Escherichia coli

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 plasmid retention by selecting clones grown in the presence of high levels of antibiotics.

Appl Environ Microbiol, 2003 Nov, 69(11), 6442 - 6
Bioaccumulation of copper ions by Escherichia coli expressing vanabin genes from the vanadium-rich ascidian Ascidia sydneiensis samea; Ueki T et al.; The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T . Ueki, T . Adachi, S . Kawano, M . Aoshima, N . Yamaguchi, K . Kanamori, and H . Michibata, Biochim . Biophys . Acta 1626:43-50, 2003) . The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro . In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space . We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed . The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 micro M copper (II) ions were initially added . The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions . These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E . coli.

Biotechnol Appl Biochem . 2003 Nov 6; {Epub ahead of print}
Evaluation of disruption methods for release of intracellular recombinant protein from Escherichia coli for analytical purposes; Tkac J et al.; The aim of the study was to find disruption methods that allow fast and reproducible measurement of intracellular recombinant proteins with potential for on-line application . Production of recombinant human superoxide dismutase (rhSOD) by Escherichia coli was used as a model . Three methods of cell disruption: sonication, osmotic shock, and chemical treatment using a non-ionic surfactant, were critically compared with respect to efficiency and reproducibility of the release of rhSOD . The release of the recombinant protein was monitored by (1) measurement of the protein content in cell culture extracts using a surface plasmon resonance (SPR) biosensor, and (2) assaying the enzyme activity with a colorimetric reagent using a spectrophotometer . Disruption by the non-ionic surfactant showed the best performance in terms of simplicity, reproducibility and efficiency of sample treatment . The surfactant did not interfere with the rhSOD binding to the antibody immobilized on the SPR-chip or with the rhSOD activity assay . When comparing the two detection methods during monitoring of an E . coli cultivation, comparable results were obtained.

J Anim Sci, 2003 Nov, 81(11), 2758 - 65
Effects of fish oil supplementation on the performance and the immunological, adrenal, and somatotropic responses of weaned pigs after an Escherichia coli lipopolysaccharide challenge; Liu YL et al.; Seventy-two crossbred pigs (7.58 +/- 0.30 kg BW) weaned at 28 +/- 3 d of age were used to investigate the effects of fish oil supplementation on pig performance and on immunological, adrenal, and somatotropic responses following an Escherichia coli lipopolysaccharide (LPS) challenge in a 2 x 2 factorial design . The main factors consisted of diet (7% corn oil {CO} or 7% fish oil {FO}) and immunological challenge (LPS or saline) . On d 14 and 21, pigs were injected intraperitoneally with either 200 microg/kg BW of LPS or an equivalent amount of sterile saline . Blood samples were collected 3 h after injection for analysis of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), cortisol, growth hormone (GH), and insulin-like growth factor (IGF)-I . On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was determined . Lipopolysaccharide challenge decreased ADG (487 vs . 586 g; P < 0.05) and ADFI (as-fed, 776 vs . 920 g; P < 0.05) from d 14 to 21 and ADG (587 vs . 652 g; P < 0.10) from d 21 to 28 . Fish oil improved ADG (554 vs . 520 g; P < 0.10) and ADFI (891 vs . 805 g; P < 0.10) from d 14 to 21 . On d 14, LPS challenge x diet interactions were observed for IL-1beta (P < 0.10), PGE2 (P < 0.001), and cortisol (P < 0.05) such that these measurements responded to the LPS challenge to a lesser extent (IL-1beta: 93 vs . 114 pg/mL, P < 0.05; PGE2: 536 vs . 1,285 pg/mL, P < 0.001; cortisol: 143 vs . 206 ng/mL, P < 0.05) in pigs receiving the FO diet than in pigs fed the CO diet . In contrast, among LPS-treated pigs, pigs fed the FO diet had higher IGF-I (155 vs . 101 ng/mL; P < 0.10) than those fed the CO diet . On d 21 among LPS-treated pigs, pigs fed FO had lower IL-1beta (70 vs . 84 pg/mL; P < 0.10) and cortisol (153 vs . 205 ng/mL; P < 0.05) than those fed CO . Pigs fed FO had lower PGE2 (331 vs . 444 pg/mL; P < 0.05) and higher IGF-I (202 vs . 171 ng/mL; P < 0.10) compared with those fed CO . Lipopolysaccharide challenge decreased GH (0.27 vs . 0.33 ng/mL; P < 0.05) on d 14, whereas it had no effect on GH on d 21 . During both LPS challenge periods, the challenge increased PBLP when these cells were incubated with 8 (1.46 vs . 1.32; P < 0.10) or 16 microg/mL (1.46 vs . 1.30; P < 0.05) of concanavalin A . Fish oil had no effect on PBLP . These results suggest that FO alters the release of proinflammatory cytokines, which might lead to improved pig performance during an immunological challenge.

J Anim Sci, 2003 Nov, 81(11), 2751 - 7
Effectiveness of an experimental consensus phytase in improving dietary phytate-phosphorus utilization by weanling pigs; Gentile JM et al.; Consensus phytase is a new biosynthetic, heat-stable enzyme derived from the sequences of multiple homologous phytases . Two experiments were conducted to determine its effectiveness, relative to inorganic P and a mutant enzyme of Escherichia coli phytase (Mutant-EP), in improving dietary phytate-P availability to pigs . In Exp . 1, 36 pigs (3 wk old, 7.00 +/- 0.24 kg of BW) were fed a low-P corn-soybean meal basal diet plus consensus phytase at 0, 250, 500, 750, 1,000, or 1,250 U/kg of feed for 5 wk . Plasma inorganic P concentration, plasma alkaline phosphatase activity, bone strength, and overall ADG and gain:feed ratio of pigs were improved (P < 0.05) by consensus phytase in both linear (R2 = 0.20 to 0.70) and quadratic (R2 = 0.30 to 0.70) dose-dependent fashions . In Exp . 2, 36 pigs (4 wk old, 9.61 +/- 0.52 kg BW) were fed the basal diet + inorganic P at 0.1 or 0.2%, consensus phytase at 750 or 450 U/kg of feed, Mutant-EP at 450 U/kg of feed, or 225 U consensus + 225 U Mutant-EP/kg of feed . Pigs fed 750 U of consensus phytase or 450 U of Mutant-EP/kg feed had plasma inorganic concentrations and bone strength that fell between those of pigs fed 0.1 or 0.2% inorganic P . These two measures were 16 to 29% lower (P < 0.05) in pigs fed 450 U of consensus phytase/kg of feed than those of pigs fed 0.2% inorganic P . Plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed Mutant-EP vs . consensus phytase at 450 U/kg at wk 2 and 3 . In conclusion, the experimental consensus phytase effectively releases phytate P from the corn-soy diet for weanling pigs . The inorganic P equivalent of 750 U of consensus phytase/kg of feed may fall between 0.1 and 0.2%, but this requires further determination.

Bioorg Khim, 2003 Sep-Oct, 29(5), 486 - 94
{Proteolysis coupled with ATP . Regulation of activity of proteolytic centers of Escherichia coli lon protease}; Pirul'nikov KB et al.; Regulation of activity of the proteolytic sites of Lon protease was studied . It was found that ATP-Mg has the properties of a noncompetitive activator of peptidase sites . The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis . It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by the native Lon protease . The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of the intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites . The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.

Pediatr Med Chir, 2003 May-Jun, 25(3), 185 - 9
Bilateral neonatal adrenal abscess . Report of two cases and review of the literature; Arena F et al.; Neonatal adrenal abscess is an extremely rare condition . 32 cases, 4 bilateral, have been described in the world literature . We treated successfully other two bilateral cases . We report on this rare condition and review the world literature . In our Department we observed two patients in the neonatal period with abdominal distension, vomiting, irritability and fever . Abdominal ultrasound (US), plain x-ray of the abdomen, intravenous pyelography and computed tomography (CT) of the abdomen were performed . In both cases bilateral suprarenal cystic masses were identified . Vanilmandelic acid, Homovanillic acid and cathecolamines were normal . The two neonates underwent a surgical exploration . Abscesses were found and drained releasing a moderate amount of haemorrhagic and purulent materials from the adrenal glands . Post-operative histology on the surgical specimen showed in both cases an abscess in partial haemorrhagic adrenal glands . No neoplastic cells were observed . The recovery was uneventful and at six months follow-up both patients were well and without signs of adrenal insufficiency . Haematogenous bacteria seeding a normal gland or abscess formation in a previous haemorrhagic gland are the most accredited theories . Neuroblastoma, Wilm's tumor and renal duplication with dilatation of the upper segment must be considered in the differential diagnosis . Percutaneous drainage (+/- biopsy) under CT or US guide should be considered the treatment of choice, followed by surgical exploration when diagnosis is not clear.

Biol Pharm Bull, 2003 Nov, 26(11), 1528 - 33
Hydrolysis of synthetic substrate, L-pyroglutamyl p-nitroanilide is catalyzed solely by pyroglutamyl aminopeptidase I in rat liver cytosol; Abe K et al.; Pyroglutamyl aminopeptidase I (PAP-I) is a cytosolic cysteine peptidase, which hydrolytically removes the L-pyroglutamate residue from the amino terminus of endogenous proteins and peptides . L-Pyroglutamyl p-nitroanilide serves as the synthetic substrate of this enzyme, while there is a possibility of other hydrolases being involved in the hydrolysis of this xenobiotic substrate . We cloned a full-length cDNA encoding rat PAP-I from a rat liver cDNA library and expressed this cDNA in Escherichia coli to obtain a recombinant PAP-I as a single protein . The cDNA encoded a sequence of 209 amino acids with a calculated molecular weight of 22913 Da . The homology of the deduced amino acid sequence of rat PAP-I was 98.6 and 94.3% to mouse and human PAP-Is, respectively . The biochemical properties of the recombinant rat PAP-I were almost identical to those of the recombinant mouse and human PAP-Is and the purified rat liver cytosolic PAP-I in terms of the molecular weight, subunit structure, affinity to the substrate, inhibitor profile and pH optimum . Immunoblot analysis using an antibody raised against recombinant rat PAP-I showed that rat PAP-I is present almost exclusively in the cytosolic fraction of the rat liver . Moreover, the hydrolyzing activity for L-pyroglutamyl p-nitroanilide in rat liver cytosolic fraction was completely inhibited by the antibody, strongly suggesting that this xenobiotic substrate is hydrolyzed solely by PAP-I.

Chem Pharm Bull (Tokyo), 2003 Nov, 51(11), 1293 - 8
Computer-aided modeling of pentachlorophenol 4-monooxygenase and site-directed mutagenesis of its active site; Nakamura T et al.; Homology modeling was used to construct a model of the three-dimensional structure of pentachlorophenol 4-monooxygenase (PcpB) . A PSI-BLAST homology search was initially performed to identify the 3D structure of proteins homologous with PcpB . The feasibility of modeled structures of PcpB was evaluated by Verify3D, which calculated structural compatibility scores based on 3D-1D profiles . The predicted structure of PcpB had an acceptable 3D-1D self-compatibility score, beyond the incorrect fold score threshold . A PcpB-pentachlorophenol (PCP) complex was then constructed utilizing the modeled PcpB structure . After energy minimization of the complex, and successive minimizations of the system that consisted of the complex and the water layer surrounding the complex, the molecular dynamics of the system were simulated . The active-site residues of PcpB were identified on the basis of the modeled structure, and PcpB mutants were then designed to change the active site residues, expressed, and purified by affinity chromatography . The mutant activity was compared with that of the wild-type to investigate the validity of the modeled structure . The experimental results suggested that Phe85, Tyr216, and Arg235 were relevant to enzyme activity, and that Tyr397 and Phe87 were important for stabilization of the structure of PcpB.

Microbiology, 2003 Nov, 149(Pt 11), 3321 - 30
(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica; Kloosterman H et al.; Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica . Deregulated, feedback-control-resistant mutants were isolated by incubation of A . methanolica on glucose mineral agar containing the toxic analogue p-fluoro-DL-phenylalanine (pFPhe) . Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation . Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites . A . methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase) . The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp . Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants . These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E . coli-type DAHP synthase), only inhibited by Tyr and Trp . DS1 and DS2 thus are well integrated in A . methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr . Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.

Microbiology, 2003 Nov, 149(Pt 11), 3257 - 63
An increase in the level of 2-oxoglutarate promotes heterocyst development in the cyanobacterium Anabaena sp . strain PCC 7120; Li JH et al.; In the filamentous cyanobacterium Anabaena sp . strain PCC 7120, a starvation of combined nitrogen induces differentiation of heterocysts, cells specialized in nitrogen fixation . How do filaments perceive the limitation of the source of combined nitrogen, and what determines the proportion of heterocysts? In cyanobacteria, 2-oxoglutarate provides a carbon skeleton for the incorporation of inorganic nitrogen . Recently, it has been proposed that the concentration of 2-oxoglutarate reflects the nitrogen status in cyanobacteria . To investigate the effect of 2-oxoglutarate on heterocyst development, a heterologous gene encoding a 2-oxoglutarate permease under the control of a regulated promoter was expressed in Anabaena sp . PCC 7120 . The increase of 2-oxoglutarate within cells can trigger heterocyst differentiation in a subpopulation of filaments even in the presence of nitrate . In the absence of a source of combined nitrogen, it can increase heterocyst frequency, advance the timing of commitment to heterocyst development and further increase the proportion of heterocysts in a patS mutant . Here, it is proposed that the intracellular concentration of 2-oxoglutarate is involved in the determination of the proportion of the two cell types according to the carbon/nitrogen status of the filament.

Microbiology, 2003 Nov, 149(Pt 11), 3193 - 202
Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis; Maslow JN et al.; In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL) . Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown . An isogenic rtfA mutant of M . avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp) . Overexpression of KatG in M . avium resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA . Plasmid loss was confirmed by screening for gfp-negative clones to select putative allelic exchange mutants . Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC) . Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core . Complementation of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724 . This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M . avium as a tool for future genetic studies involving this species.

Protein Eng, 2003 Oct, 16(10), 777 - 83
Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases; Kuhnel B et al.; Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein . In general, such fusion proteins must be cleaved to release the mature protein in its native form . The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity . We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin . DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host . Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein . The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds . Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained . The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

Hepatobiliary Pancreat Dis Int, 2003 Aug, 2(3), 387 - 90
Cloning and expression of cDNAs from hepatitis E virus structural gene; Ruan B et al.; OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection . METHODS: A 492 base cDNA was collected from 5-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China . The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme . The recombinant plasmid was transformed into E.coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E . RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively . A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2 . The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100 . CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

Fungal Genet Biol, 2003 Dec, 40(3), 271 - 86
Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa; Gupta GD et al.; Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth . This requirement may be met by the localization and/or activation of an apical SNARE-based machinery . We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa . Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants . All surviving transformants grew slowly, conidiated poorly, and were male sterile . In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation . For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores . Those that germinated produced slow-growing hyphae with abnormal branching . The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.

DNA Repair (Amst), 2003 Nov 21, 2(11), 1227 - 38
Analysis of translesion replication across an abasic site by DNA polymerase IV of Escherichia coli; Maor-Shoshani A et al.; Unrepaired replication-blocking DNA lesions are bypassed by specialized DNA polymerases, members of the Y super-family . In Escherichia coli the major lesion bypass DNA polymerase is pol V, whereas the function of its homologue, pol IV, is not fully understood . In vivo analysis showed that pol V has a major role in bypass across an abasic site analog, with little or no involvement of pol IV . This can result from the inability of pol IV to bypass the abasic site, or from in vivo regulation of its activity . In vitro analysis revealed that purified pol IV, in the presence of the beta subunit DNA sliding clamp, and the gamma complex clamp loader, bypassed a synthetic abasic site with very high efficiency, reaching 73% in 2 min . Bypass was observed also in the absence of the processivity proteins, albeit at a 10- to 20-fold lower rate . DNA sequence analysis revealed that pol IV skips over the abasic site, producing primarily small deletions . The RecA protein inhibited bypass by pol IV, but this inhibition was alleviated by single-strand binding protein (SSB) . The fact that the in vitro bypass ability of pol IV is not manifested under in vivo conditions suggests the presence of a regulatory factor, which might be involved in controlling the access of the bypass polymerases to the damaged site in DNA.

DNA Repair (Amst), 2003 Nov 21, 2(11), 1175 - 83
xni-deficient Escherichia coli are proficient for recombination and multiple pathways of repair; Lombardo MJ et al.; Single-strand-dependent DNA exonucleases play important roles in DNA repair and recombination in all organisms . In Escherichia coli the redundant functions provided by the RecJ, ExoI, ExoVII and ExoX exonucleases are required for mismatch repair, UV resistance and homologous recombination . We have examined whether the xni gene product, the single-strand exonuclease ExoIX, is also a member of this group . We find that deletion of xni has no effect on the above processes, or on resistance to oxidative damage, even in combination with other exonuclease mutations . We conclude that the xni gene product does not belong to this group of nucleases that play redundant roles in DNA recombination and repair.

