion to the gabT gene is activated by the accumulation of two self-produced extracellular signals, indole and a second unidentified signal (signal-2) . Extracellular indole contributes approximately 25% of this activation and signal-2 is responsible for the majority of activation . Using an E . coli strain unable to produce indole and containing a gabT::lacZ fusion, a genetic approach was used to search for genes involved in the production of signal-2 . A spontaneous E . coli mutant, MJ1, exhibited significantly less signal-2 activity based on the ability of spent culture supernatants from this mutant to activate the gabT::lacZ fusion . Genetic analysis of MJ1 revealed that it contained two mutations, one in thyA and a second unknown mutation, designated spl1 (signal production locus) that led to loss of signal-2 production . The spl1 second-site mutation arises at high frequency in a thyA- background because it suppresses the loss of viability . This study demonstrates that mutations in deoB and deoC were capable of suppressing the loss of viability in thyA mutants and concomitantly resulted in loss of signal-2 activity in conditioned medium . Interestingly, both deoB and deoC mutations in an otherwise wild-type background resulted in higher levels of gabT::lacZ expression in cells at low density . It is hypothesized that deoB and deoC mutations result in an enhanced rate of signal-2 uptake and thus deplete signal-2 from the external medium. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 81 - 6 FNR-mediated regulation of hyp expression in Escherichia coli; Messenger SL et al.; The hypA-E operon is involved in the maturation of all three NiFe hydrogenases in Escherichia coli . Two hyp promoters have been described; a sigma54-dependent promoter upstream of hypA, and a sigma70-dependent promoter (PhypA) within the hypA coding region . Here it is shown that the oxygen-responsive transcription factor FNR regulates PhypA under anaerobic conditions only . PhypA does not possess a canonical FNR recognition sequence, but two FNR half-sites are present . Studies using PHYPA::lacZ fusions carrying lesions in one or both FNR half-sites indicated that although some residual anaerobic activity was retained by the promoter containing only the downstream FNR half-site, both half-sites are required for maximal PhypA activity in vivo . In vitro gel retardation analysis suggested that the primary interaction occurs at the downstream FNR half-site . Possible explanations for these observations and the implications for other FNR-regulated promoters are discussed. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 63 - 71 The glyoxylate bypass of Ralstonia eutropha; Wang ZX et al.; The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli . Interposon-mutants of all three genes (DeltaaceA1, DeltaaceA2 and DeltaaceB1) were constructed, and the phenotypes of the respective mutants were investigated . Whereas R . eutropha HF39DeltaaceA1 retained only 19% of ICL activity and failed to grow on acetate, R . eutropha HF39DeltaaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded . These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle . R . eutropha HF39DeltaaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R . eutropha HF39 . Whereas in R . eutropha HF39DeltaaceA1 and R . eutropha HF39DeltaaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R . eutropha HF39DeltaaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R . eutropha HF39. Bioorg Med Chem Lett, 2003 Sep 1, 13(17), 2847 - 51 Aggregation of RecA-derived peptides on single-stranded oligonucleotides triggered by schiff base-mediated crosslinking; Sugiyama T et al.; We here show that single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine (fdU) can crosslink the peptides derived from the DNA binding site of RecA protein through a Schiff base formation . The ability of crosslinking of fdU-containing oligonucleotides was investigated using a series of peptides whose amino acid residues spanning the center of the RecA-derived peptide were sequentially replaced with lysine . Circular dichroism (CD) spectroscopy, gel mobility shift assay and sedimentation experiment demonstrated that crosslinking reaction proceeded efficiently only when the peptides bound to the oligonucleotides. Exp Mol Pathol, 2003 Dec, 75(3), 217 - 27 Function of CD80 and CD86 on monocyte- and stem cell-derived dendritic cells; Dilioglou S et al.; Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate naive CD4+ and CD8+ T cells, as well as their lack of lineage-specific markers . Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated . Human umbilical cord blood CD14+ monocytes and CD34+ stem cells were induced to differentiate into dendritic cells using 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 25 ng/mL interleukin (II)-4, 2.5 ng/mL tumor necrosis factor alpha (TNF-alpha) and 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-alpha, respectively . Differentiated dendritic cells were CD80+, CD86+, CD83+, CD54+, CD1a+, CD11b+, CD11c+, HLA-DR+, CD34-, CD3-, CD19-, CD14-, and CD16- . Reverse transcription polymerase chain reaction revealed that differentiating monocytes initially expressed CD86 mRNA while CD80 mRNA appeared on Day 2 . Differentiating stem cells expressed both CD80 and CD86 mRNA on Day 2 of culture . Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs . Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles . CD14 and CD34 derived DCs prior to the functional assay were stimulated for 18 h with 0.1 and 1.0 mg/mL Escherichia coli lipopolyssacharide, respectively . A decrease in stimulation as depicted by decreased T-cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T-cell activation by stem cell-derived DCs . The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood-isolated hematopoietic lineages . These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies. Genomics, 2003 Dec, 82(6), 596 - 605 Controlled transgene dosage and PAC-mediated transgenesis in mice using a chromosomal vector; Voet T et al.; Previously, we designed a chromosomal vector (CV) and reported germline transmission of the vector by mice and regulated expression of the human tissue factor (F3) gene present on the CV . Further characterization and development of the CV are presented here . Mice could be bred with one to four copies of the CV per cell, and it is shown that F3 expression is proportional to the CV copy number . The insertion of large sequences into the CV was investigated by the insertion of a PAC, carrying 62.5 kb of human genomic DNA containing the CSN2 and STATH genes, into the CV by means of Cre/loxP recombination (CV(PAC)) . Retrofitting the PAC with a cytomegalovirus (CMV)-5'HPRT/loxP cassette in Escherichia coli allowed efficient selection of CVs with PAC insert . Mitotic loss rates of the CV(PAC) were similar to the original CV . Furthermore, germline transmission efficiency and mitotic stability of the CV(PAC) in mice were not compromised . The human CSN2 and STATH genes were not expressed in the transchromosomal mice . In contrast, F3, already present on the CV, was expressed in CV(PAC)(+) F(1) mice similar to in CV(+) mice, suggesting that the insertion of large sequences does not interfere with transcription of genes present on the CV. BMC Infect Dis . 2003 Nov 11;3(1):26. RIDOM: comprehensive and public sequence database for identification of Mycobacterium species; Harmsen D et al.; BACKGROUND: Molecular identification of Mycobacterium species has two primary advantages when compared to phenotypic identification: rapid turn-around time and improved accuracy . The information content of the 5' end of the 16S ribosomal RNA gene (16S rDNA) is sufficient for identification of most bacterial species . However, reliable sequence-based identification is hampered by many faulty and some missing sequence entries in publicly accessible databases . METHODS: In order to establish an improved 16S rDNA sequence database for the identification of clinical and environmental isolates, we sequenced both strands of the 5' end of 16S rDNA (Escherichia coli positions 54 to 510) from 199 mycobacterial culture collection isolates . All validly described species (n = 89; up to March 21, 2000) and nearly all published sequevar variants were included . If the 16S rDNA sequences were not discriminatory, the internal transcribed spacer (ITS) region sequences (n = 84) were also determined . RESULTS: Using 5'-16S rDNA sequencing a total of 64 different mycobacterial species (71.9%) could be identified . With the additional input of the ITS sequence, a further 16 species or subspecies could be differentiated . Only Mycobacterium tuberculosis complex species, M . marinum/M . ulcerans and the M . avium subspecies could not be differentiated using 5'-16S rDNA or ITS sequencing . A total of 77 culture collection strain sequences, exhibiting an overlap of at least 80% and identical by strain number to the isolates used in this study, were found in the GenBank . Comparing these with our sequences revealed that an average of 4.31 nucleotide differences (SD +/- 0.57) were present . CONCLUSIONS: The data from this analysis show that it is possible to differentiate most mycobacterial species by sequence analysis of partial 16S rDNA . The high-quality sequences reported here, together with ancillary information (e.g., taxonomic, medical), are available in a public database, which is currently being expanded in the RIDOM project for similarity searches. Genome Biol . 