J Virol Methods, 2003 Dec, 114(1), 45 - 54
Activation of dengue protease autocleavage at the NS2B-NS3 junction by recombinant NS3 and GST-NS2B fusion proteins; Wu CF et al.; Dengue virus possesses a protease complex made up of the non-structural proteins NS2B and NS3 . This protease complex catalyzes autocleavage (cis) at the junction between NS2A and NS2B as well as between NS2B and NS3 . It also catalyzes trans cleavage at the junctions between NS3 and NS4A as well as NS4B and NS5 . The cis cleavage at the NS2B-NS3 junction has been demonstrated in Escherichia coli by linking a 40-residue hydrophilic segment of NS2B to a NS3 N-terminal protease domain carrying the NS2B-NS3 cleavage site . To explore whether the hydrophilic segment could be further shortened, residues from both N- and C-termini of the NS2B hydrophilic segment were deleted . The results indicate that the four C-terminal's consecutive Glu residues could be deleted, each one leading to a further loss of activity, whereas the N-terminal boundary needed to be absolutely preserved . To examine whether an NS2B peptide could be expressed independently and added to activate the NS3 protease domain, the hydrophilic region of NS2B was fused to the C-terminus of glutathione-S-transferase (GST) . This recombinant protein was soluble in bacteria and easily purified by affinity chromatography . Without removing the GST, the fusion protein activated the NS3 protease domain allowing it to function at the adjacent NS2B-NS3 junction . Thus, the findings reported below have produced a feasible alternative for the assay of dengue viral protease and this should facilitate the development of a screening method for inhibitors of dengue protease.

Phytochemistry, 2003 Dec, 64(7), 1203 - 11
Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis; Bertea C et al.; Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint . The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli . The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position . Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases . Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 651 - 6
In vitro studies on DNA-photosensitization by different drug stereoisomers; Lhiaubet-Vallet V et al.; The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated . The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis . Formation of single strand breaks was compared for each group of compounds . Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E . coli endonuclease III (revealing oxidized pyrimidines) and E . coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage . Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases . No pyrimidine dimers were observed . Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites . The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism . Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin . In the case of carprofen, a weak nucleoside degradation was obtained . The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin . In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer . However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

J Biomol Screen, 2002 Oct, 7(5), 433 - 40
Phage display of functional human TNF-alpha converting enzyme catalytic domain: a rapid method for the production of stabilized proteolytic proteins for assay development and high-throughput screening; Chen Y et al.; The catalytic domain of human tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS) . This would address many issues around screening of proteases in this class . The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-alpha to generate the mature TNF-alpha in vitro . To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-alpha-specific peptide in vitro . More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS . The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT . Both PDT and BET showed a similar specific cleavage profile against the defined substrates . Activity of the BET, however, was stable at 4 degrees C for less than 24 h . In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 degrees C . On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity . Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

Org Biomol Chem, 2003 Oct 21, 1(20), 3498 - 9
The mechanism of 7,8-diaminopelargonate synthase; the role of S-adenosylmethionine as the amino donor; Breen RS et al.; The product of the first transamination step in the reaction catalysed by diaminopelargonate (DAPA) synthase has been shown to be 4-(S-adenosyl)-2-oxobutanoate, which has been trapped as the corresponding alcohol.

Parasitol Res, 2004 Jan, 92(1), 43 - 7 Epub 2003 Nov 04.
Functional characterization of an alternative {lactate dehydrogenase-like} malate dehydrogenase in Plasmodium falciparum; Chan M et al.; The catalysis of malate dehydrogenase (MDH) in Plasmodium falciparum (pfMDH) which involves NAD/NADH coupling is crucial for the parasite's pathogenicity . Primers were designed based on the P . falciparum genome resource, and these facilitated the cloning of a gene coding for pfMDH from a local clinical isolate . The DNA sequence of the cloned gene revealed an open-reading frame that encodes a protein of 313 amino acids . After induction in Escherichia coli BL21, enzyme assays of the expressed pfMDH purified by affinity chromatography exhibited significant enzyme activity of about 50 U/mg, where one unit (U) of enzyme activity is defined as the amount of enzyme oxidising 1 microol NADH/min . Based on its phylogenetic status amongst MDHs and lactate dehydrogenases (LDHs), the cloned gene was clearly defined as belonging to the NADH-dependent {LDH-like} MDHs . It is noteworthy that pfMDH harbours unique structural characteristics potentially useful for screening drugs specific for disabling parasitic enzymes.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13259 - 63 Epub 2003 Nov 03.
Motility of Escherichia coli cells in clusters formed by chemotactic aggregation; Mittal N et al.; Cells of Escherichia coli under conditions of certain cellular stresses excrete attractants . Cells of chemotactic strains respond to these excreted signaling molecules by moving up their local concentration gradients and forming different types of stable multicellular structures . Multicellular clusters are the simplest among these structures . Fluorescence microscopy was used to characterize the macroscopic properties of the clusters and to track individual E . coli cells in the clusters in real time . A quantitative analysis reveals that the equilibrium cluster size is only weakly dependent on the total number of cells in the cluster . The tumble frequency of an individual cell strongly depends on the position of the cell within the cluster and its direction of movement . In the central region of the cluster, tumbles are strongly suppressed whereas near the edge of the cluster, the tumble frequency is restored for exiting cells, thereby preventing them from leaving the cluster, resulting in the maintenance of sharp cluster boundaries . A simulation based on a model of the sensory memory of E . coli reproduces the experimental data and indicates that the tumble rate and consequently the morphology of the cluster are determined by the sensory memory of cells.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13231 - 4 Epub 2003 Nov 03.
SecA-dependent quality control of intracellular protein localization; Eser M et al.; Complex secretion machineries mediate protein translocation across cellular membranes . These machines typically recognize their substrates via signal sequences, which are required for proper targeting to the translocon . We report that during posttranslational secretion the widely conserved targeting factor SecA performs a quality-control function that is based on a general chaperone activity . This quality-control mechanism involves assisted folding of signal sequenceless proteins, thereby excluding them from the secretion process . These results suggest that SecA channels proteins into one of two key pathways, posttranslational secretion or folding in the cytoplasm . Implications of this finding for intracellular protein localization are discussed.

Genome Res, 2003 Nov, 13(11), 2435 - 43
Regulatory network of Escherichia coli: consistency between literature knowledge and microarray profiles; Gutierrez-Rios RM et al.; The transcriptional network of Escherichia coli may well be the most complete experimentally characterized network of a single cell . A rule-based approach was built to assess the degree of consistency between whole-genome microarray experiments in different experimental conditions and the accumulated knowledge in the literature compiled in RegulonDB, a data base of transcriptional regulation and operon organization in E . coli . We observed a high and statistical significant level of consistency, ranging from 70%-87% . When effector metabolites of regulatory proteins are not considered in the prediction of the active or inactive state of the regulators, consistency falls by up to 40% . Similarly, consistency decreases when rules for multiple regulatory interactions are altered or when "on" and "off" entries were assigned randomly . We modified the initial state of regulators and evaluated the propagation of errors in the network that do not correlate linearly with the connectivity of regulators . We interpret this deviation mainly as a result of the existence of redundant regulatory interactions . Consistency evaluation opens a new space of dialogue between theory and experiment, as the consequences of different assumptions can be evaluated and compared.

Genome Res, 2003 Nov, 13(11), 2381 - 90
A biophysical approach to transcription factor binding site discovery; Djordjevic M et al.; Identification of transcription factor binding sites within regulatory segments of genomic DNA is an important step toward understanding of the regulatory circuits that control expression of genes . Here, we describe a novel bioinformatics method that bases classification of potential binding sites explicitly on the estimate of sequence-specific binding energy of a given transcription factor . The method also estimates the chemical potential of the factor that defines the threshold of binding . In contrast with the widely used information-theoretic weight matrix method, the new approach correctly describes saturation in the transcription factor/DNA binding probability . This results in a significant improvement in the number of expected false positives, particularly in the ubiquitous case of low-specificity factors . In the strong binding limit, the algorithm is related to the "support vector machine" approach to pattern recognition . The new method is used to identify likely genomic binding sites for the E . coli transcription factors collected in the DPInteract database . In addition, for CRP (a global regulatory factor), the likely regulatory modality (i.e., repressor or activator) of predicted binding sites is determined.

J Biol Chem, 2004 Jan 30, 279(5), 3777 - 86 Epub 2003 Nov 03.
Mechanisms of guanine nucleotide exchange and Rac-mediated signaling revealed by a dominant negative trio mutant; Debreceni B et al.; Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis . Dominant negative mutants of Rho GTPases, such as T17NRac1, that block the endogenous Rho protein activation by sequestering upstream guanine nucleotide exchange factors (GEFs) have been widely used to implicate specific members of the Rho family in various signaling pathways . We show here that such an approach could produce potentially misleading results since many Rho GEFs can interact with multiple Rho proteins promiscuously, and overexpression of one dominant negative Rho protein mutant may affect the activity of other members of the Rho family . Based on the available structural information, we have identified the highly conserved amino acid pairing of Asn(1406)Trio-Asp(65)Rac1 of the GEF-Rho GTPase interaction as the critical catalytic machinery required for the Rac1 GDP/GTP exchange reaction . The N1406A/D1407A mutant of Trio acted dominant negatively in vitro by retaining Rac1 binding activity but losing GEF catalytic activity and competitively inhibited Rac1 activation by wild type Trio . It readily blocked the platelet-derived growth factor (PDGF)-induced lamellipodia formation and inhibited the wild type Trio-induced serum response factor activation . Moreover the mutant was able to selectively inhibit Dbl-induced Rac1 activation without affecting RhoA activity in cells . In contrast to the non-discriminative inhibitory effect displayed by T17NRac1, the Trio mutant was ineffective in inhibiting PDGF-stimulated DNA synthesis and Dbl-induced transformation, revealing the Rac-independent functions of PDGF and Dbl . These studies identify a conserved pair of amino acid residues of the Trio-Rac interaction that is likely to be essential to the GEF catalysis of Rho family GTPases and demonstrate that a dominant negative mutant derived from a Rho GTPase regulator constitutes a new generation of specific inhibitors of Rho GTPase signaling pathways.

Gene, 2003 Nov 27, 320, 185 - 90
The effective number of codons for individual amino acids: some codons are more optimal than others; Fuglsang A; The aim of this study was to evaluate the codon bias using the effective number of codons for individual amino acids (N(c)(AA)) and to assess the codon bias in relation to the definition of optimal codons, using Escherichia coli as a model organism . We show that a general correlation exists between the effective number of codons (Ncirc(c)) or codon adaptation index (CAI) and N(c)(AA), but that this correlation is not equally strong for all amino acids within a degeneracy group . For example, leucine codons contribute more to Ncirc(c) and the codon adaptation index than serine codons . A possible explanation is that some optimal codons are more optimal than others, in terms of the selectional advantage they offer . This hypothesis is confirmed by further analysis on the correlations that exist between values for relative synonymous codon usage (RSCU), N(c)(AA), and the codon adaptation index.

J Biochem Biophys Methods, 2004 Jan 30, 58(1), 59 - 66
Exploring the mechanism of competence development in Escherichia coli using quantum dots as fluorescent probes; Li W et al.; The mechanism of divalent Ca2+ cation induction of Escherichia coli competence is still not fully understood, though it is a common method for introducing recombinant DNA into bacterial cells in gene engineering . Quantum dots (QDs), as a new fluorescent probe of being applied in biology research, have aroused great interest . In this paper, we explored the mechanism of E . coli competence development using QDs for the first time . Results showed that water-soluble QDs of diameter 3-4 nm could go into competent cells, but could not enter noncompetent cells . This result was further confirmed using atomic force microscopy and DNA transforming experiments, suggesting that nonphysiological, high concentrations of Ca2+ enhanced the penetrability of cell membranes so that QDs, which cannot enter cells normally due to their greater diameter (3-4 nm), can do so easily into competent cells . Therefore, we believe that, at least for E . coli, the mechanism of Ca2+-induced competence development is mediated physicochemically rather than physiologically.

J Biochem Biophys Methods, 2004 Jan 30, 58(1), 39 - 48
Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography; Dickman MJ et al.; The RuvABC resolvasome of Escherichia coli typifies nucleoprotein complexes involved in genetic transactions . This molecular assembly catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair . This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease . We have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) to rapidly identify the binding of RuvA to an immobilised synthetic Holliday junction; unambiguous identification was verified using tryptic digest of the bound protein . In conjunction with a novel fluorescent-based technique incorporating ion pair reverse phase liquid chromatography, a "footprint" of the RuvA:Holliday complex was obtained . These two complementary techniques offer a generic approach to the analysis of nucleoprotein complexes.

Gene, 2003 Nov 13, 319, 167 - 75
Identification of single nucleotide polymorphisms in the human gamma-glutamyl hydrolase gene and characterization of promoter polymorphisms; Chave KJ et al.; gamma-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action . Increased GGH activity has been found in rat hepatoma cells resistant to the cancer drug methotrexate (MTX) . The aim of this study was to identify polymorphisms in the GGH gene that modulate GGH activity and that may affect methotrexate resistance . Exons of the human gamma-glutamyl hydrolase (hGGH) gene were amplified by polymerase chain reaction (PCR) from breast cancer tissue and leukemia cell lines . Single-stranded conformational polymorphism (SSCP) analysis was performed, and PCR products containing different patterns were cloned and sequenced . Six single nucleotide polymorphisms (SNPs) were identified, at bases -401C>T, -354G>T, -124T>G, +16T>C, +452C>T, and +1102A>G, relative to the A of the translation start codon being considered as +1 . The SNP at +16, which changes codon -19 (relative to the start of the mature hGGH protein) in the endoplasmic reticulum targeting sequence of hGGH protein from cysteine to arginine, has previously been identified in this laboratory . The SNP at +452 changes the conserved hGGH protein codon 127 from threonine to isoleucine . The functions of SNPs in the promoter of the hGGH gene were studied by site-directed mutagenesis of a 516-bp region of the hGGH gene promoter in a luciferase reporter vector and transfection into HepG2 and MCF-7 cells . All of the promoter polymorphisms enhanced the production of luciferase compared to the wild-type hGGH gene promoter in HepG2 cells, and -401C>T and -124T>G enhanced luciferase expression in MCF-7 cells, suggesting that polymorphisms in the hGGH gene promoter may increase expression of hGGH protein.

Gene, 2003 Nov 13, 319, 107 - 16
Thioredoxin-mediated redox control of the transcription factor Sp1 and regulation of the thioredoxin gene promoter; Bloomfield KL et al.; In recent years, redox control has emerged as a fundamental mechanism of gene regulation through transcriptional control . Thioredoxin (Trx) is a dithiol-reducing enzyme known to be involved in the redox regulation of a number of transcription factors, and in this study, we have investigated the redox-dependent regulation of the DNA binding activity of Sp1 by thioredoxin . Electrophoretic mobility shift assays were used to show that both recombinant Sp1 produced in Escherichia coli and endogenous Sp1 expressed by MDA-MB-231 breast cancer cells is subject to redox regulation . We found that thioredoxin alone or in conjunction with the full thioredoxin system (comprising thioredoxin, thioredoxin reductase {TR}, and alpha-nicotinamide adenine dinucleotide phosphate {NADPH}) can increase Sp1 DNA binding activity in vitro to an oligonucleotide containing the Sp1 consensus sequence . Furthermore, we have provided evidence that recombinant Sp1 can bind to Sp1 consensus sequences within a 330-base pair (bp) thioredoxin promoter fragment and that this interaction can also be enhanced by the presence of thioredoxin . Luciferase reporter assays using this same minimal thioredoxin promoter region demonstrated that both Sp1 and Sp3 can bind to the promoter and act to enhance transcription . When the three identified Sp1 consensus sequences within the reporter construct were deleted, there was a loss of basal promoter activity, showing that these closely positioned sites are important for regulation of thioredoxin gene expression.

Plasmid, 2003 Nov, 50(3), 202 - 12
Type I restriction enzyme with RecA protein promotes illegitimate recombination; Kusano K et al.; Illegitimate (non-homologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs . However, we have found a type of illegitimate recombination that requires an interaction between long homologous DNA sequences . It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type I (EcoKI) restriction in vivo within a special mutant strain of Escherichia coli . In the present work, we analyzed genetic requirements for this type of illegitimate recombination in well-defined genetic backgrounds . Our analysis demonstrated dependence on RecA function and on the presence of two EcoKI sites on the substrate DNA . These results are in harmony with a model in which EcoKI restriction enzyme attacks an intermediate of homologous recombination to divert it to illegitimate recombination.

Plasmid, 2003 Nov, 50(3), 169 - 75
Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli; Park KW et al.; We have designed and constructed a new fusion expression vector (pKW32), which contains the His-tagged Vitreoscilla hemoglobin (VHb) coding gene upstream of the multiple cloning site . The pKW32 vector was designed to express target proteins as VHb fusions, which can be purified in one step by affinity chromatography . Due to the color of the heme in VHb, the VHb-fused target proteins have a red color that provides a visual aid for estimating their expression level and solubility . The red color can also be used as a visual marker throughout purification, while the concentration of the fusion protein can be determined by measuring the amount of VHb using carbon monoxide difference spectra . In addition, because of inherently high solubility of VHb, the fusion can increase the solubility of sparingly soluble target proteins . Target proteins can be easily separated from His-tagged VHb due to the presence of a thrombin-cleavage site between them . A mutant VHb, the soluble domain of Vitreoscilla cytochrome bo subunit II, and HIV integrase expressed and purified using the pKW32 system have native function . In addition, the integrase, which is known to be difficult to purify because of low solubility, was purified simply and without solubilizing agents using our system.

FEBS Lett, 2003 Nov 6, 554(1-2), 41 - 4
The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases; Capitani G et al.; Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II . Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding . This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases . A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.

Res Microbiol, 2003 Nov, 154(9), 654 - 7
Context-dependent effects of charged residues in transmembrane segments of MalF-PhoA fusions; Ehrle R et al.; Charged residues were introduced into the C-terminal transmembrane segment of a MalF-PhoA fusion to investigate the efficiency of the altered transmembrane segment to function as an export signal or a stop transfer signal . PhoA assays revealed that charges had negative effects when the transmembrane segment was part of a stop transfer signal but not when it was part of an export signal . Implications of this finding for the biogenesis of polytopic membrane proteins are discussed.

Anal Biochem, 2003 Nov 15, 322(2), 185 - 9
Detection of submicrogram quantities of Escherichia coli lipopolysaccharides by agarose-gel electrophoresis; Maccari F et al.; A sensitive agarose-gel electrophoresis method has been developed for the visualization of lipopolysaccharide (LPS) samples from several Escherichia coli serotypes, such as 026:B6, 055:B5, 0128:B12, 0111:B4, 0127:B8, and K235 . This method can detect as little as 0.5-6 microg of LPS depending on the serotype by treatment of the plates with toluidine blue, and it is able to measure sub-microgram amounts of samples, approx . 0.05-0.5 microg, when the staining with toluidine blue followed by Stains-All procedure is adopted . Treatment of LPS with alkali under anhydrous conditions removes the ester-linked fatty acids and the phosphate groups of the Lipid A component, producing the detoxified LPS . The carbohydrate neutral moiety obtained from the hydrolysate does not migrate during electrophoresis . This was utilized to monitor quantitatively the removal process of the Lipid A component for the formation of the detoxified LPS.

Anal Biochem, 2003 Nov 15, 322(2), 156 - 63
Monitoring of the heat-shock response in Escherichia coli using an optical biosensor; Vostiar I et al.; A surface plasmon resonance (SPR) method for monitoring the concentration of the chaperone DnaK and its relation to physiological stress response in a recombinant Escherichia coli strain subjected to heat shock is described . The DnaK protein, an abundantly occurring representative of the heat-shock proteins, was used as a marker of physiological stress . The SPR biosensor instrument was used for label-free immunoaffinity detection directly in cell culture lysates using an anti-DnaK monoclonal IgG antibody immobilized on the sensor surface . The SPR method provides a fast response (<8 min) and a reproducible (RSD<2%), accurate (comparison to the direct enzyme-linked immunosorbent assay), and sensitive (LOD<1 nM) assay for determination of the DnaK level in cell culture lysates . The operational stability of the method was high compared to that of other SPR assays; the sensitivity decreased at only 2.7%/h . This allowed measurement of more than 220 samples per sensor surface . Storage stability was determined at 25 degrees C (100% after 17 h) and 10 degrees C (101% after 1 month) . The method was validated by standard additions of DnaK (30, 60, and 120 nM) with recovery indices in the range 95.7-103.7%.

BMC Biochem . 2003 Nov 04;4(1):16.
Sugar recognition by human galactokinase; Timson DJ et al.; BACKGROUND: Galactokinase catalyses the first committed step of galactose catabolism in which the sugar is phosphorylated at the expense of MgATP . Recent structural studies suggest that the enzyme makes several contacts with galactose--five side chain and two main chain hydrogen bonds . Furthermore, it has been suggested that inhibition of galactokinase may help sufferers of the genetic disease classical galactosemia which is caused by defects in another enzyme of the pathway galactose-1-phosphate uridyl transferase . Galactokinases from different sources have a range of substrate specificities and a diversity of kinetic mechanisms . Therefore only studies on the human enzyme are likely to be of value in the design of therapeutically useful inhibitors . RESULTS: Using recombinant human galactokinase expressed in and purified from E . coli we have investigated the sugar specificity of the enzyme and the kinetic consequences of mutating residues in the sugar-binding site in order to improve our understanding of substrate recognition by this enzyme . D-galactose and 2-deoxy-D-galactose are substrates for the enzyme, but N-acetyl-D-galactosamine, L-arabinose, D-fucose and D-glucose are all not phosphorylated . Mutation of glutamate-43 (which forms a hydrogen bond to the hydroxyl group attached to carbon 6 of galactose) to alanine results in only minor changes in the kinetic parameters of the enzyme . Mutation of this residue to glycine causes a ten-fold drop in the turnover number . In contrast, mutation of histidine 44 to either alanine or isoleucine results in insoluble protein following expression in E . coli . Alteration of the residue that makes hydrogen bonds to the hydroxyl attached to carbons 3 and 4 (aspartate 46) results in an enzyme that although soluble is essentially inactive . CONCLUSIONS: The enzyme is tolerant to small changes at position 2 of the sugar ring, but not at positions 4 and 6 . The results from site directed mutagenesis could not have been predicted from the crystal structure alone and needed to be determined experimentally.

Biochemistry, 2003 Nov 11, 42(44), 13008 - 18
Effect of the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin on proofreading by Escherichia coli DNA polymerase I (Klenow fragment) in different sequence contexts; Kornyushyna O et al.; Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders . The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo . Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) . To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+) . For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C) . Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side . In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base . Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side . The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.

Biochemistry, 2003 Nov 11, 42(44), 12989 - 97
Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases; Nordin BE et al.; The genetic code depends on amino acid fine structure discrimination by aminoacyl-tRNA synthetases . For isoleucyl- (IleRS) and valyl-tRNA synthetases (ValRS), reactions that hydrolyze misactivated noncognate amino acids help to achieve high accuracy in aminoacylation . Two editing pathways contribute to aminoacylation fidelity: pretransfer and post-transfer . In pretransfer editing, the misactivated amino acid is hydrolyzed as an aminoacyl adenylate, while in post-transfer editing a misacylated tRNA is deacylated . Both reactions are dependent on a tRNA cofactor and require translocation to a site located approximately 30 A from the site of amino acid activation . Using a series of 3'-end modified tRNAs that are deficient in either aminoacylation, deacylation, or both, total editing (the sum of pre- and post-transfer editing) was shown to require both aminoacylation and deacylation activities . These and additional results with IleRS are consistent with a post-transfer deacylation event initiating formation of an editing-active enzyme/tRNA complex . In this state, the primed complex processively edits misactivated valyl-adenylate via the pretransfer route . Thus, misacylated tRNA is an obligatory intermediate for editing by either pathway.

Biochemistry, 2003 Nov 11, 42(44), 12784 - 91
Structural elements required for deamidation of RhoA by cytotoxic necrotizing factor 1; Buetow L et al.; Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis . This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation . Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments . We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually . We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site . Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation . In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA . None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise . In addition, we show that the specificity constant (k(cat)/K(m)) of CNF1-C for RhoA is 825 +/- 3 M(-1) s(-1) . This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets.

Epidemiol Infect, 2003 Oct, 131(2), 815 - 21
Virulence factors of Escherichia coli strains belonging to serogroups O127 and O142; Ghilardi AC et al.; A total of 102 Escherichia coli strains belonging to serogroups O127 and O142 were examined for genotypic and phenotypic characteristics . The most frequent serotypes found were O127:H21, O127:H40 and O142:H34 . The virulence properties were evaluated by adhesion to HeLa cells and hybridization with gene probes for diarrhoeagenic E . coli . Most strains in the two serogroups were categorized as enteropathogenic E . coli, but enteroaggregative E . coli was also detected in both serogroups . All strains that carried the eae sequence presented the LEE region inserted in selC . Five ribotypes were detected in serogroup O127 and four in serogroup O142 and a correlation between serotypes and ribotypes was observed mainly in serogroup O142.

Anal Sci, 2003 Oct, 19(10), 1355 - 7
Piezo electric sensor for endocrine-disrupting chemicals using receptor-co-factor interaction; Murata M et al.; In vitro screening assays are useful techniques for the determination of receptor-mediated activities in environmental samples . In order to define whether environmental chemicals act as an agonist or antagonist to the human estrogen receptor (hER), we have constructed a biosensor based on ligand-inducible interactions between hER and relative proteins on a quartz crystal microbalance (QCM) . The his-tagged proteins, which were expressed in E . coli by recombinant DNA technology, were immobilized on an Au-electrode with Ni(II)-mediated chemisorption using the histidine tag and thiol-modified iminodiacetic acid . The resonance-frequency change of the protein-modified electrode was caused by association or dissociation with the hER relative proteins on the surface in the presence of estrogen . These results suggest that this sensor is applicable as a large-scale screening tool for estrogenic compounds.

Biotechnol Bioeng, 2003 Dec 20, 84(6), 732 - 7
Metabolic characteristics of an isocitrate dehydrogenase defective derivative of escherichia coli BL21(DE3); Aoshima M et al.; A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced . Unlike the case of the K-12 strain, the e14 element was not found . The nucleotide divergence between these two strains was about 2% . Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3) . Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high . In addition, isocitrate lyase was not highly induced at the stationary phase . MA1935 was shown to be a good host strain for ICDH gene expression .

Biotechnol Bioeng, 2003 Dec 20, 84(6), 686 - 99
Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli; Canovas M et al.; The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed . Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors . In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system . The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h) . Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process . The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase . More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed .

J Immunother, 2003 Nov-Dec, 26(6), 461 - 7
Pattern of gene expression and immune responses to Semenogelin 1 in chronic hematologic malignancies; Zhang Y et al.; Normal testicular-specific proteins are frequently aberrantly expressed by tumor cells . Based on this, we have investigated Semenogelin 1, a major protein of human semen coagulum thought to be highly specific to seminal vesicles, in leukemic cells . Using reverse transcription-polymerase chain reaction, Semenogelin 1 gene was frequently expressed in chronic myeloid leukemia (5 of 8, 62.5%) and chronic lymphocytic leukemia (5 of 12, 41.7%) but rarely in multiple myeloma (2 of 30, 6.7%) . The gene was not expressed in bone marrow or peripheral blood from healthy donors . Semenogelin 1 expression is normally confined to the testis, suggesting that it is a novel Cancer-Testis (CT) antigen . Translation of the mRNA to Semenogelin 1 protein was confirmed by Western blot analysis of tumor cell lysates and by immunocytochemistry . The recombinant Semenogelin 1 protein was used with a control Escherichia coli-derived recombinant protein in ELISA and Western blot analysis to show that high titer IgG antibodies against Semenogelin 1 were detected in some patients, suggesting the in vivo immunogenicity of the protein . Immune responses predicted gene expression by the leukemia cells . Semenogelin 1 was also frequently coexpressed with other CT antigens, Sperm protein 17 and SPAN-Xb . These results therefore indicate that Semenogelin 1 is a novel CT antigen capable of inducing B-cell responses in vivo in chronic leukemias.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13179 - 83 Epub 2003 Oct 31.
Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane; Angevine CM et al.; The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F1Fo ATP synthase is poorly understood . In the membrane-bound Fo sector of the enzyme, H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c . Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a . Aqueous access pathways have previously been mapped to surfaces of aTMH4 . Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag+ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices . Access to cAsp-61 at the center of the membrane may be mediated in part by Ag+-sensitive residues 248, 249, 251, and 252 in aTMH5 . From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2 . The Ag+-sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane . In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2-5 . The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H+ transport cycle.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13219 - 24 Epub 2003 Oct 31.
Distinct peptide signals in the UmuD and UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the ClpXP protease; Neher SB et al.; The Escherichia coli UmuD' protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates . The intracellular concentration of UmuD' is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD', and by ClpXP degradation . UmuD' is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degradation to occur . Here, we show that UmuD functions as a UmuD' delivery protein for ClpXP . UmuD can also deliver a UmuD partner for degradation . UmuD resembles SspB, a well-characterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP . The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD/D' in the cell . Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.

J Biol Chem, 2004 Jan 23, 279(4), 3096 - 110 Epub 2003 Nov 01.
Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton; Kumar N et al.; Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo . Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin . However, direct association of villin with PIP(2) has never been characterized . In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin . Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1 . We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics . Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin . Synthetic peptides analogous with these sites confirmed the binding domains . Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2) . By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin . In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin . Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin . Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin . This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).

J Bacteriol, 2003 Nov, 185(22), 6741 - 5
Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon; Sota M et al.; The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury . The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751 . Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids . Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.

J Bacteriol, 2003 Nov, 185(22), 6719 - 22
Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation; Butkus ME et al.; SecA is an ATPase and motor protein that drives protein translocation across the bacterial plasma membrane . In Escherichia coli SecA levels are regulated by the secretion needs of the cell utilizing secM, which encodes a secreted protein . Previous studies demonstrated that this regulation requires a translational pause within secM, whose duration regulates the accessibility of the secA Shine-Dalgarno sequence on secM secA mRNA . Here we provide evidence that translocon "pulling" of nascent SecM is what regulates the duration of the secM translational pause, and thus secA expression levels, thereby providing direct support for this model.

J Bacteriol, 2003 Nov, 185(22), 6707 - 11
Secretion defects that activate the phage shock response of Escherichia coli; Jones SE et al.; The phage shock protein (psp) operon of Escherichia coli is induced by membrane-damaging cues . Earlier studies linked defects in secretion across the inner membrane to induction of the psp response . Here we show that defects in yidC and sec secretion induce psp but that defects in tat and srp have no effect . We have also determined the cellular location of PspB and PspD proteins.

J Bacteriol, 2003 Nov, 185(22), 6695 - 701
Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli; Hwang J et al.; The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes . BFP filament biogenesis and function are encoded by the 14-gene bfp operon . The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography . Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex . Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space . A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.

J Bacteriol, 2003 Nov, 185(22), 6624 - 32
SoxRS-regulated expression and genetic analysis of the yggX gene of Escherichia coli; Pomposiello PJ et al.; Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage . Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli . Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS . Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins . Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS . Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site . Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription . A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain . Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain . These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

J Bacteriol, 2003 Nov, 185(22), 6609 - 14
Temperature sensing by the dsrA promoter; Repoila F et al.; Synthesis of the small regulatory RNA DsrA is under temperature control . The minimal dsrA promoter of 36 bp contains sufficient information to ensure such regulation . In vivo, we have analyzed the critical elements responsible for the temperature control of dsrA by using a collection of chimeric promoters combining various elements of the dsrA promoter and the lacUV5 promoter, which does not respond to temperature . Our results favor an RNA polymerase-DNA interaction model instead of a trans-acting factor for temperature regulation . While all of the elements of the dsrA promoter contribute to temperature-sensitive expression, the sequence of the -10 box and the spacer region are the essential elements for the thermal response of the dsrA promoter . The proper context for these promoter elements, including at least one of the flanking elements, the -35 region or the start site region, is also required . Point mutations demonstrate that the sequence of the -10 box imposes constraints on the length and the sequence of the spacer and/or its AT richness, even at low temperature . These results show a complex interdependence of different regions in the promoter for temperature regulation.

J Bacteriol, 2003 Nov, 185(22), 6600 - 8
A novel family of Escherichia coli toxin-antitoxin gene pairs; Brown JM et al.; Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability . We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12 . Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition . In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins . Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain . Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs . Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.

J Bacteriol, 2003 Nov, 185(22), 6556 - 61
Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli; Iyer R et al.; The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach . Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine alpha-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm . The arginine system afforded significant protection to wild-type E . coli cells in our acid shock experiments . The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC . Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine . Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli . Evidence for a lysine-dependent XAR system in E . coli is also presented . The protection by lysine, however, is milder than that by arginine.

J Bacteriol, 2003 Nov, 185(22), 6540 - 7
Assembly of TolC, a structurally unique and multifunctional outer membrane protein of Escherichia coli K-12; Werner J et al.; TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel alpha-beta-barrel conformation absent in the other model outer membrane proteins used in assembly studies . The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway . During its assembly, the newly translocated nascent TolC monomers are released in the periplasm . Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane . The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer . None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins . Two assembly-defective TolC mutants were isolated and characterized . One of the mutants (TolC(I106N)) was defective in the folding of nascent monomers, while the other (TolC(S350F)) was impaired in steps involving trimerization and membrane insertion of folded monomers.

J Bacteriol, 2003 Nov, 185(22), 6530 - 9
Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming; Hand NJ et al.; Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect . The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1 . Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes . Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation . Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes . We speculate that the common properties of the suppressors relate to secretion defects . The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase . Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion . Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.

J Bacteriol, 2003 Nov, 185(22), 6522 - 9
Cosmid-based system for transient expression and absolute off-to-on transcriptional control of Escherichia coli genes; Cronan JE; Cosmids are plasmids that contain the phage lambda sequences (cos) required for packaging of the phage DNA into the virion . Induction of a lambda prophage in an Escherichia coli strain carrying a cosmid results in lysates containing phage particles that are filled with cosmid DNA . However, the lysates also contain a large excess of infectious phage particles which complicate use of the packaged cosmids . I report that cosmids packaged by induction of a strain carrying a prophage with an altered cos region results in lysates containing very high levels (>10(10)/ml) of particles that contain cosmid DNA together with very few infectious phage particles . These lysates can be used to transduce cosmid DNA into all of the cells of a growing culture with minimal physiological disturbance . When the cosmid carries a conditionally active origin of replication, transductional introduction of the cosmid under nonreplicative conditions provides a system of transient expression . Transient expression has been used to make a recA strain temporarily recombination proficient and to temporarily introduce a site-specific recombinase . Transductional introduction of a cosmid also allows absolute off-to-on transcriptional control of nonessential genes . Two examples are given showing that when a strain carrying a null mutation in the gene of interest is transduced with a packaged cosmid carrying a functional copy of that gene, the expression of the gene rapidly goes from absolutely off to high-level expression . Additional possible uses of in vivo-packaged cosmids are proposed.