2003;4(11):235 . Epub 2003 Oct 28. Extending knowledge of Escherichia coli metabolism by modeling and experiment; Voit EO et al.; One of the challenges for 'post-genomic' biology is the integration of data from many different sources . Two recent studies independently take steps towards this goal for Escherichia coli, using mathematical modeling and a combination of gene expression and protein levels to predict new gene functions and metabolic behaviors. Appl Spectrosc, 2003 Jan, 57(1), 51 - 7 Radiation dose distribution in polymer gels by Raman spectroscopy; Rintoul L et al.; The Raman spectroscopy of polymer gel dosimeters has been investigated with a view to developing a novel dosimetry technique that is capable of determining radiation dose within a micrometer of spatial resolution . The polymer gel dosimeter, known as the PAG dosimeter, is typically made up of acrylamide, N,N'-methylene-bis--acrylamide, gelatin, and water . A polyacrylamide network within the gelatin matrix forms in response to an absorbed dose . The loss of monomers may be monitored by corresponding changes to the Raman spectrum . Principal component analysis offers a simple method of quantifying the absorbed radiation dose from the Raman spectrum of the polymer gel . The background luminescence in the spectrum increased significantly with dose and is shown to originate in the glass of the sample vial . The competing effects of elastic scatter, which increases with dose due to the formation of polymer, and sample absorption were quantified and found to introduce errors of up to 5% under certain conditions . Raman spectra as a function of distance from the air-surface interface have been measured for samples that were subjected to doses delivered by a clinical linear accelerator . The depth dose profile thus obtained compared favorably with "gold standard" ion-chamber measurements. J Plant Physiol, 2003 Oct, 160(10), 1219 - 31 Characterization of a hydroperoxide lyase gene and effect of C6-volatiles on expression of genes of the oxylipin metabolism in Citrus; Gomi K et al.; A number of C6-volatile products of the lipoxygenase (LOX) pathway was examined for their antifungal activity and a potential role as a signal molecule in citrus . trans-2-Hexenal induced the rough lemon lipoxygenase gene (RlemLOX), hydroperoxide lyase gene (RlemHPL) and AOS gene, but hexanal, and hexanol suppressed them . cis-3-Hexenol and trans-2-hexenol increased expression of the AOS gene but not RlemLOX and RlemHPL . Transcripts of the RlemHPL and AOS gene were detected constitutively in leaves by northern blot, but wounding or inoculation with nonpathogenic Alternaria alternata rapidly increased the transcript accumulation . Transcripts of the RlemHPL and AOS genes were also induced with pathogenic A . alternata, which produces the host-selective ACR-toxin, but the signal declined rapidly after inoculation . An increase in enzymatic activity of HPL after wounding or inoculation with nonpathogen was suppressed in leaves infected with the pathogen . Interestingly, vapor treatment with trans-2-hexenol delayed necrotic spot formation in the leaves inoculated with the pathogenic A . alternata . Since trans-2-hexenol has no antifungal activity to A . alternata and also did not inhibit necrosis formation by ACR-toxin alone, the delay of symptoms may be caused by activation of AOS in the LOX pathway to produce oxylipin derivatives such as methyl jasmonate for activation of defense related genes with antifungal activity. J Vet Sci, 2003 Aug, 4(2), 167 - 73 Localization of antigenic sites at the amino-terminus of rinderpest virus N protein using deleted N mutants and monoclonal antibody; Choi KS et al.; The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection . Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants . Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells . In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149 . Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79 . Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus. J Biol Chem, 2004 Jan 30, 279(5), 3348 - 53 Epub 2003 Nov 10. Role of His-16 in turnover of T4 pyrimidine dimer glycosylase; Meador MG et al.; Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding . This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain . Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine . The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed . The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein . The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme . This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle. J Biol Chem, 2004 Feb 6, 279(6), 4386 - 93 Epub 2003 Nov 10. Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II . Uncoupling the beta and DNA binding activities of the gamma complex; Snyder AK et al.; Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis . The Escherichia coli gamma complex loads the beta sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the gamma complex for beta and DNA . This is the second of two reports (Williams, C . R., Snyder, A . K., Kuzmic, P., O'Donnell, M., and Bloom, L . B . (2004) J . Biol . Chem . 279, 4376-4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and beta . Mutations were made to an Arg residue in a conserved SRC motif in the delta' and gamma subunits that interacts with the ATP site of the neighboring gamma subunit . Mutation of the delta' subunit reduced the ATP-dependent beta binding activity, whereas mutation of the gamma subunits reduced the DNA binding activity of the gamma complex . The gamma complex containing the delta' mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type gamma complex . A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the gamma mutations, consistent with the reduced DNA binding activity of this complex . The differential effects of these mutations suggest that ATP binding at the gamma1 site may be coupled to conformational changes that largely modulate interactions with beta, whereas ATP binding at the gamma2 and/or gamma3 site may be coupled to conformational changes that have a major role in interactions with DNA . Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for beta and DNA. J Biol Chem, 2004 Feb 6, 279(6), 4376 - 85 Epub 2003 Nov 10. Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I . Two distinct activities for individual ATP sites in the gamma complex; Williams CR et al.; The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis . ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA . This is the first of two reports (Snyder, A . K., Williams, C . R., Johnson, A., O'Donnell, M., and Bloom, L . B . (2004) J . Biol . Chem . 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta . Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes . Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding . In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly . In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly . These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta. Biochemistry, 2003 Nov 18, 42(45), 13269 - 79 Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli; Baldwin J et al.; The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides . Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster . Substitution of key active site residues results in perturbations of the normal oxygen activation pathway . Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway . Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mossbauer and X-ray absorption spectroscopy . These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2 . An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate . As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance . In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A) . Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster. Biochemistry, 2003 Nov 18, 42(45), 13202 - 11 Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli; Planson AG et al.; The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine . Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection . F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein . F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds . F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide . Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges . On the contrary, when produced as a fusion protein with E . coli MBP, the F19 moiety was natively folded . Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast . These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo . Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein. Biochemistry, 2003 Nov 18, 42(45), 13106 - 12 Spectroscopic evidence that osmolytes used in crystallization buffers inhibit a conformation change in a membrane protein; Fanucci GE et al.; BtuB is a bacterial outer-membrane protein that transports vitamin B(12) . Spectroscopic studies using site-directed spin labeling (SDSL) indicate that the N-terminus of BtuB undergoes a dramatic structural change from a docked (folded) to an undocked (unfolded) configuration upon substrate binding . However, this dramatic conformational change is not observed in the crystal structures of BtuB . Here, we make an attempt to resolve this discrepancy and find that the effects of solutes can explain the discrepancy between the results obtained using these two methods . Specifically, if SDSL is performed with the buffers used for the crystallization of BtuB, the substrate-induced order-disorder transition of the N-terminal Ton box observed in intact membranes is blocked . Moreover, poly(ethylene glycol) 3350, which is a component of the crystallization and soaking buffers, is shown to inhibit this structural transition . It is likely that the crystal structure of BtuB in its holo form represents an osmotically trapped conformation . Conformational changes involving relatively modest energy differences and significant hydration changes may be sensitive to solutes used during crystallization, and this example demonstrates the value of combining multiple structural methods in the examination of protein structure and function. Vet Res Commun, 2003 Oct, 27(7), 539 - 48 Effect of infusing lactoferrin hydrolysate into bovine mammary glands with subclinical mastitis; Kawai K et al.; The therapeutic effect of administering lactoferrin hydrolysate (LFH) into the mammary glands of cows with subclinical mastitis was evaluated . Seven millilitres of a preparation of LFH (7% protein) was infused into 35 quarters of 25 cows with subclinical mastitis . The numbers of bacteria in the milk from infected quarters decreased, and bacteria disappeared by the 14th day after the administration of LFH . The mean somatic cell counts (SCC) peaked one day after administration of LFH and the counts were significantly p < 0.01) decreased on days 7, 14 and 21 compared to those before the administration of LFH . The mean lactoferrin concentration in the milk peaked on days 2 or 3 and then gradually decreased to day 14, returning to the level before the administration of LFH . It appears that administration of LFH may have a therapeutic effect when infused into the quarters of cows with subclinical mastitis. IUBMB Life, 2003 Aug, 55(8), 473 - 81 Rotary movements within the ATP synthase do not constitute an obligatory element of the catalytic mechanism; Berden JA; After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism . On the other hand, the experimental evidence in favor of a two-sites catalytic mechanism, derived from various approaches and not compatible with a three-sites rotary mechanism, appear to be very solid . Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation. Radiats Biol Radioecol, 2003 Jul-Aug, 43(4), 464 - 9 {Role of nitric oxide in the SOS response in Escherichia coli}; Vasil'eva SV et al.; Having one electron with unpaired spin, nitric oxide (NO) shows high reactivity and activates or inhibits free radical chain reactions . NO toxic and genotoxic effects appear to be the result of intracellular formation of peroxinitrite that can induce some cellular damages, including DNA strand breaks, DNA base oxidation, destruction of the key enzymes, etc . Taking into account the character of DNA damages being formed under NO activity, we proposed a formation of the SOS signal and induction the SOS DNA repair response in E . coli cells treated with NO physiological donors--DNIC and GSNO . The ability of NO donor compounds to induce the SOS DNA response in E . coli PQ37 with sfiA::lacZ operon fusion is reported here at the first time . So, the SOS DNA repair response induction is one of the function of nitric oxide. Radiats Biol Radioecol, 2003 Jul-Aug, 43(4), 400 - 3 {Effect of a dose rate of chronic low-intensity irradiation with 60Co gamma emission and media tonicity on the dynamics of ageing and dying off in Escherichia coli BS-1}; Morozov II et al.; The experimental data on radiation reduction of the dynamics of Escherichia coli BS-1 ageing and dying-off with the increase of intensity of chronic exposure to 60Co gamma-Rays in the range of dose rates from 0.1 to 7.6 x 10(2) microGe/h are presented . This phenomenon takes place only under cell irradiation in isotonic and hypotonic suspension medium. EMBO Rep, 2003 Dec, 4(12), 1169 - 74 Epub 2003 Nov 07. The ubiquitin-like protein HUB1 forms SDS-resistant complexes with cellular proteins in the absence of ATP; Luders J et al.; Ubiquitin and ubiquitin-like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)-termini and epsilon-amino groups of lysine residues of acceptor proteins . A hallmark of UBLs is a protruding C-terminal tail with a terminal glycine residue, which is required for ATP-dependent conjugation . Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C-terminal processing . HUB1 exhibits sequence similarity with ubiquitin but lacks a C-terminal tail bearing a glycine residue . Here we show that HUB1 can form SDS-resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C-terminal conjugation of HUB1 to substrates . The adducts are still formed when the C-terminus of HUB1 was altered by epitope tagging, amino-acid exchange or deletion, or when cells were depleted of ATP . We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins. J Biochem (Tokyo), 2003 Oct, 134(4), 567 - 74 Genetic, enzymatic, and structural analyses of phenylalanyl-tRNA synthetase from Thermococcus kodakaraensis KOD1; Shiraki K et al.; Phenylalanyl-tRNA synthetase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-PheRS) was cloned . The open reading frames for both the alpha-subunit (Tk-pheRSA) and beta-subunit (Tk-pheRSB) genes were 1,503 bp (501 amino acids) and 1,722 bp (574 amino acids), respectively . Tk-pheRSB located 879 bp downstream from Tk-pheRSA with a putative TATA box, suggesting that these two subunits are transcribed and regulated independently in KOD1 cells . Tk-PheRS and its respective subunits were expressed in Escherichia coli cells and the proteins were purified . Tk-PheRS showed an optimum enzymatic activity at around 95 degrees C and retained its tertiary structure at 98 degrees C . The estimated isoelectric point (pI) for the alpha-subunit is 9.4 and that for the beta-subunit is 4.6, the largest difference among the 12 kinds of PheRSs reported . The considerable thermostability of Tk-PheRS may be responsible for the electrostatic interaction between the alpha- and beta-subunits. J Immunol, 2003 Nov 15, 171(10), 5521 - 8 CiC3-1a-mediated chemotaxis in the deuterostome invertebrate Ciona intestinalis (Urochordata); Pinto MR et al.; Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms . However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown . To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C . intestinalis hemocytes . We found that the migration of C . intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab . As is true for mammalian C3a, the chemotactic activity of C . intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a(59-76)) also promoted hemocyte chemotaxis . Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a(59-75)) retained most of the directional hemocyte migration activity . The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is G(i) protein coupled . Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3'-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells . Our findings indicate that CiC3-1a mediates chemotaxis of C . intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes. J Immunol, 2003 Nov 15, 171(10), 4984 - 9 Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos; Agrawal S et al.; Dendritic cells (DCs) are pivotal in determining the class of an adaptive immune response . However, the molecular mechanisms within DCs that determine this decision-making process are unknown . Here, we demonstrate that distinct Toll-like receptor (TLR) ligands instruct human DCs to induce distinct Th cell responses by differentially modulating mitogen-activated protein kinase signaling . Thus, Escherichia coli LPS and flagellin, which trigger TLR4 and TLR5, respectively, instruct DCs to stimulate Th1 responses via IL-12p70 production, which depends on the phosphorylation of p38 and c-Jun N-terminal kinase 1/2 . In contrast, the TLR2 agonist, Pam3cys, and the Th2 stimulus, schistosome egg Ags: 1) barely induce IL-12p70; 2) stimulate sustained duration and magnitude of extracellular signal-regulated kinase 1/2 phosphorylation, which results in stabilization of the transcription factor c-Fos, a suppressor of IL-12; and 3) yield a Th2 bias . Thus, distinct TLR agonists differentially modulate extracellular signal-regulated kinase signaling, c-Fos activity, and cytokine responses in DCs to stimulate different Th responses. Clin Diagn Lab Immunol, 2003 Nov, 10(6), 1090 - 5 Characterization of the cytokine immune response in children who have experienced an episode of typical hemolytic-uremic syndrome; Westerholt S et al.; The lipopolysaccharide (LPS) of enterohemorrhagic Escherichia coli (EHEC) and Shiga toxin together substantially contribute to the pathophysiology of typical hemolytic-uremic syndrome (HUS) . Both factors have been shown to be immune stimulators and could play a key role in the individual innate immune response, characterized by proinflammatory and anti-inflammatory cytokines . By use of a whole blood stimulation model, we therefore compared the LPS- and superantigen-induced cytokine responses in children who had been having recovering from an acute episode of typical HUS for at least 6 months (group 1) with those in controls, who consisted of patients seen in the pediatric neurology outpatient department for routine examination (group 2) . Samples were analyzed for cytokine protein levels and the levels of mRNA production . LPS stimulation revealed lower levels of interleukin 10 (IL-10) (P < 0.05) and increased levels of gamma interferon (P < 0.05) and increased ratios of pro- and anti-inflammatory cytokines (P < 0.05 for the IL-1beta/IL-10 ratio; P < 0.05 for the tumor necrosis factor alpha/IL-10 ratio) in group 1 . In addition superantigen stimulation showed decreased IL-2 levels in group 1 (P < 0.01) . Our results suggest an alteration of the cytokine response characterized by high proinflammatory cytokine levels and low anti-inflammatory cytokine levels as well as low levels of IL-2 production in children who have experienced an episode of typical HUS . We hypothesize that this altered immune response is not a residual effect of the infection but a preexisting characteristic of the patient . This could be one reason why individuals infected with EHEC are potentially predisposed to a systemic disease (HUS). Clin Diagn Lab Immunol, 2003 Nov, 10(6), 1051 - 8 Analysis of the shotgun expression library of the Mycobacterium tuberculosis genome for immunodominant polypeptides: potential use in serodiagnosis; Bisen PS et al.; A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance . An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector . DNA from a virulent M . tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides . beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies . Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21 . These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis . The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%. J Biol Chem, 2004 Jan 30, 279(5), 3142 - 50 Epub 2003 Nov 07. Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa . New insights into the specificity of thioredoxin function; Jeong W et al.; We have identified and characterized a 14-kDa human thioredoxin (Trx)-related protein designated TRP14 . This cytosolic protein was expressed in all tissues and cell types examined, generally in smaller amounts than Trx1 . Although TRP14 contains five cysteines, only the two Cys residues in its WCPDC motif were exposed and redox sensitive . Unlike Trx1, which was an equally good substrate for both Trx reductase 1 (TrxR1) and TrxR2, oxidized TRP14 was reduced by TrxR1 but not by TrxR2 . Biochemical characterization of TRP14 suggested that, like Trx1, TRP14 is a disulfide reductase; its active site cysteine is sufficiently nucleophilic with the pK(a) value of 6.1; and its redox potential (-257 mV) is similar to those of other cellular thiol reductants . However, although TRP14 reduced small disulfide-containing peptides, it did not reduce the disulfides of known Trx1 substrates, ribonucleotide reductase, peroxiredoxin, and methionine sulfoxide reductase . These results suggest that TRP14 and Trx1 might act on distinct substrate proteins. J Biol Chem, 2004 Feb 20, 279(8), 6943 - 51 Epub 2003 Nov 07. Differential effects of Parkinson's disease-associated mutations on stability and folding of DJ-1; Gorner K et al.; Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease . In some patients the gene is deleted . The molecular basis of disease in patients with point mutations is less obvious . We have investigated the molecular properties of {L166P}DJ-1 and the novel variant {E64D}DJ-1 . When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of {L166P}DJ-1 were dramatically lower than wild-type {WT}DJ-1 and {E64D}DJ-1 . Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of {L166P}DJ-1 were because of accelerated protein turnover . Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable {L166P}DJ-1 mutant . Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism . However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1 . To gain further insight into the structural defects of DJ-1 mutants, human {WT}DJ-1 and both mutants were expressed in Escherichia coli . As in eukaryotic cells, expression levels of {L166P}DJ-1 were dramatically reduced compared with {WT}DJ-1 and {E64D}DJ-1 . Circular dichroism spectrometry revealed that the solution structures of {WT}DJ-1 and {E64D}DJ-1 are rich in beta-strand and alpha-helix conformation . Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and {WT}DJ-1 was more flexible in this regard than {E64D}DJ-1 . Thus, structural defects of {E64D}DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation. J Biol Chem, 2004 Feb 13, 279(7), 5555 - 64 Epub 2003 Nov 07. Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein; Vaysse L et al.; The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors . We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide . The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes . The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold . The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells . Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding . In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA . However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid . Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible. Hepatobiliary Pancreat Dis Int, 2002 May, 1(2), 172 - 5 Living related liver transplantation for an infant with biliary atresia; Zheng SS et al.; OBJECTIVE: To sum up the preliminary experience in living related liver transplantation (LRLT) . METHODS: A 9-month-old male infant with biliary atresia (BA) who had undergone an unsuccessful Kasai operation was defined as a candidate for LRLT . The donor was his 30-year-old mother . Her lateral lobe of the left liver was transplanted into the infant's body as the graft . The left branches of the portal vein, left hepatic artery and left hepatic vein of the graft were end-to-end anastomosed to the portal vein, hepatic artery proper and hepatic vein of the recipient respectively . Biliary drainage was reestablished via Roux-en-Y operation . RESULTS: The donor retained her liver function within 2 weeks after the operation . Steroid and FK506 were prescribed in immunosuppressive therapy for the recipient . The blood bilirubin level of the infant decreased to normal 2 weeks after operation . No acute rejection occurred . Biliary leakage in the early period after the transplantation was controlled by drainage, and E.coli infection was effectively treated with antibiotics . The donor and recipient are in satisfactory condition to the present . CONCLUSION: LRLT is advisable for children with biliary atresia. Hepatobiliary Pancreat Dis Int, 2002 Nov, 1(4), 587 - 91 Mononuclear macrophages in pathogenesis of acute lung injury during acute obstructive cholangitis; Feng HY et al.; OBJECTIVE: To determine the role of mononuclear macrophages in the pathogenesis of acute lung injury during acute obstructive cholangitis . METHODS: Sixty Wistar rats were used to study the correlation between the behavior of mononuclear macrophages and acute pulmonary injury during acute obstructive cholangitis (AOC) . Animal model of AOC was made according to the method that the common bile duct was injected with Escherichia coli and ligated . The rats were killed at 6 h, 12 h, 24 h and 48 h after operation . The phagocytic function of Kupffer cells (KCs), the number of alveolar macrophages (AMs) in bronchoalveolar lavage liquid, and the extravascular water content of lung tissue were measured . The levels of lipid peroxide (LPO) and supperoxide dismutase (SOD) were determined too . Pathological alterations of liver and lung tissue were observed under light and electron microscopes . RESULTS: KCs phagocytic function was significantly elevated at the 6th hour but markedly decreased from the 24th hour to the 48th hour in the AOC group as compared with the control (P<0.05) . From the 12th to the 48th hour, the number of AMs, the extravascular water content of lung tissue, and the content of LPO significantly increased, but the SOD level of lung tissue decreased greatly (P<0.05) . Morphologically, KCs proliferated diffusely in the early period in livers of the AOC group, but decreased markedly in the late period . Mitochondria of KCs were swollen or even vacuolated; focal cytoplasmic degeneration and many myeli like figures could be seen in the cytoplasm . The changes of injury such as disturbance of pulmonary capillary blood circulation, degeneration and/or necrosis of the lung tissue and endothelium, and inflammatory reactions could be observed . In other two groups, no evident morphological changes were observed . CONCLUSIONS: KCs phagocytic function is decreased, whereas AM is activated by the invading bacteria to release such inflammatory mediators as free radicals, resulting in acute pulmonary injury . It seems that there is a close relationship between the functional status of mononuclear macrophages and the development of acute lung injury . The dysfunction of mononuclear macrophages may play an important role in the pathogenesis of multiple organ damage, especially acute pulmonary injury. Biomaterials, 2004 Feb, 25(4), 617 - 24 Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin; Asakura T et al.; There are a variety of silkworms and silk fibroins produced by them . Silks have many inherent suitable properties for biomaterials . In this paper, a novel silk-like hybrid protein, {DGG(A)(12)GGAASTGRGDSPAAS}(5), which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced . The genes encoding the hybrid protein were constructed and expressed in Escherichia coli . The main conformation of the polyalanine region, that is, either alpha-helix or beta-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively . This structural change was monitored with 13C CP/MAS NMR . Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained. J Am Coll Cardiol, 2003 Nov 5, 42(9), 1656 - 62 Inflammation-induced vasoconstrictor hyporeactivity is caused by oxidative stress; Pleiner J et al.; OBJECTIVES: We sought to determine the role of oxidative stress in the development of vascular dysfunction in inflammation . BACKGROUND: Hyporeactivity to catecholamines and other vasoconstrictors is present in acute inflammation . Because oxidative stress plays a significant role in inflammation, impaired responsiveness may be overcome by anti-oxidants . METHODS: In randomized, double-blind, cross-over studies, forearm blood flow (FBF) responses to norepinephrine (NE), angiotensin II (ANG II), and vasopressin (VP) were assessed before and 4 h after induction of systemic inflammation by low doses of Escherichia coli endotoxin (lipopolysaccharide {LPS}, 20 IU/kg intravenously) or after placebo in healthy volunteers . Furthermore, the effect of intra-arterial vitamin C (24 mg/min) or placebo on NE-induced or ANG II-induced vasoconstriction was studied after LPS . RESULTS: Administration of LPS caused systemic and forearm vasodilation, increased white blood cell count, elevated body temperature, and reduced vitamin C plasma concentrations . Lipopolysaccharide decreased the responses of FBF to NE by 59%, to ANG II by 25%, and to VP by 51% (n = 9, p < 0.05, all effects) . Co-administration of vitamin C completely restored the response to NE and to ANG II, which was comparable to that observed under baseline conditions (n = 8) . CONCLUSIONS: E . coli-endotoxemia reduces FBF responsiveness to vasoconstrictors . The hyporeactivity can be corrected by high doses of vitamin C, suggesting that oxidative stress may represent an important target for inflammation-induced impaired vascular function. Bull Math Biol, 2003 Nov, 65(6), 1025 - 51 Identification of all steady states in large networks by logical analysis; Devloo V et al.; The goal of generalized logical analysis is to model complex biological systems, especially so-called regulatory systems, such as genetic networks . This theory is mainly characterized by its capacity to find all the steady states of a given system and the functional positive and negative circuits, which generate multistationarity and a cycle in the state sequence graph, respectively . So far, this has been achieved by exhaustive enumeration, which severely limits the size of the systems that can be analysed . In this paper, we introduce a mathematical function, called image function, which allows the calculation of the value of the logical parameter associated with a logical variable depending on the state of the system . Thus the state table of the system is represented analytically . We then show how all steady states can be derived as solutions to a system of steady-state equations . Constraint programming, a recent method for solving constraint satisfaction problems, is applied for that purpose . To illustrate the potential of our approach, we present results from computer experiments carried out on very large randomly-generated systems (graphs) with hundreds, or even thousands, of interacting components, and show that these systems can be solved using moderate computing time . Moreover, we illustrate the approach through two published applications, one of which concerns the computation times of all steady states for a large genetic network. J Mol Biol, 2003 Nov 21, 334(2), 205 - 13 Role of the non-template strand of the elongation bubble in intrinsic transcription termination; Ryder AM et al.; Intrinsic transcription terminators of Escherichia coli and other bacteria, consisting primarily of an RNA hairpin preceding a terminal uridine-rich RNA segment, suffice to dissociate the otherwise stable elongation complex of core RNA polymerase . The essential functions of the hairpin and U-rich segments have been established, although the precise mechanism of termination is unknown . We identify another element of the terminator, namely the non-template DNA strand in the region of the terminal transcription bubble . Failure of the terminal bubble to rewind through complementary base-pairing strongly reduces the efficiency of terminator function, suggesting that the natural pathway of termination consists of coupled rewinding of the DNA template and unwinding of the RNA/DNA hybrid at the site of release. J Mol Biol, 2003 Nov 21, 334(2), 197 - 204 The coherent feedforward loop serves as a sign-sensitive delay element in transcription networks; Mangan S et al.; Recent analysis of the structure of transcription regulation networks revealed several "network motifs": regulatory circuit patterns that occur much more frequently than in randomized networks . It is important to understand whether these network motifs have specific functions . One of the most significant network motifs is the coherent feedforward loop, in which transcription factor X regulates transcription factor Y, and both jointly regulate gene Z . On the basis of mathematical modeling and simulations, it was suggested that the coherent feedforward loop could serve as a sign-sensitive delay element: a circuit that responds rapidly to step-like stimuli in one direction (e.g . ON to OFF), and at a delay to steps in the opposite direction (OFF to ON) . Is this function actually carried out by feedforward loops in living cells? Here, we address this experimentally, using a system with feedforward loop connectivity, the L-arabinose utilization system of Escherichia coli . We measured responses to step-like cAMP stimuli at high temporal resolution and accuracy by means of green fluorescent protein reporters . We show that the arabinose system displays sign-sensitive delay kinetics . This type of kinetics is important for making decisions based on noisy inputs by filtering out fluctuations in input stimuli, yet allowing rapid response . This information-processing function may be performed by the feedforward loop regulation modules that are found in diverse systems from bacteria to humans. Jpn J Physiol, 2003 Aug, 53(4), 253 - 8 Role of prostaglandin E2 and indomethacin in the febrile response of pigeons; Nomoto S; Intravenous (i.v.) injection of 10 microg/kg Escherichia coli lipopolysaccharide (LPS), applied at 13:00, evoked in pigeons a biphasic rise of core temperature (T(core)), so that LPS induced with a latency of 30 min first a decrease of T(core), and 90 min after LPS, T(core) increased, obtaining maximum values from 18:00 to 20:00 . Prostaglandins have been considered to be importantly involved in fevers in mammals . To investigate an involvement of prostaglandins in the cyclic variations of T(core) in