J Bacteriol, 2003 Nov, 185(22), 6507 - 12
Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity; Hesse L et al.; Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans . Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity . The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis . However, chlamydiae do not synthesize detectable peptidoglycan . The paradox created by these observations is known as the chlamydial anomaly . The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity . In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E . coli mutant possessing a temperature-sensitive lesion in MurC . The recombinant MurC domain was overexpressed in and purified from E . coli . It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM) . Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained . Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule . However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

J Biol Chem, 2004 Jan 30, 279(5), 3708 - 16 Epub 2003 Oct 30.
Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel; Ryazanova LV et al.; Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called alpha-kinases that display no sequence homology to conventional protein kinases . It is an unusual bifunctional protein that contains an alpha-kinase domain fused to an ion channel . The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis . However, little is known about its protein kinase activity . To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria . ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues . The kinase is specific for ATP and cannot use GTP as a substrate . ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin . Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity . The kinase requires Mg(2+) (optimum at 4-10 mM) or Mn(2+) (optimum at 3-5 mM), with activity in the presence of Mn(2+) being 2 orders of magnitude higher than in the presence of Mg(2+) . Zn(2+) and Co(2+) inhibited ChaK1-cat kinase activity . Ca(2+) at concentrations up to 1 mM did not affect kinase activity . Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg(2+) can directly modulate TRPM7/ChaK1 kinase activity in vivo.

J Biol Chem, 2004 Jan 16, 279(3), 1794 - 800 Epub 2003 Oct 31.
Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors; Zhao J et al.; Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction . Here we have determined the physical interaction between plant PLD and the only canonical alpha-subunit (Galpha) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction . PLDalpha1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Galpha . PLDalpha1 contains a sequence motif analogous to the G alpha-interacting DRY motif normally conserved in G-protein-coupled receptors . Mutation of the central Lys residue PLD(K564A) of this motif abolished the PLDalpha1-Galpha binding, whereas mutation of the two flanking residues PLD(E563A) and PLD(F565A) decreased the binding . Addition of Galpha to PLDalpha1 inhibited PLDalpha1 activity, whereas the PLD(K564A) mutation that disrupted the Galpha-PLDalpha1 binding abolished the inhibition . GTP relieved the Galpha inhibition of PLDalpha1 activity and also inhibited the binding between PLDalpha1 and Galpha . Meanwhile, the PLDalpha1-Galpha interaction stimulated the intrinsic GTPase activity of Galpha . Therefore, these results have demonstrated the direct binding between Galpha and PLDalpha1, identified the DRY motif on PLDalpha1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDalpha1 and Galpha.

J Biol Chem, 2004 Jan 16, 279(3), 1801 - 9 Epub 2003 Oct 31.
Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase; Wolfe MD et al.; Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly . One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism . We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E . coli tRNA . This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln) . Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB . Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium . Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme . In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA . Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity . These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

Mol Biol (Mosk), 2003 Sep-Oct, 37(5), 893 - 9
{Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein}; Rau VA et al.; Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit . Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration . This was considered as indirect evidence for complexing of RNA polymerase with TBP . In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit . It was assumed that E . coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.

Mol Biol (Mosk), 2003 Sep-Oct, 37(5), 885 - 92
{Inactivation of pgsA gene affects biosynthesis and secretion of Escherichia coli alkaline phosphatase}; Golovastov VV et al.; Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidylglycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli . A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process . A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA . In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.

Mol Biol (Mosk), 2003 Sep-Oct, 37(5), 810 - 9
{Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes}; Lunina NA et al.; The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli . The recombinant proteins AglB and AglA were purified to homogeneity and characterized . Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C . AglB is an oligomer of identical 55-kDa subunits capable of aggregation . This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity . The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB . Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme . AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain . It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link . Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate . For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required . Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.

J Infect Dis, 2003 Nov 1, 188(9), 1295 - 309 Epub 2003 Oct 13.
Escherichia coli K1 invasion increases human brain microvascular endothelial cell monolayer permeability by disassembling vascular-endothelial cadherins at tight junctions; Sukumaran SK et al.; We investigated the permeability changes that occur in the human brain microvascular endothelial cell (HBMEC) monolayer, an in vitro model of the blood-brain barrier, during Escherichia coli K1 infection . An increase in permeability of HBMECs and a decrease in transendothelial electrical resistance were observed . These permeability changes occurred only when HBMECs were infected with E . coli expressing outer membrane protein A (OmpA) and preceded the traversal of bacteria across the monolayer . Activated protein kinase C (PKC)-alpha interacts with vascular-endothelial cadherins (VECs) at the tight junctions of HBMECs, resulting in the dissociation of beta-catenins from VECs and leading to the increased permeability of the HBMEC monolayer . Overexpression of a dominant negative form of PKC-alpha in HBMECs blocked the E . coli-induced increase in permeability of HBMECs . Anti-OmpA and anti-OmpA receptor antibodies exerted inhibition of E . coli-induced permeability of HBMEC monolayers . This inhibition was the result of the absence of PKC-alpha activation in HBMECs treated with the antibodies.

J Infect Dis, 2003 Nov 1, 188(9), 1276 - 83 Epub 2003 Oct 14.
Identification and characterization of a phospholipase D-superfamily gene in rickettsiae; Renesto P et al.; The completion of the sequencing of the genomes of both Rickettsia conorii and R . prowazekii provides the opportunity to identify putative virulence factors within these strictly intracellular pathogens . A role for a phospholipase A(2) (PLA(2)) in rickettsial pathogenicity was hypothesized, but the corresponding gene has not been identified . We have identified a gene that encodes a putative phospholipase D (PLD) and that has been detected by Southern blotting in 11 analyzed strains of rickettsiae . The recombinant protein is dimeric and has PLD activity, as demonstrated by its capacity to release {(3)H}-choline from phosphatidyl {(3)H}-choline . This PLD is present in whole rickettsial lysates and likely is a virulence factor, because incubation of rickettsiae with an anti-PLD antibody reduced their cytotoxic activity against Vero cells . This enzyme might account for the activity previously attributed to PLA(2) and might be critical for the intracellular life of these bacteria.

Pediatr Nephrol, 2003 Dec, 18(12), 1229 - 35 Epub 2003 Oct 31.
Risk factors for poor renal prognosis in children with hemolytic uremic syndrome; Gianviti A et al.; Many factors have been proposed as predictors of poor renal prognosis in children with hemolytic uremic syndrome (HUS), but their role is still controversial . Our aim was to detect the most reliable early predictors of poor renal prognosis to promptly identify children at major risk of bad outcome who could eventually benefit from early specific treatments, such as plasmapheresis . Prognostic factors identifiable at onset of HUS were evaluated by survival analysis and a proportional hazard model . These included age at onset, prodromal diarrhea (D), leukocyte count, central nervous system (CNS) involvement, and evidence of Shiga toxin-producing Escherichia coli (STEC) infection . Three hundred and eighty-seven HUS cases were reported; 276 were investigated for STEC infection and 189 (68%) proved positive . Age at onset, leukocyte count, and CNS involvement were not associated with the time to recovery . Absence of prodromal D and lack of evidence of STEC infection were independently associated with a poor renal prognosis; only 34% of patients D(-)STEC(- )recovered normal renal function compared with 65%-76% of D(+)STEC(+), D(+)STEC(-) and D(-)STEC(+ )patients . In conclusion, absence of both D and evidence of STEC infection are needed to identify patients with HUS and worst prognosis, while D(-) but STEC(+) patients have a significantly better prognosis.

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 359 - 66 Epub 2003 Oct 31.
Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis; Kataoka M et al.; The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced . The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively . The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily . However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2 . The two CPR genes were overexpressed in Escherichia coli . The E . coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Planta, 2004 Jan, 218(3), 466 - 73 Epub 2003 Oct 31.
The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells; Lu Y et al.; Transitory starch is stored during the day inside chloroplasts and then broken down at night for export . Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown . An amylomaltase gene ( malQ) cloned from Escherichia coli is necessary for maltose metabolism in E . coli . We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants . Two mutants of Arabidopsis thaliana (L) Heynh . were identified in which the gene encoding a putative amylomaltase enzyme { disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)} was disrupted by a T-DNA insertion . Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose . In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts . At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants . These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol . We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts . As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants . Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E . coli.

J Mol Microbiol Biotechnol, 2003, 6(1), 41 - 56
Dual control by regulators, GntH and GntR, of the GntII genes for gluconate metabolism in Escherichia coli; Tsunedomi R et al.; Escherichia coli possesses two systems, GntI and GntII, for gluconate uptake and catabolism, whose genes are regulated by GntR as a repressor and GntH as an activator, respectively . Additionally, GntH exerts negative control of the GntI genes via the same binding element as that of GntR . We thus examined whether GntR involves regulation of the GntII genes or not . This regulation and the control by GntH were examined by using single-copy LACZ operon fusions and by RT-PCR, suggesting positive and negative regulation by GntR and positive regulation by GntH . Moreover, the introduction of mutations into possible GntR-binding elements revealed that both regulators share at least one of the elements . The results presented allow us to speculate that GntR initiates expression of the GntII genes, followed by their large induction by GntH when cells were grown in gluconate minimum medium . As in the case of the GntI genes, such a cross-regulation between the GntI and GntII via the two regulators may be important for cells to grow with gluconate .

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13270 - 3 Epub 2003 Oct 30.
Solution structure of a de novo protein from a designed combinatorial library; Wei Y et al.; Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins . Randomly generated sequences, however, rarely fold into well ordered protein-like structures . To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures . We have constructed focused libraries of de novo sequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent . Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designed a priori . Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure . We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences . This library is "naive" in that it has not been subjected to high-throughput screens or directed evolution . The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design . As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent . The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 is not a molten globule and forms a unique structure . These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures . These findings validate the binary-code strategy as an effective method for producing vast collections of well folded de novo proteins.

J Biol Chem, 2004 Jan 16, 279(3), 1627 - 36 Epub 2003 Oct 30.
Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit; Breitinger U et al.; The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding alpha- and gephyrin anchoring beta-subunits . To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (alpha1-(1-219)) and the large intracellular TM3-4 loop (alpha1-(309-392)) of the human GlyR alpha1-subunit were individually expressed in HEK293 cells and in Escherichia coli . The extracellular domain obtained from E . coli expression was purified in its denatured form and refolding conditions were established . Circular dichroism and Fourier-transform-infrared spectroscopy suggested approximately 25% alpha-helix and approximately 48% beta-sheet for the extracellular domain, while no alpha-helices were detectable for the TM3-4 loop . Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight . For the extracellular domain from E . coli, we found an apparent molecular weight compatible with a 15mer by gel filtration . The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of approximately 5 and 15 subunits . Likewise, for the intracellular domain from E . coli, a single molecular mass peak of approximately 49 kDa indicated oligomerization in a defined native structure . As shown by {(3)H}strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding . Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein . These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.

J Biol Chem, 2004 Jan 30, 279(5), 3121 - 31 Epub 2003 Oct 30.
Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues; Amitai G et al.; Inteins are protein-splicing domains present in many proteins . They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond . The C-terminal residue of inteins is typically an asparagine (Asn) . Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts . We studied protein-splicing activity of two inteins with atypical C-terminal residues . One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy) . Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions . We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism . Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein . All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage . The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue . We present and discuss several new models for reactions in the protein-splicing pathway.

EMBO J, 2003 Nov 3, 22(21), 5941 - 50
Sequences that direct significant levels of frameshifting are frequent in coding regions of Escherichia coli; Gurvich OL et al.; It is generally believed that significant ribosomal frameshifting during translation does not occur without a functional purpose . The distribution of two frameshift-prone sequences, A_AAA_AAG and CCC_TGA, in coding regions of Escherichia coli has been analyzed . Although a moderate level of selection against the first sequence is evident, 68 genes contain A_AAA_AAG and 19 contain CCC_TGA . The majority of those tested in their genomic context showed >1% frameshifting . Comparative sequence analysis was employed to assess a potential biological role for frameshifting in decoding these genes . Two new candidates, in pheL and ydaY, for utilized frameshifting have been identified in addition to those previously known in dnaX and nine insertion sequence elements . For the majority of the shift-prone sequences no functional role can be attributed to them, and the frameshifting is likely erroneous . However, none of frameshift sequences is in the 306 most highly expressed genes . The unexpected conclusion is that moderate frameshifting during expression of at least some other genes is not sufficiently harmful for cells to trigger strong negative evolutionary pressure.

EMBO J, 2003 Nov 3, 22(21), 5904 - 17
Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition; Skelding Z et al.; The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7 . This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA . Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion . We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA . We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.

EMBO J, 2003 Nov 3, 22(21), 5883 - 92
Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp; Bunting KA et al.; Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases . The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations . These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases . The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli . The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors . However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.

Endocrinology, 2004 Feb, 145(2), 809 - 16 Epub 2003 Oct 30.
Development of a murine model of autoimmune thyroiditis induced with homologous mouse thyroid peroxidase; Ng HP et al.; Autoimmune thyroid disease (AITD) is a common autoimmune disease . Thyroid peroxidase (TPO) is a well characterized autoantigen in AITD . Autoantibodies and autoreactive T lymphocytes to TPO are believed to play a major role in the pathogenesis of lymphocytic thyroiditis . To understand the pathogenic mechanisms of AITD and the role of TPO, we have established a mouse model of lymphocytic thyroiditis by immunizing C57Bl/6 (H-2(b)), CBA (H-2(k)), and C57Bl/6 x CBA F1 mice with recombinant murine TPO (rmTPO) ectodomain comprising amino acid residue 1-837 produced in Escherichia coli . Mice were immunized with 30 microg purified ectodomain in complete Freund's adjuvant . Antibodies against rmTPO were detected in the serum of all mice from day 21 onward . Draining lymph node cells from rmTPO-immunized animals showed dose-dependent proliferation to TPO stimulation . Mice killed at d 50 and 90 revealed variable degrees of thyroiditis with infiltration of mononuclear cells and destruction of thyroid follicles . C57Bl/6 and the F1 mice, in comparison with CBA mice, showed a greater degree of thyroiditis . There was a lack of correction between the intensity of thyroiditis and the anti-TPO response . Immunotyping of the thyroid cellular infiltrates showed predominantly CD4+ T cells and B220+ B cells but scanty CD8+ T cells . None of the control mice injected with the purified fusion partner developed anti-TPO antibodies and thyroiditis . In conclusion, a genuine autoimmune mouse model of lymphocytic thyroiditis was established using autologous mouse TPO . This new model induced with autologous TPO will lead to a better understanding of the mechanisms in destructive thyroiditis and will assist in the development of new strategies for modulating the pathogenic immune response.

Am J Physiol Gastrointest Liver Physiol, 2004 Mar, 286(3), G385 - 94 Epub 2003 Oct 30.
Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat; Yeh KY et al.; Hepcidin has been implicated as the iron stores regulator: a hepatic signaling molecule that regulates intestinal iron absorption by undefined mechanisms . The possibility that hepcidin regulates the expression of ferroportin 1 (FPT1), the basolateral iron transporter, was examined in rats after administration of LPS, an iron chelator, or His-tagged recombinant hepcidin (His-rHepc) . In the liver, LPS stimulated a biphasic increase of hepcidin mRNA with peaks of mRNA at 6 and 36 h . Concurrently, hepatic FPT1 mRNA expression decreased to minimal level at 6 h and then increased with a peak at 24-36 h . LPS also induced biphasic changes in intestinal FPT1 mRNA expression, with decreased levels at 6 h and increased expression at 48 h . Whereas the initial decrease of FPT1 coincides with an LPS-induced decrease in serum iron, both intestinal and hepatic FPT1 expression recovered, whereas serum iron concentration continued to decrease for at least 24 h . Dietary iron ingestion increased intestinal ferritin protein production but did not reduce intestinal FPT1 mRNA expression . The iron chelator pyrrolidinedithiocarbamate (PDTC) stimulated hepatic hepcidin without suppressing intestinal FPT1 expression . In PDTC-treated rats, LPS stimulated no additional hepatic hepcidin expression but did increase intestinal FPT1 expression . Administration of HisrHepc induced significant reduction of intestinal FPT1 expression . Taken together, these data suggest that hepcidin mediates LPS-induced downregulation of intestinal FPT1 expression and that the hepcidin signaling pathway involves a PDTC-sensitive step.

Virology, 2003 Oct 10, 315(1), 10 - 9
Genetic and phylogenetic characterization of the type II cyclobutane pyrimidine dimer photolyases encoded by Leporipoxviruses; Bennett CJ et al.; Shope fibroma virus and myxoma virus encode proteins predicted to be Type II photolyases . These are enzymes that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs) . When the Shope fibroma virus S127L gene was expressed in an Escherichia coli strain lacking functional CPD repair pathways, the expressed gene protected the bacteria from 70-75% of the ultraviolet (UV) light-induced cytotoxic DNA damage . This proportion suggests that Leporipoxvirus photolyases can only repair CPDs, which typically comprise approximately 70% of the damage caused by short wavelength UV light . To test whether these enzymes can protect virus genomes from UV, we exposed virus suspensions to UV-C light followed by graded exposure to filtered visible light . Viruses encoding a deletion of the putative photolyase gene were unable to photoreactivate UV damage while this treatment again eliminated 70-90% of the lethal photoproducts in wild-type viruses . Western blotting detected photolyase protein in extracts prepared from purified virions and it can be deduced that the poxvirion interior must be fluid enough to permit diffusion of this approximately 50-kDa DNA-binding protein to the sites where it catalyzes photoreactivation . Photolyase promoters are difficult to categorize using bioinformatics methods, as they do not obviously resemble any of the known poxvirus promoter motifs . By fusing the SFV promoter to DNA encoding a luciferase open reading frame, the photolyase promoter was found to exhibit very weak late promoter activity . These data show that the genomes of Leporipoxviruses, similar to that of fowlpox virus, encode catalytically active photolyases . Phylogenetic studies also confirmed the monophyletic origin of poxviruses and suggest an ancient origin for these genes and perhaps poxviruses.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 295 - 301
Identification of a triad of arginine residues in the active site of the ArsC arsenate reductase of plasmid R773; Shi J et al.; ArsC from plasmid R773 catalyzes reduction of arsenate in Escherichia coli . Arg-60, Arg-94 and Arg-107 are near the active site residue Cys-12, suggesting that they form an anion binding pocket in the active site and/or participate in catalysis . These three arginine residues were altered to a variety of other residues by site-directed mutagenesis . Only mutants with arginine-to-lysine substitutions conferred arsenate resistance in vivo, although purified R60A, R60E, R60K exhibited varying levels of enzymatic activity . The data support the hypothesis that this triad of arginine residues is involved in arsenate binding and transition-state stabilization.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 249 - 53
Prevalence of secreted autotransporter toxin gene among diffusely adhering Escherichia coli isolated from stools of children; Taddei CR et al.; In this report, we analyzed the prevalence of the sat gene in 336 Escherichia coli samples collected from stools of children with and without diarrhea in Brazil and in 100 uropathogenic E . coli strains . The results show a high correlation between diffusely adhering E . coli (DAEC) and the presence of sat (44%) in intestinal isolates . DAEC strain FBC114 expresses a 107-kDa protein, which showed 98% homology with Sat.

Bioorg Med Chem Lett, 2003 Nov 17, 13(22), 4139 - 41
An efficient enzymatic synthesis of thiamin pyrophosphate; Melnick JS et al.; Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase . This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.

Bioorg Med Chem Lett, 2003 Nov 17, 13(22), 4129 - 32
A mechanism for substrate-Induced formation of 6-hydroxyflavin mononucleotide catalyzed by C30A trimethylamine dehydrogenase; Lu X et al.; Experiments are described to determine the origin of the 6-hydroxyl group of 6-hydroxyFMN produced by the substrate-induced transformation of FMN in the C30A mutant of trimethylamine dehydrogenase . The conversion of FMN to 6-hydroxyFMN is carried out in the presence of H(2)(18)O and 18O(2), and the results clearly show that the 6-hydroxyl group is derived from molecular oxygen and not from water.

Arch Biochem Biophys, 2003 Nov 15, 419(2), 214 - 21
Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis; Carletti E et al.; The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined . Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues . The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST . To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys . The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.

Arch Biochem Biophys, 2003 Nov 15, 419(2), 139 - 46
Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase; Cho H et al.; NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins . Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme . Site-directed mutagenesis was used to examine the important roles of these residues . Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography . Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+ . Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type . However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R . Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type . Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+ . The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2) . Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type . These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.

Arch Biochem Biophys, 2003 Nov 15, 419(2), 120 - 8
Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine-palmitoyl transferase; Lightle S et al.; Recent studies have indicated that ceramide generated in the liver is secreted into the bloodstream as component of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) . This manuscript investigates the effect of host acute phase response to inflammation on lipoprotein ceramide levels . In humans, two different patterns of responses were found . One group of volunteers experienced transient increases in serum ceramide at 1.5h after LPS administration . Second group showed prolonged increases that reached up to 10-fold above the basal level and continued for up to 24h . Increases in ceramide were found only in VLDL and LDL particles . LPS administration induced similar increases in mice . These increases were accompanied by activation of secreted sphingomyelinase in serum and serine-palmitoyl transferase in liver . ASMase knockout mice retained LPS-induced increases in serum ceramide, thus suggesting that the elevation of VLDL and LDL ceramide content is attributed at least in part to activation of de novo synthesis of ceramide in the liver.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 495 - 500
A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity; Zhan J et al.; omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons . Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia . In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function . The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA . The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC) . The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L . The analgesic function was detected by using mouse hot-plate assay . After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed . The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 307 - 12
A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells; Xie Z et al.; Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms . The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments . In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced . This BsAb possesses bivalent arms specifically binding to the extracellular domain of erbB2 and monovalent Fab fragment redirecting NK cells . The recombinant protein could be expressed and purified from Escherichia coli as native proteins without refolding . It was fully functional in bispecific binding to SKBR3 and NK cells . The molecular size of this trivalent BsAb protein is larger than diabody and smaller than whole antibody and expected to have advantages for both high penetration of small antibody fragments and the slow circulation clearance of whole antibody . This novel protein may be an attractive target for further improvement and evaluation.

Planta, 2004 Jan, 218(3), 417 - 26 Epub 2003 Oct 30.
Sulfur assimilation in soybean ( Glycine max {L.} Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase; Chronis D et al.; A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a (32)P-labeled expressed sequence tag . Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide . The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases . Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family . The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine . Expression of soybean SATase in E . coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography . The purified protein exhibited SATase activity, indicating that the E . coli-expressed protein is a functionally active SATase . The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway . Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds . Reverse transcriptase-polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development . In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 682 - 5 Epub 2003 Oct 29.
Purification and partial characterization of the Pyrococcus horikoshii methylmalonyl-CoA epimerase; Bobik TA et al.; Methylmalonyl-CoA epimerase (MCE) from the hyperthermophilic archaeon, Pyrococcus horikoshii, was expressed at high levels in Escherichia coli, purified, and partially characterized . The P . horikoshii MCE enzyme was a homodimer with an apparent molecular mass of 31,700 Da . The K(m) of the enzyme for methylmalonyl-CoA was 79 microM and the k(cat) was 240 s(-1) . The P . horikoshii enzyme was extremely heat-stable and withstood boiling for 60 min without detectable loss in activity.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 698 - 704 Epub 2003 Oct 28.
Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2; Vadali RV et al.; The gene coding for alcohol acetyltransferase ( ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli . This genetically engineered strain of E . coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium . Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway . Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied . Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts . Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels . The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.

Drugs Today (Barc), 2003 Sep, 39(9), 673 - 93
STX-liposome conjugates as candidate vaccines; Uchida T; Infection with Shiga toxin-producing (Stx) Escherichia coli (STEC) currently represents a serious public health problem due to its life-threatening complications: hemorrhagic colitis and hemolytic uremic syndrome . An inability to induce neutralizing antibody in response to primary STEC infection has been reported in STEC-infected humans . Therefore, active immunization with detoxified Stx to induce the production of neutralizing antibodies against Stx is currently an attractive option . Although this would not prevent the spread of infection, it would protect against death caused by cytotoxin-producing E . coli infection . Stx coupled with liposomes effectively induced protection against challenge with lethal doses of Stx in mice and in monkeys . Unique characteristics of antigen-liposome conjugates found in our investigations are reviewed, and the possible application of Stx-liposome conjugates in vaccines for the prevention of life-threatening systemic complications caused by STEC infection is discussed . (c) 2003 Prous Science . All rights reserved.

Invest New Drugs, 2003 Nov, 21(4), 421 - 8
Effect of novel benzoylphenylurea derivatives on DNA polymerase alpha activity using the synthesome-based in vitro model system; Abdel-Aziz W et al.; Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S . National Cancer Institute . They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models . One of these agents, NSC639829, has now entered clinical trials . Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization . The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity . In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha.Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E . coli DNA polymerase I and calf thymus DNA polymerase alpha . Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds . NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E . coli and calf thymus enzymes . All of the four compounds did not show inhibitory effect on DNA polymerase delta . The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2167 - 75
A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression; Suzuki T et al.; We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7 . About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0% . REN-7-PPO showed broad substrate specificity toward various aromatic compounds . Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase . Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C . The gene encoding the PPO was cloned . Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence . We constructed a high-level expression system of this gene in Escherichia coli . The properties of the recombinant enzyme were identical that of wild-type . In conclusion, this PPO is a thermostable laccase.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2145 - 53
Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp . strain KNK65MA, and cloning of the gene; Nanba H et al.; Thiobacillus sp . strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate . The enzyme was also resistant to SH reagents . The enzyme, purified to homogeneity, was a dimer composed of identical subunits . The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively . The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E . coli cells . The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13247 - 52 Epub 2003 Oct 29.
Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease; Postel EH et al.; Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations . It is also one of several recently discovered DNases belonging to the NM23/NDK family . E . coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases . Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway . Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA . We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3'-blocking residues from the ends of duplex DNA . All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein . Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13213 - 8 Epub 2003 Oct 29.
A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs; Li Y et al.; The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43 degrees C because of a temperature-sensitive mutation in C5 protein, the protein subunit of the enzyme . Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43 degrees C compared with 30 degrees C . These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors . An analysis of the tna, secG, rbs, and his operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13561 - 6 Epub 2003 Oct 29.
Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica; Dey I et al.; The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide . However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon . Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E . histolytica that is responsible for the biosynthesis of prostaglandin (PG)E2 . PGE2 produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate . COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E . histolytica . The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR . Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels . Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba . Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE2 but not into PGD2 or PGF2alpha . COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors . Taken together, these studies reveal that E . histolytica produces PGE2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.

Virology, 2003 Oct 25, 315(2), 415 - 24
Functional analysis of the cloverleaf-like structure in the 3' untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement; Chen IH et al.; The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail . The coat protein accumulation level of the mutant, BaMV40A/DeltaABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana . This suggested that ABC domain might play an important role in BaMV RNA replication . To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/DeltaA, -/DeltaB, and -/DeltaC each lacking stem-loop A, B, and C, respectively, were created . Our results showed that accumulation of viral products of mutants BaMV40A/DeltaB and -/DeltaC were not as efficient as wild-type . On the contrary, level of accumulation of viral products of BaMV/DeltaA was similar to that of wild-type in protoplasts and inoculated leaves . Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation . Using UV cross-linking and competition experiments, it was demonstrated that the E . coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain.

Virology, 2003 Oct 25, 315(2), 313 - 21
Epitope selection from an uncensored peptide library displayed on avian leukosis virus; Khare PD et al.; Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities . The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship . To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform . In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells . In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies . A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity . The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed . No significant sequence censorship was observed in producing the virus display library from the plasmid library . Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target . The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.

AIDS Res Hum Retroviruses, 2003 Sep, 19(9), 743 - 53
Drug resistance profiles of recombinant reverse transcriptases from human immunodeficiency virus type 1 subtypes A/E, B, and C; Quan Y et al.; We have expressed purified recombinant reverse transcriptase (RT) from clinical isolates of human immunodeficiency virus subtypes B, C, and A/E in Escherichia coli . The drug sensitivities of these RTs were then determined for both nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) in cell-free RT assays . Although A/E and C viruses contained numerous polymorphisms relative to subtype B (i.e., naturally occurring variations unrelated to drug resistance), the wild-type enzymes prepared from these or subtype A/E clinical isolates displayed <2-fold differences in drug sensitivities with regard to the active triphosphate active forms of NRTIs, as compared with RT expressed from BH-10 recombinant virus . Recombinant RTs from clinical isolates of subtypes B, C, and A/E that contained multiple resistance-associated mutations displayed expected variations in levels of resistance to the intracellular active forms of 3TC, ddI, ddC, and PMPA, that is, 3TCTP, ddATP, ddCTP, and PMPApp, respectively . Subtype A/E and C RT enzymes contained only minor NNRTI polymorphisms that distinguished them from wild-type subtype B enzymes and wild-type RTs from these various subtypes showed only 1- to 4-fold variability in IC(50) values for each of nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), and calanolide A . In contrast, RT enzymes from subtype B and C viruses harboring specific NNRTI mutations were highly resistant to all four tested NNRTIs . Subtype C variants containing the novel V106M resistance codon showed cross-resistance to all approved NNRTIs in cell-free RT assays.

Biotechnol Lett, 2003 Oct, 25(19), 1643 - 5
High-throughput screening for gene libraries expressing carbohydrate hydrolase activity; Leemhuis H et al.; A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes . The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly measured using liquid cultures grown in microtiter plates without the need of separation or purification steps.

Biotechnol Lett, 2003 Oct, 25(19), 1625 - 8
Controlled rotation of biological microscopic objects using optical line tweezers; Dasgupta R et al.; Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam . By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved . Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile . We have used this approach for rotation of biological objects varying in size from 2 to 40 microm . At 25 mW trapping beam power at the object plane E . coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz . To our knowledge, this is the first report for rotation of an intracellular object.

Biotechnol Lett, 2003 Oct, 25(19), 1597 - 603
Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility; Park HJ et al.; HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death . To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E . coli . We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively . We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi . Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.

Microbiol Immunol, 2003, 47(9), 661 - 8
Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13; Borgan MA et al.; In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced . All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes . To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E . coli . Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24) . Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain . MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays . MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1 . Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes . These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.

J Cell Physiol, 2004 Jan, 198(1), 125 - 32
Gingival and dermal fibroblasts produce interleukin-1 beta converting enzyme and interleukin-1 beta but not interleukin-18 even after stimulation with lipopolysaccharide; Tardif F et al.; Epithelial cells play a critical role in periodontal disease through the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) . However, the role played by fibroblasts is still unclear . The rationale of this study was to throw light on the role of gingival fibroblasts in periodontal disease . We thus investigated the expression of IL-1 beta, IL-18, and ICE mRNA and the secretion of the corresponding proteins by human normal gingival fibroblasts before and after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli . IL-1 beta, IL-18, and ICE mRNA expression was evaluated by RT-PCR . Proteins were analyzed by Western blot and ELISA . We demonstrated that gingival fibroblasts expressed ICE mRNA . Basal expression of ICE was modulated following cell stimulation with lipopolysaccharide (5 mug/ml) . However, gingival fibroblasts expressed low levels of IL-1 beta mRNA . The expression was potentiated by LPS . The expression of IL-1 beta mRNA was followed by the secretion of IL-1 beta but not IL-18 protein . Our study suggests that fibroblasts may be involved in the defense against infections via an IL-1 beta-mediated but not an IL-18-mediated mechanism .

Jpn J Infect Dis, 2003 Aug, 56(4), 158 - 60
Safe and easy monitoring of anti-rabies antibody in dogs using his-tagged recombinant N-protein; Inoue S et al.; The virus neutralization (VN) test is a reliable indicator of adequate vaccination in animals . However, the VN test is tedious and complicated to perform . Enzyme-linked immunosorbent assay (ELISA), though rapid and simple compared to the VN test, is complicated and hazardous during preparation of the viral antigen . In an effort to overcome the disadvantage of ELISA, the recombinant His-tagged nucleoprotein (His-rNP) expressed in Escherichia coli was used as a safe antigen for ELISA (i.e., live virus was not used) . Anti-rabies antibody levels were determined by fluorescent ELISA (FELISA) using His-rNP as an antigen . The presence of anti-rabies VN antibody was determined by the rapid fluorescent focus inhibition test (RFFIT) . The VN titers by RFFIT were found to correlate well with the FITC-signal determined by the FELISA (r = 0.616) . The sensitivity and specificity of the FELISA were 91.7 and 100%, respectively . This study showed that the His-rNP could be useful as an antigen of ELISA to test for anti-rabies antibody in vaccinated dogs . Several studies in Japan have investigated the antibody level in the sera of vaccinated dogs . A safe and convenient test using His-rNP would contribute to our understanding of the status of herd immunity among not only domestic dogs but also stray dogs in Japan.

J Biol Chem, 2004 Jan 9, 279(2), 1176 - 83 Epub 2003 Oct 28.
Diacylglycerols containing Omega 3 and Omega 6 fatty acids bind to RasGRP and modulate MAP kinase activation; Madani S et al.; We elucidated the effects of different diacylglycerols (DAGs), i.e . 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG), on {3H}PDBu binding to RasGRP . The competition studies with these DAGs on {3H}PDBu binding to RasGRP revealed different Ki values for these DAG molecular species . Furthermore, we transfected human Jurkat T cells by a plasmid containing RasGRP and assessed the implication of endogenous DAGs on activation of MAP kinases ERK1/ERK2, induced by phorbol-12-myristate-13-acetate (PMA) . In control cells, GF109203X, a protein kinase C inhibitor, inhibited ERK1/ERK2 activation . However, this agent curtailed but failed to completely diminish ERK1/ERK2 phosphorylation in RasGRP-overexpressing cells, though calphostin C, a DAG binding inhibitor, suppressed the phosphorylation of MAP kinases in these cells . In cells incubated with arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), PMA induced the production of endogenous DAGs containing these fatty acids, respectively: DAG-AA, DAG-DHA, and DAG-EPA . The inhibition of production of DAG-AA and DAG-DHA significantly inhibited MAP kinase activation in RasGRP overexpressing, but not in control, cells . Our study demonstrates that three DAG molecular species bind to RasGRP, but only DAG-AA and DAG-DHA participate in the modulation of RasGRP-mediated activation of MAP kinases in Jurkat T cells.