birds, pigeons were injected i.v . with either 10 mg/kg indomethacin (INDO) or 100 mg/kg aspirin, or they were treated with intracerebroventricular (i.c.v.) injections of 100 microg/kg INDO at various times before or after LPS . When INDO or aspirin was i.v . injected 30 or 15 min before LPS, it diminished the initial decrease of T(core) by more than 50%, whereas the i.v . injection of these drugs 2 and 4 h after LPS did not affect the febrile rise of T(core) . i.c.v . injections of INDO given either before or after LPS neither influenced the initial drop of T(core) nor the following febrile hyperthermia . Both the i.v . injection of 1 mg/kg prostaglandin E(2) (PGE(2)) and the i.c.v . injection of 1 microg/kg PGE(2) lowered T(core) . Our observations suggest that prostaglandins are not involved in the febrile elevation of T(core) in pigeons, but appear to participate in the decrease of T(core), which shortly follows the i.v . injection of LPS . This initial drop of T(core) following LPS may be caused by a peripheral action of prostaglandins because it was not influenced by the i.c.v . injection of indomethacin. Biochemistry (Mosc), 2003 Sep, 68(9), 988 - 93 Characterization of recombinant integrase of human immunodeficiency virus type 1 (isolate Bru); Semenova EA et al.; Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virus-encoded integrase protein . The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene . Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli) . The main kinetic parameters of HIV-1 integrase (K(m) = (3.7 +/- 0.2).10(-10) M, k(cat) = (1.2 +/- 0.3).10(-7 )sec(-1)) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate . Inhibition of integrase by aurintricarbonic acid ({I}(50) = 6.3 +/- 0.4 microM) and dependence of integrase activity on Mg2+ and Mn2+ concentration were studied. Biomacromolecules, 2003 Nov-Dec, 4(6), 1680 - 5 Mechanism for the phase transition of a genetically engineered elastin model peptide (VPGIG)40 in aqueous solution; Yamaoka T et al.; The concentration dependence of the pressure- and temperature-induced cloud point transition (Pc and Tc, respectively) of aqueous solutions of an elastin-like polypeptide with a repeating pentapeptide Val-Pro-Gly-Ile-Gly sequence (MGLDGSMG(VPGIG)40VPLE) was investigated by using apparent light scattering, differential scanning calorimetry, and circular dichroism methods . In addition, the effects of salts and surfactants on these properties were investigated . The Pc and Tc of the present peptide in aqueous solution were strongly concentration dependent . The calorimetric measurements showed that the enthalpy of transitions was 300-400 kJ/mol, i.e., 7-10 kJ/mol per VPGIG pentamer . The Tc of the (VPGIG)40 solution was highly affected by the addition of inert salts or SDS . The effects of salts were consistent with those observed in the lyotropic series or Hoffmeister series . The CD spectrum at low peptide concentrations indicated that the present peptide forms type II beta-turn-like structure(s) at higher temperatures, but the temperature dependence of random coil diminishment (195 nm) and beta-turn formation (210 nm) were not exactly coincident . A hypothetical mechanism of the (VPGIG)40 phase transition that could account for these observations was postulated . Observations suggest that the temperature-responsive properties of the elastin model peptides occur via a mechanism involving conformational change-association-aggregation and that the first two are strongly interactive. Prep Biochem Biotechnol, 2003 Nov, 33(4), 321 - 39 Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins; Nam SH et al.; The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E . coli strains, NM522 and BL21 . After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea . The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis . Western analysis identified five E . coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates . Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity . The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column . A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain. Prep Biochem Biotechnol, 2003 Nov, 33(4), 253 - 68 Comparative study of three methods for non-radioactive in vivo DNA labeling in Escherichia coli using nucleoside analogs; Perez-Bello D et al.; In the present work, a comparative study of 5-FdUrd, thy-, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E . coli DH5alpha cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd . According to the colorimetric immunoenzymatic results, we found that the minimal detectable labeled DNA (MDLD) was 312pg with the 5-FdUrd and thy- methods for 5-BrdUrd labeled plasmid DNA . 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy- and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA . Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA. Parasitol Res, 2004 Jan, 92(1), 58 - 64 Epub 2003 Nov 06. Protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2; Yang CD et al.; The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2 . The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2 . The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1 . The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2 . The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns . After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability . The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2 . Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice . Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days . Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%) . These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T . gondii infections in mice. Cancer Chemother Pharmacol, 2004 Feb, 53(2), 107 - 15 Epub 2003 Nov 07. Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase; Nivens MC et al.; PURPOSE: Gene transfer of cDNA sequences that confer drug resistance can be used (1) to protect hematopoietic cells against the toxic effects of chemotherapy, (2) for in vivo enrichment of genetically engineered cells and (3) to protect cytotoxic T lymphocytes in drug-resistant immunotherapy approaches for the treatment of cancer . We have previously developed strategies to confer resistance to agents targeting thymidylate synthase (TS) and have now expanded our drug resistance strategies to include retroviral expression of tyrosyl-DNA phosphodiesterase (TDP-I), an enzyme recently implicated in the repair of topoisomerase-I (Top-I)/DNA lesions induced by camptothecin (CPT) . The combination of TS and Top-I inhibition has been shown to be an effective treatment for several types of cancer . MATERIALS AND METHODS: Retroviral vectors were generated that individually encoded TS and TDP-I or that coexpressed both enzymes . Murine fibroblast and Chinese hamster lung transfectants were generated with the vectors and resistance to TS- and Top-I-directed inhibitors was tested . Murine bone marrow progenitor cells were also transduced using recombinant retroviruses encoding TS and TDP-I and the degree of drug resistance conferred to gene-modified cells was tested . RESULTS: Enforced expression of TDP-I increased TDP-I activity in gene-modified cells and conferred up to threefold resistance to CPT . The degree of resistance was dependent on the duration of drug treatment . Simultaneous expression of the TS gene encoding E . coli TS optimized for expression in mammalian cells (optecTS) and TDP-I conferred extremely high-level resistance to concurrent treatment with the TS-inhibitor BW1843U89 and CPT . Furthermore, by direct analysis of DNA fragmentation using the comet assay, substantial protection was conferred (fourfold) against DNA fragmentation associated with combination drug treatments by dual enzyme expression compared to non-modified cells . Hematopoietic progenitor assays of murine bone marrow cells transduced with retroviral vectors encoding TS and TDP-I showed that bone marrow cells could be protected from the cytotoxic effects of TS and Top-I inhibition . CONCLUSIONS: Enforced expression of optecTS and TDP-I conferred antifolate and CPT resistance to genetically modified cells . Additionally, this work further illustrated a role for TDP-I in the repair of dead-end Top-I complexes and implied that TDP-I expression analysis may aid in predicting the therapeutic effectiveness of the CPT class of compounds. Microbiol Immunol, 2003, 47(10), 735 - 43 Peptidase activity of dipeptidyl aminopeptidase IV produced by Porphyromonas gingivalis is important but not sufficient for virulence; Kumagai Y et al.; Porphyromonas gingivalis is a pathogen associated with adult periodontitis, which is a chronic inflammatory disease characterized by breakdown of the periodontal tissue . Dipeptidyl aminopeptidase IV (DPPIV) produced by P . gingivalis has been considered to be a potential virulence factor based on the finding that the virulence was reduced by disruption of the gene (dpp ) coding for DPPIV . In the present study, we constructed a shuttle vector that is mobilized from Escherichia coli to P . gingivalis and is maintained stably in both bacteria, and we showed that the virulence was restored by introducing the cloned wild-type dpp gene into the null mutant of P . gingivalis using our vector system . To assess the implications of the peptidase activity in the virulence, mutant DPPIV with the catalytic Ser mutagenized to Ala (DPPSA) was produced . The P . gingivalis strain expressing DPPSA exhibited an intermediate virulence between the strain expressing wild-type DPPIV and the strain harboring a vector . From these results, it is suggested that peptidase activity is very important but not sufficient for virulence. Microbiol Immunol, 2003, 47(10), 717 - 25 Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin; Ohmura-Hoshino M et al.; A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains . The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced . The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively . The variant toxin designated as Stx1v52 was purified to homogeneity . Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1 . In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum . Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate . However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA. Science, 2003 Nov 7, 302(5647), 1046 - 9 Roles for mating and environment in C . elegans sex determination; Prahlad V et al.; In Caenorhabditis elegans the two sexes, hermaphrodites and males, are thought to be irreversibly determined at fertilization by the ratio of X chromosomes to sets of autosomes: XX embryos develop as hermaphrodites and XO embryos as males . We show instead that both sex and genotype of C . elegans can be altered postembryonically and that this flexibility requires sexual reproduction . When grown in specific bacterial metabolites, some XX larvae generated by mating males and hermaphrodites develop as males and lose one X chromosome . However, XX larvae produced by hermaphrodite self-fertilization show no such changes . We propose that sexual reproduction increases developmental flexibility of progeny, allowing for better adaptation to changing environments. Plant Physiol, 2003 Dec, 133(4), 1453 - 63 Epub 2003 Nov 06. Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean; Kinoshita T et al.; Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily . They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL . In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening . However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking . Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase . Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors . We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation . Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2 . We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata . The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses. J Clin Microbiol, 2003 Nov, 41(11), 5277 - 81 Association of cytolethal distending toxin locus cdtB with enteropathogenic Escherichia coli isolated from patients with acute diarrhea in Calcutta, India; Pandey M et al.; Among Escherichia coli strains isolated from stool specimens from patients with acute diarrhea, 1.4% were found to harbor cdtB by use of enrichment cytolethal distending toxin (CDT) PCR . These isolates were identified as being enteropathogenic E . coli (EPEC) . In a retrospective study using a probe hybridization assay, 6 of 138 EPEC strains were found to harbor the cdtB locus . cdtB-positive isolates mostly belong to the O86a and O127a serogroups, with the former being associated with higher expression of CDT . Pulsed-field gel electrophoresis profiles showed that the EPEC strains harboring cdtB strains are genetically diverse. J Clin Microbiol, 2003 Nov, 41(11), 5022 - 32 Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources; Ramachandran V et al.; The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E . coli (STEC) strains from all other pathotypes of diarrheagenic E . coli . EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis . Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types . We designated these new intimin genes Int- micro, Int-nu, and Int-xi . The PCR-RFLP assay was used to screen 213 eae-positive E . coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes . Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates . Int-beta, the most commonly identified eae subtype (82 of 213 {38.5%} isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 {18.3%} isolates; 11 serotypes), Int-theta (25 of 213 {11.7%} isolates; 15 serotypes), Int-gamma (19 of 213 {8.9%} isolates; 9 serotypes), and Int-epsilon (21 of 213 {9.9%} isolates; 5 serotypes) . Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected . Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta) . Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E . coli strains. J Biol Chem, 2004 Jan 30, 279(5), 3265 - 72 Epub 2003 Nov 05. Mutation E252C increases drastically the Km value for Na+ and causes an alkaline shift of the pH dependence of NhaA Na+/H+ antiporter of Escherichia coli; Tzubery T et al.; A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km for Na(+) (50-fold) of the Na(+)/H(+) antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range . In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX . Thus, although both the Na(+)/H(+) antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys(249) in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8 . Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys(249), and the rate of the pH-induced conformational change is increased specifically by the presence of Na(+) or Li(+), the specific ligands of the antiporter . Chemical modification of E252C by N-ethylmaleimide, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; {2-(trimethylammonium)ethyl}methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K(m) of the mutant antiporter. J Biol Chem, 2004 Jan 30, 279(5), 3292 - 9 Epub 2003 Nov 06. Diversity in the rates of transcript elongation by single RNA polymerase molecules; Tolic-Norrelykke SF et al.; Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations . We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates . Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy . The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription . With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian . However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s) . The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers . These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution. J Theor Biol, 2003 Dec 7, 225(3), 383 - 8 Can tRNAs act as antisense RNA? The case of mutA and dnaQ; Dorazi R; Many mutator genes have been characterized in E . coli, but the realization that mutA, the most recent mutator pathway described, encodes for a missense suppressor glycine tRNA caused a real surprise . The connection between expression of mutA and a 10 times increase in the spontaneous mutation rate is not readily explainable . The first attempt to describe the mechanism of action suggested a direct mistranslation of one subunit of polymerase III (PolIII) and the ideal candidate was the epsilon subunit carrying the 3'-->5' exonuclease activity . This subunit increases PolIII accuracy about 100 times . However, such direct mistranslation of epsilon was later ruled out when it became clear that all mutA cells express an error-prone form of PolIII . This result could not be reconciled with the very low level of mistranslation (1%) caused by mutA . But there is no need to invoke amino acid misincorporation in epsilon to destroy its activity . On the contrary, I suggest a new way to regulate epsilon amount, based on the reinterpretation of the mutA pathway through the new and puzzling observation that several tRNAs (including mutA which encodes for a glycine missense suppressor tRNA) are complementary to the 5' end of dnaQ mRNA . Accordingly, I propose that uncharged tRNAs can act as antisense RNAs, decreasing translation of dnaQ and possibly other genes . This could represent a new regulatory function for tRNAs and of course gives a direct and unrecognized link between starvation and mutation rate. Structure (Camb), 2003 Nov, 11(11), 1381 - 92 Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method; Takeuchi K et al.; We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment . The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel . Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel . Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins . The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution. Structure (Camb), 2003 Nov, 11(11), 1359 - 67 Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing; Strub MP et al.; A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described . This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine . The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase . Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine . Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds . In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122 . This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography. Structure (Camb), 2003 Nov, 11(11), 1310 - 1 A mob of reps; Dyda F et al.; Emerging structural results confirm that the large Rolling Circle Replication initiator superfamily is composed of two classes of proteins that are circularly permutated with respect to each other, as initially suggested by sequence analysis . The two classes are united by the same endonucleolytic mechanism and a conserved Mg(2+) binding site containing multiple histidine ligands unique to this superfamily. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14247 - 52 Epub 2003 Nov 05. Induction of frameshift and base pair substitution mutations by the major DNA adduct of the endogenous carcinogen malondialdehyde; VanderVeen LA et al.; Instability of repetitive sequences is a hallmark of human cancer, and its enhancement has been linked to oxidative stress . Malondialdehyde is an endogenous product of oxidative stress that reacts with guanine to form the exocyclic adduct, pyrimido{1,2- alpha}purin-10(3H)-one (M1G) . We used site-specifically modified single- and double-stranded vectors to investigate the mutagenic potential of M1G in bacteria and mammalian cells . M1G induced frameshift mutations (-1 and -2) when positioned in a reiterated (CpG)4 sequence but not when positioned in a nonreiterated sequence in Escherichia coli and in COS-7 cells . The frequency of frameshift mutations was highest when M1G was placed at the third G in the sequence . M1G induced base pair substitutions at comparable frequencies in both sequence contexts in COS-7 cells . These studies indicate that M1G, an endogenously generated product of oxidative stress, induces sequence-dependent frameshift mutations and base pair substitutions in bacteria and in mammalian cells . This finding suggests a potential role for the M1G lesion in the induction of mutations commonly associated with human diseases. Nucleic Acids Res, 2003 Nov 15, 31(22), 6663 - 73 Molecular flip-flops formed by overlapping Fis sites; Hengen PN et al.; The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart . Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication . Only one of these sites is bound by Fis at a time, so the structure is a molecular flip-flop that could direct alternative firing of replication complexes in opposite directions . Alternatively, the flip-flop could represent part of an on-off switch for replication . Because they can be used to create precise switched states, molecular flip-flops could be used as the basis of a novel molecular computer. Nucleic Acids Res, 2003 Nov 15, 31(22), 6598 - 609 Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor; Galan B et al.; The HpaR-mediated regulation of the hpa-meta operon (Pg promoter) of the 4-hydroxyphenylacetic acid catabolic pathway of Escherichia coli has been studied . The HpaR regulator was purified to homogeneity showing that it is able to bind selectively to 4-hydroxyphenylacetic, 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids, which act as inducers of the system . The role of HpaR as a repressor and the requirement for cAMP receptor protein for maximal activity have been confirmed by in vitro transcription analyses . Two DNA operators, OPR1 and OPR2, have been identified in the intergenic region located between the hpa-meta operon and the hpaR gene . The OPR1 operator contains a perfect palindromic sequence overlapping the transcriptional +1 start site of the Pg promoter . The OPR2 operator shows a similar but imperfect palindromic sequence and is located far downstream of the +1 start site of the Pr promoter . The binding of HpaR to OPR2 displays a clear cooperativity with OPR1 binding . Based on the above observations and the results of permanganate footprinting experiments, a repression mechanism for HpaR is postulated . A 3-dimensional model of HpaR, generated by comparison with the crystal structures of the homologous regulators, MarR and MexR, suggests that HpaR is a dimer that contains a typical winged-helix DNA binding motif in each subunit. J Biol Chem, 2004 Jan 30, 279(5), 3273 - 9 Epub 2003 Nov 05. The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper; Ruan W et al.; Structural and functional studies recently indicated that the erythropoietin receptor exists as a preassembled homodimer whose activation by ligand binding requires self-interaction of its transmembrane segment . Here, we probed the interface formed by the transmembrane segments by asparagine-scanning mutagenesis in a natural membrane . We show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids . The strongest impact of asparagine was observed at position 241, suggesting the highest packing density around this position, which is in agreement with results obtained upon mutation to alanine . Interestingly, the same face of the transmembrane helix had previously been shown to enter a heterophilic interaction with the transmembrane segment of gp55-P, a viral membrane protein that leads to ligand-independent receptor activation in infected cells . Further, functional characterization of an erythropoietin receptor mutant with asparagine at position 241 in a hematopoietic cell line showed that this protein could still be activated by erythropoietin yet was not constitutively active . This suggests that forced self-interaction of the transmembrane segments does not suffice to induce signaling of the erythropoietin receptor. J Biol Chem, 2004 Feb 6, 279(6), 4465 - 70 Epub 2003 Nov 05. The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics; Tomashek JJ et al.; The F1F0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically . Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts . However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay . We have developed an optimized assay and have collected synthase kinetic data over a substrate concentration range of 2 orders of magnitude for both ADP and Pi from the synthase enzyme of E . coli . Negative and positive cooperativity of substrate binding and positive catalytic cooperativity were all observed . ATP synthesis displayed biphasic kinetics for ADP indicating that 1) the enzyme is capable of catalyzing efficient ATP synthesis when only two of three catalytic sites are occupied by ADP; and 2) occupation of the third site further activates the rate of catalysis. Appl Environ Microbiol, 2003 Nov, 69(11), 6688 - 97 Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp . strain A5; Kasai Y et al.; Cycloclasticus sp . strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes . A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo . This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs) . Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes . The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase . The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene . PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the {2Fe-2S} cluster ligands were conserved . Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms . Significantly, the E . coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases. Appl Environ Microbiol, 2003 Nov, 69(11), 6605 - 9 Viability of and plasmid retention in frozen recombinant Escherichia coli over time: a ten-year prospective study; Koenig GL; The long-term viability and plasmid retention of recombinant Escherichia coli strains were investigated by real-time testing of master cell banks (MCBs) stored at the Roche Molecular Systems Culture Collection (RMSCC) . MCBs at the RMSCC were cryogenically frozen and stored at -80 degrees C for long-term preservation . At regular intervals during a period of 5 to more than 10 years, representative cryovials of each MCB were tested for viability and plasmid retention . Plasmid retention and viability for all 30 MCBs were stable over time . Twenty-seven MCBs maintained high levels of plasmid retention (at or near 100%), while three MCBs showed lower plasmid retention rates (ranging from 13.9 to 96.5%) that were consistent over time . New MCBs with high plasmid retention were created from two of the MCBs with lower plasmid retention by selective pressure with high levels of antibiotics . These new MCBs have shown stable viability and high plasmid retention over the first 5 months of storage . In conclusion, this study shows that properly selected, frozen and stored MCBs retain viability and maintain plasmid retention over time . Moreover, it is possible to recover cultures with high plasmid retention from MCBs with low p