J Biol Chem, 2004 Jan 23, 279(4), 2341 - 9 Epub 2003 Oct 28.
An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli; Hoshi H et al.; The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides . As for the synthetic mechanism, there are two alternative manners for the sugar elongation process . Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers . On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end . Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs . Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected . Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E . coli as the active form . It was demonstrated that an engineered HAS2 expressed in E . coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16) . Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end . According to the present results, large scale production of engineered recombinant HASs is to be performed using E . coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.

J Biol Chem, 2004 Jan 16, 279(3), 2316 - 23 Epub 2003 Oct 28.
Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica; Kumagai M et al.; Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized . Deduced amino acid sequences of the alpha- and beta-subunit of E . histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively . They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms . Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL . Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT . This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine . EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT . These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.

J Biol Chem, 2004 Jan 9, 279(2), 1184 - 90 Epub 2003 Oct 28.
N-terminal extension of canine glutamine synthetase created by splicing alters its enzymatic property; Shin D et al.; It was found that an extra exon exists in the first intron of glutamine synthetase gene, generated by means of alternative splicing . Inclusion of this exon decreased the translation of glutamine synthetase (GS) in human, dog, and mouse . When translated in vitro with the canine GS transcript containing the exon, we obtained two different species of GS enzymes . Besides the known 45-kDa protein, the extended form of GS was identified with additional 40 amino acids on its N-terminal end . An upstream ATG in the extra exon served as a translation initiator for the long form of GS . When the long transcript was translated in vivo in animal cells, only the long GS was expressed . On the other hand, the long GS is less predominant relative to the short one in canine tissues including brain and liver . Subcellular fractionation of canine brain revealed that the long GS is present in all cellular compartments as is the short one, which is consistent with fluorescence microscopy data obtained with green fluorescent protein fused to GS . The short (SGS) and long (LGS) forms of canine GS were purified in Escherichia coli and shown to have similar Km values for l-glutamate and hydroxylamine . However, the Km values for ATP were slightly altered, 1.3 and 1.9 mm for the short and long GSs, respectively . The Kis for l-methionine-S-sulfoximine (MSOX), a highly potent ATP-dependent inactivator of GS, were considerably different such that the values are 0.067 and 0.124 mm for the short and long forms, respectively . When the intrinsic fluorescences of tryptophans were monitored upon bindings of chloride and metal ions without any effect on the oligomeric state, the pattern of quenching in LGS was significantly different from that of SGS . Taken together, the N-terminal extension in the long isoform of GS induces a conformational change of core enzyme, leading to a change in affinity to its substrates as well as in the effector-induced conformational alterations.

J Biol Chem, 2004 Jan 30, 279(5), 3758 - 65 Epub 2003 Oct 28.
The structure of the AXH domain of spinocerebellar ataxin-1; Chen YW et al.; Spinocerebellar ataxia type 1 is a late-onset neurodegenerative disease caused by the expansion of a CAG triplet repeat in the SCA1 gene . This results in the lengthening of a polyglutamine tract in the gene product ataxin-1 . This produces a toxic gain of function that results in specific neuronal death . A region in ataxin-1, the AXH domain, exhibits significant sequence similarity to the transcription factor HBP1 . This region of the protein has been implicated in RNA binding and self-association . We have determined the crystal structure of the AXH domain of ataxin-1 . The AXH domain is dimeric and contains an OB-fold, a structural motif found in many oligonucleotide-binding proteins, supporting its proposed role in RNA binding . By structure comparison with other proteins that contain an OB-fold, a putative RNA-binding site has been identified . We also identified a cluster of charged surface residues that are well conserved among AXH domains . These residues may constitute a second ligand-binding surface, suggesting that all AXH domains interact with a common yet unidentified partner.

J Biol Chem, 2004 Jan 30, 279(5), 3212 - 7 Epub 2003 Oct 28.
Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa . A novel secreted bioluminescent reporter enzyme; Markova SV et al.; Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli . We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm) . Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase . The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885 . The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion . On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase . The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected . These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.

Spectrochim Acta A Mol Biomol Spectrosc, 2003 Nov, 59(13), 3185 - 91
Study on Escherichia coli alkaline phosphatase conformation by phosphorimetry in the presence of denaturant; Zhang HR et al.; The influence of different denaturants on the phosphorescence spectrum and lifetime decay of Escherichia coli alkaline phosphatase (AP) was investigated . Phosphorescence intensity and lifetime of tryptophan residue (Trp-109) decrease upon addition of guanidine hydrochloride, ethylene diamine tetraacetic acid, and urea or decreasing acidity . The experiments show that AP undergoes different pathways with different denaturants and that the activation energy data, DeltaS degrees (not equal) and deltaH degrees (not equal) further confirm that there is a stable intermediate state between the folded and unfolded AP states in solution.

J Mol Biol, 2003 Nov 7, 333(5), 931 - 49
Insights into catalysis by a knotted TrmD tRNA methyltransferase; Elkins PA et al.; The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution . TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer . Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain . The two bound AdoHcy moieties are buried at the bottom of deep clefts . The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur . In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis . The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring . Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.

Viral Immunol, 2003, 16(3), 321 - 33
Induction of hepatitis C virus-specific humoral and cellular immune responses in mice and rhesus by artificial multiple epitopes sequence; Li Q et al.; The investigation of antigenic epitopes in hepatitis C virus (HCV) protein suggests that a central sequence combined with multiple antigenic epitopes of HCV might be significant as a potential vaccine candidate . This artificial sequence of combined and modified multiple antigenic epitopic peptides (Hc-B2), containing three B and four T cell epitopes, was constructed and expressed in E . coli . Antigen analysis indicated that this peptide antigen was capable of interacting with anti-sera collected from hepatitis C patients infected by three genotypes of HCV from three different geographic areas of China, respectively . The immunological analysis of this peptide antigen in mice and rhesus suggested that its immunogenicity was effective . However, a complete evaluation of this peptide could not be made as an effective animal model for HCV infection (such as in the chimpanzee) was not available for this study.

OMICS, 2003 Fall, 7(3), 301 - 16
From annotated genomes to metabolic flux models and kinetic parameter fitting; Segre D et al.; Significant advances in system-level modeling of cellular behavior can be achieved based on constraints derived from genomic information and on optimality hypotheses . For steady-state models of metabolic networks, mass conservation and reaction stoichiometry impose linear constraints on metabolic fluxes . Different objectives, such as maximization of growth rate or minimization of flux distance from a reference state, can be tested in different organisms and conditions . In particular, we have suggested that the metabolic properties of mutant bacterial strains are best described by an algorithm that performs a minimization of metabolic adjustment (MOMA) upon gene deletion . The increasing availability of many annotated genomes paves the way for a systematic application of these flux balance methods to a large variety of organisms . However, such a high throughput goal crucially depends on our capacity to build metabolic flux models in a fully automated fashion . Here we describe a pipeline for generating models from annotated genomes and discuss the current obstacles to full automation . In addition, we propose a framework for the integration of flux modeling results and high throughput proteomic data, which can potentially help in the inference of whole-cell kinetic parameters.

J Proteome Res, 2003 Sep-Oct, 2(5), 543 - 52
Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins; Quach TT et al.; Hydrophobic membrane proteins often have complex functions and are thus of great interest . However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation . We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis . CNBr selectively cleaves methionine residues . But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces . To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel . The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied . We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature . This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples . The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.

Biofizika, 2003 Sep-Oct, 48(5), 812 - 3
{On some properties of spectral portraits of nucleotide sequences, computed my a matrix Fourier analysis method}; Kravatskaia GI et al.; The quantitative characteristics of a spectral portrait of the nucleotide sequence of E . coli oriC were determined by the method of matrix Fourier analysis.

Mol Cell Biol, 1982 Dec, 2(12), 1463 - 71
New properties of simian virus 40 large T antigen; Seif R; An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40 . The 8K T antigen was weakly labeled in vivo with {35S}methionine or 32Pi . A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen . The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen . Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin . The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences . Antibodies against large T antigen interacted with native recA protein.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13235 - 40 Epub 2003 Oct 27.
The two rotor components of yeast mitochondrial ATP synthase are mechanically coupled by subunit delta; Duvezin-Caubet S et al.; The mitochondrial ATP synthase is made of a membrane-integrated F0 component that forms a proton-permeable pore through the inner membrane and a globular peripheral F1 domain where ATP is synthesized . The catalytic mechanism is thought to involve the rotation of a 10-12 c subunit ring in the F0 together with the gamma subunit of F1 . An important and not yet resolved question is to define precisely how the gamma subunit is connected with the c-ring . In this study, using a doxycycline-regulatable expression system, we provide direct evidence that the rest of the enzyme can assemble without the delta subunit of F1, and we show that delta-less mitochondria are uncoupled because of an F0-mediated proton leak . Based on these observations, and taking into account high-resolution structural models, we propose that subunit delta plays a key role in the mechanical coupling of the c-ring to subunit gamma.

J Virol, 2003 Nov, 77(22), 12319 - 30
Analysis of herpes simplex virus ICP0 promoter function in sensory neurons during acute infection, establishment of latency, and reactivation in vivo; Thompson RL et al.; We have begun an analysis of the functional architecture of the ICP0 promoter in neurons in vivo with the ultimate goal of determining how this gene is regulated during reactivation in vivo . Promoter/reporter mutants in which the Escherichia coli beta-galactosidase (beta-Gal) gene was driven by various permutations of the ICP0 promoter were employed to permit the analysis of promoter function without the added complications that would arise due to inappropriate regulation of ICP0 protein levels . A whole-ganglion immunohistochemical staining procedure (N . M . Sawtell, J . Virol . 77:4127-4138, 2003) was used for direct comparisons of the expression of the promoter/reporter gene to expression of the native protein in the same cell . In this way, the expression of the putative wild-type promoter could be validated and results for mutant promoters could be compared to expression of the native gene . We found that a DNA fragment from bp -562 through the methionine start codon of the ICP0 gene contained all sequences required for properly regulated ICP0 expression in diverse cell types (including sensory neurons of the trigeminal ganglia {TG}) in vitro and in vivo, as indicated by colocalization of ICP0 and beta-Gal . Truncation of the ICP0 promoter to bp -145 or -129 resulted in the loss of immediate-early (alpha) kinetics . The truncated promoters expressed high levels of the reporter gene with leaky late (gamma1) kinetics in vitro and in some cell types in vivo . Unexpectedly, the truncated promoters did not express in TG neurons . Thus, TAATGARAT or other sequences upstream of bp -145 in the ICP0 promoter are required for basal expression of ICP0 in neurons but are not required for basal expression in other cells in vivo . There was a >95% concordance between reporter and native protein expression detected with the 562-bp promoter in neurons during the acute stage . However, this was not the case during reactivation from latency in vivo, as nearly twice as many neurons contained detectable beta-Gal as contained detectable ICP0 . This same 562-bp promoter/reporter cassette, when placed in the context of a latency-associated transcript (LAT) null mutant, resulted in >95% concordance of expression of beta-Gal and ICP0 during reactivation in vivo . These last results strongly suggest that there is a posttranscriptional constraint on the expression of ICP0 protein during reactivation from latency and that this constraint is mediated by LAT.

J Biol Chem, 2004 Jan 9, 279(2), 945 - 51 Epub 2003 Oct 26.
A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state; West JM et al.; A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution . Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy . Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C . The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering . Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate . The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission . Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission . The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical . Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate . These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

J Biol Chem, 2004 Jan 9, 279(2), 859 - 65 Epub 2003 Oct 27.
Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase activity; Covo S et al.; DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination . Pol mu is induced by ionizing radiation and exhibits low fidelity . Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism . It can also replicate across two tandem abasic sites, using the same mechanism . Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site . Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template . This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions . Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase . To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties . These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.

Biophys J, 2003 Nov, 85(5), 2977 - 87
Glycerol conductance and physical asymmetry of the Escherichia coli glycerol facilitator GlpF; Lu D et al.; The aquaglyceroporin GlpF is a transmembrane channel of Escherichia coli that facilitates the uptake of glycerol by the cell . Its high glycerol uptake rate is crucial for the cell to survive in very low glycerol concentrations . Although GlpF allows both influx and outflux of glycerol, its structure, similar to the structure of maltoporin, exhibits a significant degree of asymmetry . The potential of mean force characterizing glycerol in the channel shows a corresponding asymmetry with an attractive vestibule only at the periplasmic side . In this study, we analyze the potential of mean force, showing that a simplified six-step model captures the kinetics and yields a glycerol conduction rate that agrees well with observation . The vestibule improves the conduction rate by 40% and 75% at 10- micro M and 10-mM periplasmic glycerol concentrations, respectively . In addition, neither the conduction rate nor the conduction probability for a single glycerol (efficiency) depends on the orientation of GlpF . GlpF appears to conduct equally well in both directions under physiological conditions.

Biophys J, 2003 Nov, 85(5), 2884 - 99
Electrostatic tuning of permeation and selectivity in aquaporin water channels; Jensen MO et al.; Water permeation and electrostatic interactions between water and channel are investigated in the Escherichia coli glycerol uptake facilitator GlpF, a member of the aquaporin water channel family, by molecular dynamics simulations . A tetrameric model of the channel embedded in a 16:0/18:1c9-palmitoyloleylphosphatidylethanolamine membrane was used for the simulations . During the simulations, water molecules pass through the channel in single file . The movement of the single file water molecules through the channel is concerted, and we show that it can be described by a continuous-time random-walk model . The integrity of the single file remains intact during the permeation, indicating that a disrupted water chain is unlikely to be the mechanism of proton exclusion in aquaporins . Specific hydrogen bonds between permeating water and protein at the channel center (at two conserved Asp-Pro-Ala "NPA" motifs), together with the protein electrostatic fields enforce a bipolar water configuration inside the channel with dipole inversion at the NPA motifs . At the NPA motifs water-protein electrostatic interactions facilitate this inversion . Furthermore, water-water electrostatic interactions are in all regions inside the channel stronger than water-protein interactions, except near a conserved, positively charged Arg residue . We find that variations of the protein electrostatic field through the channel, owing to preserved structural features, completely explain the bipolar orientation of water . This orientation persists despite water translocation in single file and blocks proton transport . Furthermore, we find that for permeation of a cation, ion-protein electrostatic interactions are more unfavorable at the conserved NPA motifs than at the conserved Arg, suggesting that the major barrier against proton transport in aquaporins is faced at the NPA motifs.

Biochim Biophys Acta, 2003 Nov 3, 1652(1), 75 - 81
Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp . PCC6803; Yin X et al.; The dxr gene encoding the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp . PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR . The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration . The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively . By native PAGE, both enzymes were dimers under reducing conditions . The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme . Both enzymes had similar properties to previously characterized reductoisomerases from other sources . The K(m)'s for the metal ions Mn(2+), Mg(2+), and Co(2+) were determined for native DXR for the first time, with the K(m) for Mg(2+) being approximately 200-fold higher than the K(m)'s for Mn(2+) and Co(2+).

J Immunol Methods, 2003 Oct 1, 281(1-2), 177 - 85
Displaying and epitope mapping of CD147 on VCSM13 phages: influence of Escherichia coli strains; Tayapiwatana C et al.; The external domain of a human leukocyte surface molecule, CD147 was displayed on the surface of phage . Two Escherichia coli laboratory strains, XL-1 Blue and TG-1, were chosen to separately propagate the recombinant phages . By sandwich enzyme linked immunosorbent assay (ELISA), CD147 on phage particles were individually captured by six CD147 mAbs and subsequently detected by anti-M13 conjugated HRP . All mAbs specifically bound the CD147 on phage particles derived from TG-1 . On the contrary, only four of them could recognize the CD147 on phages produced by XL-1 Blue . The results indicate that the environment in the TG-1 periplasm is more appropriate than that of XL-1 Blue for promoting the suitable folding of CD147 . This finding emphasizes the importance of selecting the appropriate E . coli host for display of a complex protein . The epitopes of CD147 displayed on the phage were further mapped by competitive inhibition ELISA, which is a reliable and economical method . Certain clusters of mAb recognition areas were identified and will provide valuable information for the discovery of the ligand for CD147.

J Immunol Methods, 2003 Oct 1, 281(1-2), 143 - 8
Tandem IMAC-HPLC purification of a cocaine-binding scFv antibody; Moss JA et al.; Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression . However, the high purity demands of certain applications are occasionally unattainable through a single IMAC separation . GNC92H2scFv is a cocaine-binding single-chain antibody fragment that is unstable during long-term storage in aqueous solution . To circumvent this problem, a reversed-phase HPLC separation was performed following IMAC purification of GNC92H2scFv from Escherichia coli cell culture supernatant . The resulting HPLC effluent was then freeze-dried to afford a salt-free lyophilizate amenable to long-term storage with minimal loss in binding activity . HPLC purification also effectively removed an 80-kDa protein contaminant that co-eluted with the IMAC-purified protein . Of special importance for in vivo applications of recombinantly expressed protein therapeutics, an HPLC purification step afforded a 1000-fold reduction in lipopolysaccharide (LPS) endotoxin contamination in the final GNC92H2scFv product.

Cryobiology, 2003 Oct, 47(2), 93 - 101
Impact of high pressure freezing on DH5alpha Escherichia coli and red blood cells; Suppes GJ et al.; The impact of high pressure and freezing on survivability of Escherichia coli and human red blood cells was evaluated to determine the utility of high-pressure transitions for preserving living cells . Based on microscopy and survivability, high pressures did not directly impact physical damage to living cells . E . coli studies showed that increased cell death is due to indirect phenomena with decreasing survivability at increasingly high pressures and exposure times . Pressurization rates up to 1.4kbar/min had negligible effects relative to exposures of >5min at high pressures.Both glycine and control of pH near 7.0 were successful in reducing the adverse impacts of high pressure . Survivability increased from <1% at 5min exposure to 2.1kbar of pressure to typical values >20% . The combination of glycine and the buffer salt led to even further improvements in survivability . Pressure changes were used to traverse temperature and pressures consistent with Ice I and Ice III phase boundaries of pure water.

Biochim Biophys Acta, 2003 Oct 20, 1630(1), 41 - 6
tRNA hopping: effects of mutant tRNAs; O'Connor M; Movement of tRNA and mRNA through the ribosome is coupled . However, selection for suppression of a -1 frameshift mutation in Escherichia coli has yielded a class of mutant tRNAs that can violate this mechanism and "hop" or disengage from their cognate codons and re-pair downstream in the mRNA . Previously described tRNA mutants of this class included those with insertions in the anticodon of tRNA(Val)1 . This report describes further tRNA(Val)1 alterations that enhance hopping; these include a novel insertion in the anticodon loop, base substitutions in the anticodon stem and a base deletion in the variable loop . These results indicate that several different features of a tRNA are important for maintaining stable codon-anticodon interactions and coupled movement of tRNA and mRNA during the elongation phase of protein synthesis.

Mol Cell, 2003 Oct, 12(4), 959 - 70
Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli; Bregeon D et al.; Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription . Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription . Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine . Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion . Genetic analyses showed that, in E . coli, 8-oxoguanine is subject to transcription-coupled repair . Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.

Mol Cell, 2003 Oct, 12(4), 947 - 57
The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch; Hernday AD et al.; The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch . In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites . We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites . Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites . Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.

Mol Cell, 2003 Oct, 12(4), 937 - 46
Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli; Alami M et al.; The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence . TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown . Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles . TatA association is observed only in the presence of a transmembrane H(+) gradient . TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI . Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif . The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.

Mol Cell, 2003 Oct, 12(4), 903 - 11
Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translational quality control; Hayes CS et al.; Cells employ many mechanisms to ensure quality control during protein biosynthesis . Here, we show that, during the pausing of a bacterial ribosome, the mRNA being translated is cleaved at a site within or immediately adjacent to the A site codon . The extent of this A site mRNA cleavage is correlated with the extent of ribosome pausing as assayed by tmRNA-mediated tagging of the nascent polypeptide . Cleavage does not require tmRNA, the ribosomal alarmone (p)ppGpp, or bacterial toxins such as RelE which have been shown to stimulate a similar activity . Translation is required for cleavage, suggesting that the ribosome participates in the reaction in some fashion . When normal protein synthesis is compromised, A site mRNA cleavage and the tmRNA system provide a mechanism for reducing translational errors and the production of aberrant and potentially harmful polypeptides.

Mol Cell, 2003 Oct, 12(4), 799 - 800
Better late than never for repair of miscoding lesions within a transcribed template; Seeberg E et al.; Transcription does not always stall at base damage in DNA and can create mutated transcripts from miscoding lesions . In this issue of Molecular Cell, present genetic analysis of E . coli to indicate that the highly mutagenic purine modification, 8-oxoguanine, is subject to transcription-coupled repair despite transcriptional bypass and generation of mutant transcripts.

Biochemistry, 2003 Nov 4, 42(43), 12682 - 90
Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase; Watson JN et al.; Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile . Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide . The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose . A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised . For the wild-type enzyme, the value of the Bronsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups . Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining . The Bronsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively) . These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1) . Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.

Biochemistry, 2003 Nov 4, 42(43), 12676 - 81
Mechanism of substrate inhibition in Escherichia coli phosphofructokinase; Fenton AW et al.; Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP . In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition . Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms . We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3) . The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates . In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer . Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon . The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.

Biochemistry, 2003 Nov 4, 42(43), 12654 - 61
Effects of DNA adduct structure and sequence context on strand opening of repair intermediates and incision by UvrABC nuclease; Zou Y et al.; DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps . In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site . The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC . In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts . Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo{a}pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides . This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC . We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates . The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site . We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion . The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.

Biochemistry, 2003 Nov 4, 42(43), 12610 - 6
Evidence in Escherichia coli that N3-methyladenine lesions and cytotoxicity induced by a minor groove binding methyl sulfonate ester can be modulated in vivo by netropsin; Shah D et al.; The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer . We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts . While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate . We report herein the relationship between the in vivo formation of N3-methyladenine and toxicity in wild-type and base excision repair defective mutant Escherichia coli . In addition, it is demonstrated that both N3-methyladenine adduction and cytotoxicity can be inhibited in vivo with netropsin, a potent competitive inhibitor of binding of lex to DNA . The results show a clear relationship between the levels of N3-methyladenine and toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome . For methyl methanesulfonate, which does not sequence selectively methylate DNA, a relationship between the formation of N3-methyladenine and toxicity is also observed . However, netropsin affects neither the level of N3-methyladenine nor the toxicity of methyl methanesulfonate in E . coli.

Biochemistry, 2003 Nov 4, 42(43), 12596 - 609
Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine; Cayley S et al.; To better understand the biophysical basis of osmoprotection by glycine betaine (GB) and the roles of cytoplasmic osmolytes, water, and macromolecular crowding in the growth of osmotically stressed Escherichia coli, we have determined growth rates and amounts of GB, K(+), trehalose, biopolymers, and water in the cytoplasm of E . coli K-12 grown over a wide range of high external osmolalities (1.02-2.17 Osm) in MOPS-buffered minimal medium (MBM) containing 1 mM betaine (MBM+GB) . As osmolality increases, we observe that the amount of cytoplasmic GB increases, the amounts of K(+) (the other major cytoplasmic solute) and of biopolymers remain relatively constant, and the growth rate and the amount of cytoplasmic water decrease strongly, so concentrations of biopolymers and all solutes increase with increasing osmolality . We observe the same correlation between the growth rate and the amount of cytoplasmic water for cells grown in MBM+GB as in MBM, supporting our proposal that the amount of cytoplasmic water is a primary determinant of the growth rate of osmotically stressed cells . We also observe the same correlation between cytoplasmic concentrations of biopolymers and K(+) for cells grown in MBM and MBM+GB, consistent with our hypothesis of compensation between the anticipated large perturbing effects on cytoplasmic protein-DNA interactions of increases in cytoplasmic concentrations of K(+) and biopolymers (crowding) with increasing osmolality . For growth conditions where the amount of cytoplasmic water is relatively large, we find that cytoplasmic osmolality is adequately predicted by assuming that contributions of individual solutes to osmolality are additive and using in vitro osmotic data on osmolytes and a local bulk domain model for cytoplasmic water . At moderate growth osmolalities (up to 1 Osm), we conclude that GB is an efficient osmoprotectant because it is almost as excluded from the biopolymer surface in the cytoplasm as it is from native protein surface in vitro . At very high growth osmolalities where cells contain little cytoplasmic water, predicted cytoplasmic osmolalities greatly exceed observed osmolalities, and the efficiency of GB as an osmolality booster decreases as the amount of cytoplasmic water decreases.

Biochemistry, 2003 Nov 4, 42(43), 12562 - 9
Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that TM VIII faces an aqueous channel; Winkler HH et al.; The contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water-filled channel through which ATP is transported was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli . A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used . Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport . The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity . Five patterns of inhibition of ATP transport by the MTS reagents were observed . (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C . (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents . (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C) . (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C . However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate . (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES . Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition . Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles . The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.

Biotechniques, 2003 Oct, 35(4), 750 - 2, 754, 756 passim
Generation of a phagemid mouse recombinant antibody fragment library by multisite-directed mutagenesis; Kelley LL et al.; A nonimmune phagemid recombinant antibody fragment (rFab) library was generated with a nominal diversity of 1.16 x 10(7) using the QuikChange Multi Site-Directed Mutagenesis kit . Two degenerate primers spanning the third complementarity-determining region (CDR) loops of the antibody fragment light and heavy chain were mutated such that eight or nine amino acids were randomly changed per CDR loop . Seven proteins were used to evaluate the library quality . Protein-specific rFab antibodies were selected after three panning cycles . From 12% to 64% of the randomly selected colonies produced positive ELISA signals to the phagemid rFabs . Multisite-directed mutagenesis allowed a diverse rFab library to be rapidly constructed while retaining the structural framework of a Fab that had been optimized for production in Escherichia coli.

Biotechniques, 2003 Oct, 35(4), 724 - 6, 728, 730 passim
Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5; Dudley A et al.; The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin . Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis . Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases . This led us to suspect the presence of a contaminant in the purified samples . Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography . Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity . These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.

Proteins, 2003 Nov 15, 53(3), 758 - 67
The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded; Zeev-Ben-Mordehai T et al.; Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier . The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography . Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size . Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation . Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation . Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure . In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro) .

Proteins, 2003 Nov 15, 53(3), 740 - 7
The importance of explicit chain representation in protein folding models: an examination of Ising-like models; Karanicolas J et al.; A class of models that represents a protein chain as a sequence of "folded" and "unfolded" residues has recently been used to correlate rates and mechanisms of protein folding with the protein native structure . In order to better understand the conditions under which these "Ising-like" models apply, we compare results from this model to those obtained from an off-lattice model which uses the same potential function . We find that Ising-like models by construction impose folding via a highly sequential nucleation-condensation mechanism, which in turn leads to more rugged energy landscapes, fewer "pathways" to the native state, and in the specific case examined here, the cold shock protein A from Escherichia coli, a qualitative difference in the most likely order of events in folding .

Eur J Immunol, 2003 Nov, 33(11), 3186 - 95
The B subunit of Escherichia coli heat labile enterotoxin abrogates oral tolerance, promoting predominantly Th2-type immune responses; Plant A et al.; Mucosal antigen encounter usually results in a state of systemic non-responsiveness (tolerance) . This failure to mount a protective response is a major hurdle to mucosal vaccine development . Hence, the identification of safe and effective mucosal adjuvants promoting protective immunity is of critical importance . The non-toxic B subunit of Escherichia coli heat labile enterotoxin(EtxB) is a potent nasal adjuvant; however, its usefulness following oral delivery is unconfirmed . We used DO11.10 chimeric mice to assess whether EtxB could abrogate tolerance to oral OVA . We show that admixing EtxB with OVA for oral immunization abrogates oral tolerance and results in a weak anti-OVA immune response . Importantly, EtxB profoundly modulated the nature of the response to subsequent parenteral challenge, promoting IgG1 in favor of IgG2a antibodies and depressing IFN-gamma production while elevating TGF-beta secretion . The addition of EtxB promoted T cell division, as assessed by loss of staining with carboxyfluorescein diacetate succinimidyl ester . Enhanced cell division promoted by EtxB was associated with T cell differentiation (increased numbers of CD45RBlow cells) in vivo, although dividing OVA-specific T cells were CD25- . These data suggest that although EtxB is a weak oral adjuvant, it can profoundly modulate the nature of the immune response to admixed antigen.

Eur J Immunol, 2003 Nov, 33(11), 2980 - 6
Human dendritic cells respond to Porphyromonas gingivalis LPS by promoting a Th2 effector response in vitro; Jotwani R et al.; Understanding how mucosal pathogens modulate the immune response may facilitate the development of vaccines for disparate human diseases . In the present study, human monocyte-derived DC (MDDC)were pulsed with LPS of the oral pathogen Porphyromonas gingivalis and Escherichia coli 25922 and analyzed for: (i) production of Th-biasing/inflammatory cytokines; (ii) maturation/costimulatory molecules; and (iii) induction of allogeneic CD4+ and naive CD45RA+ T cell proliferation and release of Th1 or Th2 cytokines . We show that E . coli LPS-pulsed MDDC released Th1-biasing cytokines - consisting of high levels of IL-12 p70, IFN-gamma-inducible protein 10 (IP-10) - but also TNF-alpha, IL-10, IL-6 and IL-1beta . In contrast, no IL-12 p70 or IP-10, and lower levels of TNF-alpha and IL-10 were induced by P . gingivalis LPS . These differences were sustained at LPS doses that yielded nearly equivalent maturation of MDDC; moreover the T cell response was consistent: E . coli LPS-pulsed MDDC induced higher T cell proliferation, and T cells released more IFN-gamma and IL-2, but less IL-5 than T cells co-cultured with P . gingivalis LPS pulsed-MDDC . IL-13 was secreted by naive CD45RA+CD45RO-CD4+ T cells in response to P . gingivalisLPS-pulsed MDDC . These results suggest that human MDDC can be polarized by LPS from the mucosal pathogen P . gingivalis to induce a Th2 effector response in vitro.

Plant Cell Rep, 2004 Jan, 22(6), 422 - 9 Epub 2003 Oct 25.
Cloning and expression of 1-aminocyclopropane-1-carboxylate synthase cDNA from rosa (Rosa x hybrida); Wang D et al.; The role of 1-aminocyclopropane-1-carboxylate (ACC) synthase in rose flower petal senescence was investigated . A cDNA library from senescing petals of rose ( Rosa x hybrid cv . Kardinal) prepared in lambdacDNA ZAP Express Vector was probed with a rose-specific 400-bp probe, and seven putative positive ACC synthase clones were isolated . Except for differences in length, the sequences of these clones were identical . A full-length clone, RKacc7, 1,750 bp long, coded for an open reading frame of 480 amino acids that contained the 11 conserved amino acid residues, the substrate and pyridoxal 5'-phosphate binding sites, all of which are characteristic of all ACC synthases . The transcripts prepared in vitro from the full-length clone when translated in rabbit reticulocyte lysates exhibited a 55-KDa polypeptide that comigrated with a polypeptide synthesized from a mRNA fraction isolated from senescing petals, and both were immunoselected by anti-ACC synthase antibodies . Reverse transcriptase-PCR-based studies showed that in planta RKacc7 is specifically expressed in rose petals, ovary and sepals . The expression of ACC synthase increased dramatically as the flower matured to senescence and also correlated positively with ethylene levels . The results of genomic Southern blots probed with RKacc7 are consistent with a pattern expected from a multigene family.

Transplantation, 2003 Oct 27, 76(8), 1201 - 7
Selection of lowly immunogenic and highly tolerogenic donor and recipient allochimeric class I major histocompatibility complex proteins; Perez J et al.; BACKGROUND: Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1.An rat alloantigen . We examined the potential of alpha1h-RT1.An versus alpha2h-RT1.An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts . METHODS: The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1.An AAs . Allochimeric and wild-type (WT)-RT1.An proteins were generated in an Escherichia coli expression system . RESULTS: A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1.An protein; alpha2h148-179n-RT1.Ac protein was ineffective . However, subcutaneous injection of 100 mug WT-RT1.An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts . Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay . In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting . Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation . CONCLUSIONS: The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

J Physiol . 2003 Oct 24; {Epub ahead of print}
Transference of Recombinant VE-cadherin Cytoplasmic Domain Alters Endothelial Junctional Integrity and Microvascular Permeability; Guo M et al.; VE-cadherin constitutes endothelial adherens junctions through a homophilic binding of its extracellular domain and by anchoring of its intracellular domain to actin cytoskeleton via catenins . The aim of this study was to determine the functional importance of VE-cadherin- cytoskeleton association in the maintenance of endothelial junctional integrity . A recombinant VE- cadherin cytoplasmic domain (rVE-cad CPD) was expressed in E . coli and purified through Ni-NTA spin columns . Immunoprecipitation assays showed that rVE-cad CPD was able to bind beta-catenin in vitro and to compete with endogenous VE-cadherin for binding of beta- catenin in human umbilical vein endothelial cells . A significant increase in the transendothelial flux of albumin was observed in the endothelial cell monolayers transfected with rVE-cad CPD . Importantly, transfection of rVE-cad CPD into intact isolated coronary venules markedly elevated albumin permeability of the venular endothelium . In addition, immunofluorescence microscopic analysis revealed a conformational change of VE-cadherin from a uniform, continuous distribution along the cell membrane under control conditions to a diffuse, grid-like pattern after rVE-cad CPD- transfection . The effects were likely due to an attenuated anchorage of endogenous VE-cadherin to the cytoskeleton, as evidenced by a decreased partitioning of VE-cadherin in the detergent-insoluble cytoskeletal pool . The results suggest that the intracellular association of VE-cadherin with beta-catenin-linked cytoskeleton is essential to the maintenance of endothelial junctional integrity and microvascular permeability.

Mol Cancer Ther, 2003 Oct, 2(10), 949 - 59
Mechanistic studies of a novel human fusion toxin composed of vascular endothelial growth factor (VEGF)121 and the serine protease granzyme B: directed apoptotic events in vascular endothelial cells; Liu Y et al.; The serine protease granzyme B (GrB; 25 kDa) is capable of inducing apoptosis through both caspase-dependent and caspase-independent mechanisms . We designed a novel vascular-targeting fusion construct designated as GrB/vascular endothelial growth factor (VEGF)121, which is composed of a non-heparin-binding isoform of VEGF and the proapoptotic pathway enzyme GrB fused via a short, flexible tether (G4S) . The chimeric fusion gene was then cloned into a bacterial vector, and the protein was expressed in Escherichia coli and purified by nickel-NTA metal affinity chromatography . Western blotting confirmed incorporation of both VEGF121 and GrB proteins into the construct . GrB/VEGF121 specifically bound (ELISA) to porcine aortic endothelial (PAE)/FLK-1 cells overexpressing the FLK-1/KDR receptor but not to cells overexpressing the FLT-1 receptor . Immunofluoresence studies showed that the GrB moiety of GrB/VEGF121 was delivered efficiently and rapidly into the cytosol of PAE/FLK-1 cells but not into that of PAE/FLT-1 cells after 4 h treatment with GrB/VEGF121 . Treatment of cells with GrB/VEGF121 showed that the IC50 was approximately 10 nM against PAE/FLK-1 cells; however, there were no cytotoxic effects observed on PAE/FLT-1 cells at doses up to 200 nM . GrB/VEGF121 induced apoptotic events specifically on PAE/FLK-1 as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, DNA laddering, and cytochrome c release from mitochondria . In addition, the fusion construct mediated the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase in target endothelial cells within 4 h after treatment . In conclusion, delivery of the human proapoptotic pathway enzyme GrB to tumor vascular endothelial cells or to tumor cells may have significant therapeutic potential and represents a potent new class of targeted therapeutic agents with a unique mechanism of action.

J Biol Chem, 2004 Jan 16, 279(3), 1659 - 64 Epub 2003 Oct 25.
SecYEG proteoliposomes catalyze the Deltaphi-dependent membrane insertion of FtsQ; van der Laan M et al.; In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase . Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway . We now demonstrate that the signal recognition particle-dependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG . SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect . These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.

J Endotoxin Res, 2003, 9(5), 313 - 6
The role of STAT1 in viral sensitization to LPS; Durbin J et al.; The phenomenon of endotoxin sensitization by virus infection is well documented but not yet well understood . Infection by virtually any viral agent will quickly induce expression of type I interferons (IFN-alpha/beta), and type II IFN-gamma production will follow as NK cells and T cells are activated . It has been well established that type II IFN pretreatment can intensify the effects of endotoxin . We have recently demonstrated that type I IFN induction by lymphocytic choriomeningitis virus (LCMV) infection dramatically increases TNF-alpha production following LPS treatment, and that this sensitization by type I IFN is STAT1 dependent . Taken together these data suggest that the STAT1-mediated, MyD88-independent, arm of the LPS signaling pathway plays an important role in endotoxin toxicity, and that this pathway mediates a major component of virus-enhanced LPS sensitization.

Mol Cell Biochem, 2003 Oct, 252(1-2), 273 - 8
Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity; Kelley PB et al.; alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins . The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress . Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins . F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E . coli and subsequent purification of the protein by size-exclusion chromatography . Secondary and tertiary structure analyses showed some structural changes in the mutant . Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure . The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

Yi Chuan Xue Bao, 2003 Sep, 30(9), 830 - 4
Research on the genetic variations of a1-fucosytransferase (FUT1) gene in 26 pig breeds; Yan XM et al.; Enterotoxigenic Escherichia coli F18(ECF18) is a main pathogen that causes edema disease and post-weaning diarrhoea in piglets, and al-fucosytransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ECF18 bacteria . The genetic variations at position 307 nucleotide in open reading frame of FUT1 gene in 26 pig breeds (total 1458 individuals) from 5 western commercial pig breeds and 21 Chinese native pig breeds were investigated by PCR-RFLP . The results showed that the genetic polymorphisms of the FUT1 locus were only detected in 5 western pig breeds and the Chinese Lingao pig breed, 5 western pig breeds possessed 3 different genotypes, and Lingao pig breed had two susceptible genotypes GG and AG, while all the other 20 Chinese native pig breeds only presented the susceptible genotype GG . The results indicated that if M307G-A point mutation in the coding region of FUT1 gene was the key factor determining the expression of the ECF18 receptor, most of Chinese native pig breeds were absent of the genetic background on the resistance to ECF18 bacteria . In this case, it was inferred that the resistance gene to ECF18 might be originated from western pig breeds . In addition, it is of great importance for the conservation of Lingao pig breed as it is the only found Chinese native pig breed possessing resistance M307A allele in FUT1 gene . Generally, compared with exotic pig breeds, Chinese native pig breeds have stronger resistance to edema disease and post-weaning diarrhoea in piglets . The results suggested that further study should be done to identify and characterize putative QTL (quantitative trait locus) or/and the functional gene responsible for the resistance to ECF18 in Chinese native pig breeds.

Ukr Biokhim Zh, 2003 May-Jun, 75(3), 95 - 8
{Effect of colloid gold on physiologic-biochemical processes in Escherichia coli 1257}; Gruzina TG et al.; The influence of colloid gold on the growth processes, ATP-ase activity and extrusion of protons in Escherichia coli 1257 was studied . The particles of colloid gold exert nonmonotonous influence on these processes with different direction is such a way that small concentration of this metal (5 x 10(-7)-5 x 10(-6) mg/ml) exert stimulative effect, while higher concentrations of colloid gold result in the suppression of biological activity of the bacterial cells . The discovered peculiarities of colloid gold influence of E . coli strain may be determined by specificity of contact interaction of metal particles with the surface of bacterial cells.

Ukr Biokhim Zh, 2003 May-Jun, 75(3), 88 - 94
{Effect of ADP and GroES on interaction of molecular chaperonin GroEL with non-native lysozyme}; Marchenko NIu et al.; The interaction of the molecular chaperonin GroEL with fluorescein-labeled lysozyme in the presence of high concentrations of thiol reagent--dithiothreitol (DTT) has been studied . In case of high concentrations of DTT lysozyme loses the native conformation due to the disruption of the intramolecular disulfide bonds stabilizing its structure and effectively aggregates . It has been shown that in the presence of high concentrations of DTT and two-fold molar excess of GroEL the lysozyme tightly interacts with GroEL that essentially decreases the efficiency of its aggregation . The addition of ADP to the complex of GroEL with nonnative lysozyme noticeably decreases the interaction of the chaperonin with nonnative protein target resulting in some increase of the efficiency of its aggregation . However, the addition of the co-chaperonin GroES together with ADP (i.e . the formation of the complex of GroEL with GroES) leads to drastic weakness of the interaction of GroEL with nonnative lysozyme and the efficiency of its aggregation becomes comparable with that in the absence of GroEL.

J Leukoc Biol, 2004 Feb, 75(2), 207 - 13 Epub 2003 Oct 23.
Autologous stem-cell transplantation restores the functional properties of CD14+CD16+ monocytes in patients with myeloma and lymphoma; Dayyani F et al.; The CD14+CD16+ monocytes appear to be important to immune defense against infection, as these cells are very potent with respect to tumor necrosis factor (TNF) production, phagocytosis, and antigen presentation . Myeloablative high-dose chemotherapy (HDT) and subsequent autologous stem-cell transplantation (ASCT) are being used increasingly for therapy of hematological malignancies, but the pronounced immunosuppression renders the patients prone to infection . To determine the functional properties of CD14+CD16+ monocytes under these conditions, 15 patients with lymphoma or myeloma were examined . Before HDT, the ratio of CD14+CD16+ cells to the population of the classical CD14++ monocytes was 0.28 +/- 0.12; this ratio changed during the course of HDT and ASCT in favor of the CD14+CD16+ monocytes to a maximum of 12.4 +/- 7.8 (P<0.001) on day 3.5 +/- 1.6 after transplanation (Tx) and returned to 0.11 +/- 0.07 (P<0.001) after engraftment on day 11.3 +/- 2.2 . Although the absolute number of classical CD14++ monocytes declined to less than 1/microl at the nadir, the number of CD14+CD16+monocytes fell from 29.7 +/- 9.8/microl to 4.5 +/- 3.0/microl at the nadir and increased to 13.8 +/- 9.8/microl at the day of discharge from the hospital . Flow cytometric analysis of phagocytosis of fluorescein isothiocyanate (FITC)-labeled Escherichia coli showed that 30 +/- 10% CD14+CD16+ monocytes of patients were FITC-positive before Tx, and at engrafment, the percentage of FITC-positive cells had doubled to 60 +/- 6% (healthy controls, 41+/-7%) . When determining generation of reactive oxygen species after E . coli ingestion, the CD14+CD16+ monocytes showed a decreased response before Tx (32+/-12% positve cells), which increased to 53 +/- 24% after ASCT . The median fluorescence intensity of human leukocyte antigen (HLA)-DR expression on the CD14+CD16+ monocytes increased from 11 +/- 6 before Tx to 17 +/- 11 after Tx, and the production of TNF after lipopolysaccharide showed no remarkable difference (46+/-13 vs . 49+/-14 channels) . At the same time, expression of TNF and of HLA-DR showed a dramatic decrease in the CD14++ monocytes . Taken together after stem-cell Tx, the function of the CD14++ monocytes is impaired, and the functional properties of CD14+CD16+ monocytes recover, indicating that these cells may be important for defense against infections post-ASCT.

Nucleic Acids Res . 2003 Nov 1;31(21):e128.
Exploiting features of adenovirus replication to support mammalian kinase production; Cotten M et al.; Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products . We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system . Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity . The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells . Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

Nucleic Acids Res, 2003 Nov 1, 31(21), 6127 - 38
Transcriptional response to DNA damage in the archaeon Sulfolobus solfataricus; Salerno V et al.; Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression . Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) light-induced DNA damage . However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea . We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are efficiently repaired in vivo in the dark, suggesting that a NER pathway is active . DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB . The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics . Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D . Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.

Nucleic Acids Res, 2003 Nov 1, 31(21), 6104 - 16
Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1; Ajuh P et al.; CDC5L and PLRG1 are both spliceosomal proteins that are highly conserved across species . They have both been shown to be part of sub- spliceosomal protein complexes that are essential for pre-mRNA splicing in yeast and humans . CDC5L and PLRG1 interact directly in vitro . This interaction is mediated by WD40 regions in PLRG1 and the C-terminal domain of CDC5L . In order to determine whether this interaction is important for the splicing mechanism, we have designed peptides corresponding to highly conserved sequences in the interaction domains of both proteins . These peptides were used in in vitro splicing experiments as competitors to the cognate sequences in the endogenous proteins . Certain peptides derived from the binding domains of both proteins were found to inhibit in vitro splicing . This splicing inhibition could be prevented by preincubating the peptides with the corresponding partner protein that had been expressed in Escherichia coli . The results from this study indicate that the interaction between CDC5L and PLRG1 is essential for pre-mRNA splicing and further demonstrate that small peptides can be used as effective splicing inhibitors.

Glycobiology, 2004 Feb, 14(2), 147 - 55 Epub 2003 Oct 23.
Hydrolysis, lactonization, and identification of alpha(2 --> 8)/alpha(2 --> 9) alternatively linked tri-, tetra-, and polysialic acids; Cheng MC et al.; Alpha-(2 --> 8)/alpha(2 --> 9) alternatively linked polysialic acid (PSA) can be identified by controlled hydrolysis followed by the analysis with capillary electrophoresis (CE) . Due to the different stability of alpha(2 --> 8) and alpha(2 --> 9) linkages in acidic hydrolysis, oligosialic acids (OSAs) from the hydrolysis of alpha(2 --> 8)/alpha(2 --> 9) OSA/PSA could be classified into two groups in the CE profile . The group with an odd numerical degree of polymerization (DP) had two peaks in the CE profile, and the other group, with even number of DP, showed one peak . Each alternating alpha(2 --> 8)/alpha(2 --> 9) linked OSA contains two isomers: one starts with the alpha(2 --> 8) linkage from the nonreducing end and the other starts with the alpha(2 --> 9) linkage from the nonreducing end . Trimers and tetramers were isolated by using a Mono Q column with an HPLC system . The two trimer isomers are alpha(2 --> 8)/alpha(2 --> 9) and alpha(2 --> 9)/alpha(2 --> 8) linkages and only showed partial separation by CE . After lactonization, sialidase hydrolysis, and alkaline treatment, the two trimer isomers could be separated and identified by CE analysis, but only the alpha(2 --> 8)/alpha(2 --> 9) trimer could be converted to the dilactone in glacial acetic acid . The two tetramer isomers could be converted to four monolactones and three dilactones . These lactonized species could be identified on the basis of several principles in sialidase hydrolysis and lactonization . In conclusion, regioselectivity on the lactonization of oligosialic acids proceeds under several principles: (1) Lactonization takes place more easily in the alpha(2 --> 8) linkage than in the alpha(2 --> 9) linkage; (2) all of the positions of alpha(2 --> 8) linkages in alpha(2 --> 8)/alpha(2 --> 9) alternatively linked OSA can be lactonized regardless of external or internal carboxyl groups involved; and (3) for the site of alpha(2 --> 9) linkage, only internal carboxyl groups can be lactonized.

J Biol Chem, 2004 Jan 30, 279(5), 3743 - 8 Epub 2003 Oct 23.
Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport; Murakami S et al.; We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A . (2002) Nature 419, 587-593) . The extramembrane headpiece of the AcrB trimer contains a central pore composed of three alpha-helices . Each pore helix belongs to a different monomer . In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix . Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity . These residues are localized on one side of the pore helix, i.e . on the wall of the pore . These observations strongly indicate the important role of this pore in the drug transport process . A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 alpha-helical turns . Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.

J Biol Chem, 2004 Jan 2, 279(1), 274 - 81 Epub 2003 Oct 22.
Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase; Gross R et al.; The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits . The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site . Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding . Variant HydABC complexes were produced in W . succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2 . Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography . The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups . The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC . Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group . This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group . The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E . coli FdnI.

J Biol Chem, 2004 Jan 9, 279(2), 1090 - 9 Epub 2003 Oct 23.
Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation; Figueiredo L et al.; In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins . As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol . Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec) . We find that the groESgroEL operon of M . mazei is unable to functionally replace groESgroEL in E . coli . However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo . The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system . This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium . Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro . In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction . These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea . Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.

Vaccine, 2003 Nov 7, 21(31), 4532 - 8
Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin; Watanabe I et al.; For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (LT) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens . However, these toxin also are the causative agents of diarrhea . There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants . In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, C3d, which is fused to the secreted form of hemagglutinin (sHA-mC3d3) in the influenza-BALB/c mouse model . The fusion protein sHA-mC3d3, the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant . After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA . In addition, viral infection was completely inhibited in these mice . In contrast, in the absence of the adjuvant, only sHA-mC3d3-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge . Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination.

Biochem Biophys Res Commun, 2003 Nov 7, 311(1), 112 - 20
Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB; Chopra P et al.; The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms . These processes are carried out by specific protein kinases and phosphatases . In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein . Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues . It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB . We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB . Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum . PknA has been shown, whereas PknB has been proposed to play a role in cell division . The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M . tuberculosis.

Zhonghua Liu Xing Bing Xue Za Zhi, 2003 Oct, 24(10), 917 - 9
{Recombinant OspC identification and antigenicity detection from Borrelia burgdorferi PD91 in China}; Chen J et al.; OBJECTIVE: To recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E . coli for early diagnosis of Lyme disease . METHODS: The OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D . The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing . The antigenicity was verified with Western Blot . RESULTS: OspC gene was cloned correctly into vector PET-11D . The resultant sequence was definitely different from the published sequence . The recombinant OspC seemed to have had strong antigenicity . CONCLUSION: The findings laid basis for the studies on early diagnosis of Lyme disease.

Immunobiology, 2003, 207(5), 305 - 13
Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus; Li H et al.; In previous studies, we suggested that epitope-vaccine might be a new strategy against virus infection . Based on this hypothesis, we designed and expressed a recombinant immunogen (multi-epitope-peptide) comprising repeats of three neutralizing-epitopes (neutralizing epitopes: aa92-105, 127-133 and 183-195) of hemagglutininin (HA) of influenza virus (H3N2) in E . coli . After vaccination, the recombinant multi-epitope protein could induce a high level of antibodies with predefined multi-epitope-specificity in mice and rabbits . The epitope-specific antibodies in sera were tested using three different epitope-peptides (synthetic peptides) in ELISA assay, and the serum dilutions from 1 : 6400 to 1 : 25600 were confirmed . In western blot analysis, both the antiserum and the antibodies purified by synthetic epitope-peptide coupled sepharose columns could recognize natural HA from influenza virus particles (strain A/Wuhan/359/95 H3N2) . In hemagglutination inhibition (HI) tests, these three antisera at the dilutions from 1 : 20 to 1 : 80 showed inhibitory activity . Interestingly, antisera and purified antibodies induced by the epitope-vaccine could partially inhibit plaque-formation of influenza virus (strain A/Wuhan/359/95) on MDCK cell monolayers . These results suggest that the recombinant multi-epitope vaccine can simultaneously induce multi-antiviral activities against influenza virus, which may provide a new way to develop effective vaccines against influenza virus.

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