Anal Biochem, 2002 Jun 1, 305(1), 68 - 81 Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase; VerBerkmoes NC et al.; Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products . By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained . The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products . Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step . Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products . For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein . In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions . The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture . The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time . (c) 2002 Elsevier Science (USA). Anal Biochem, 2002 Jun 1, 305(1), 55 - 67 Immunofluorescence microchamber technique for characterizing isolated organelles; Murray JW et al.; We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers . Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays . Several studies are presented to demonstrate the utility of this technique . Kinesin was visualized in postnuclear supernatants . Golgi and endoplasmic reticulum bound quantitatively to chambers . Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules . Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor . The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange . (c) 2002 Elsevier Science (USA). Mol Cells, 2002 Apr 30, 13(2), 296 - 301 Cloning, analysis, and expression of the gene for inorganic pyrophosphatase of Aquifex pyrophilus and properties of the enzyme; Hoe HS et al.; The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced . The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase . The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase . The gene was expressed under the control of a tac promoter in E . coli . The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg(-1) protein . The native enzyme has a homotetramer of 177 amino acids . The enzyme shows optimal activity in pH 7.5 . The optimum temperature was approximately 70 degrees C . A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective. Mol Cells, 2002 Apr 30, 13(2), 259 - 63 Expression, purification, and characterization of the oligomeric states of rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum; Chae YK; The rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum was successfully overexpressed in Escherichia coli with 6x His-Tag and purified by using Ni-NTA technology . It was characterized by SDS and native polyacrylamide gel electrophoresis (PAGE), as well as size-exclusion chromatography . The protein was pure, judged by SDS-PAGE, but three or more oligomeric species were observed by native PAGE and size-exclusion chromatography . The smallest rubredoxin oxidoreductase species is the dimer . The multiple species are stable and remain in their respective oligomeric states, judged by the chromatographic and electrophoretic results . A model is proposed in order to explain the structural basis for these results. Mol Cells, 2002 Apr 30, 13(2), 185 - 93 Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis); Lim HS et al.; Autonomously replicating sequences (ARSs) are thought to occur within, or adjacent to, the matrix attachment regions (MARs) . To identify fish ARSs, MARs of the mud loach fish were obtained from nuclear matrices using a modified LIS method . These DNA fragments were screened for their ability to act as ARSs by being cloned into the ARS cloning vector, pURY19, and transformed into Saccharomyces cerevisiae . Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive control vector, pURY19-2 microm, which contained the 2 microm circle origin of yeast . In particular, one clone, pURY19-ARS223, was 18 times more efficient in back-transforming E . coli than the positive control vector . Therefore, ARS223, which has strong ARS activity in yeast, could be a good candidate for inclusion in expression vehicles that are used to transfect fish cell lines or embryos . A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus sequences, and are predicted to have hairpin loop structures, or curved or kinked DNA . In addition, the MAR-Finder program suggested that ARSs also contain MAR motifs . These include AT tracts, ORI patterns, kinked DNA, ATC tracts, and Topoisomerase II consensus sequences . The in vitro matrix binding assay confirmed that all of the cloned ARSs could associate with the nuclear matrix . This indicates that ARSs elements may be located in or near the MARs . This is the first study that has identified and characterized ARSs in fish. Mol Cells, 2002 Apr 30, 13(2), 175 - 84 Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry; Lee K et al.; Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects . In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting . We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins . Internal calibration always gave better results . However, for a large number of samples, two step calibrations (i.e . database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient . From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel. Antivir Chem Chemother, 2001 Nov, 12(6), 367 - 73 Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate; Bukhtiyarova M et al.; Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection . Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins . NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1' . The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells . The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration . The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates . A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro . The compound, designated DPC-AB9144-00, inhibited approximately 75% of the NS3/4 activity at 10 microM with the NS4A/B substrate . However, no antiviral activity was detected with DPC-AB9144-00 in BVDV-infected Madin-Darby bovine kidney cells at concentrations as great as 90 pM, suggesting permeability or that other cellular-derived limitations were present. J Med Microbiol, 2002 Jun, 51(6), 484 - 90 Role of the respiratory burst in co-operative reduction in neutrophil survival by influenza A virus and Escherichia coli; Engelich G et al.; Influenza A virus (IAV)-induced impairment of neutrophil function or survival may be a cause of bacterial superinfection of IAV-infected subjects . This study was performed to determine the mechanism through which the combination of IAV and Escherichia coli co-operatively reduces neutrophil survival . Neutrophil binding of annexin-V and caspase-3 activation was significantly increased by either IAV or E . coli, supporting the concept that the micro-organisms accelerate neutrophil apoptosis . The anti-apoptotic agent granulocyte-macrophage colony stimulating factor (GM-CSF) did not improve, but further reduced, survival of neutrophils treated with IAV and E . coli . As addition of E . coli resulted in greater neutrophil uptake of IAV and greater neutrophil respiratory burst responses to IAV, this study tested whether respiratory burst activation by IAV and E . coli contributes to reducing neutrophil survival . The cell-permeant NADPH oxidase inhibitor, diphenylene iodonium, significantly increased survival of neutrophils treated with either E . coli alone or the combination of IAV and E . coli . In contrast, catalase, which is not cell permeant, did not alter survival of E . coli- and IAV-treated neutrophils . Azide enhanced neutrophil hydrogen peroxide responses to IAV and E . coli, and reduced survival of these cells . These results indicate that co-operative induction of intracellular respiratory burst responses by IAV and E.coli mediates the reduced neutrophil survival caused by these pathogens in vitro. J Protein Chem, 2002 Mar, 21(3), 177 - 85 Expression, characterization, and reaction of recombinant monkey metallothionein-1 and its C33M mutant; Yu WH et al.; After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system . The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant) . The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra . The reactions of MI-1 and C33M mutant with 5,5'-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied . The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra . The mutation of cysteine-to-methionine at position 33 mostly maintains the alpha-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain. J Protein Chem, 2002 Mar, 21(3), 169 - 75 Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin; Watanabe Y; Reassembly of OmpF porin from its denatured monomer into the sodium dodecyl sulfate-resistant species was investigated by using 27 kinds of mild surfactants . Polyethyleneoxide-type surfactants with a hydrophilic-lipophilic balance value of 10.8-14.6 induced the trimerization of denatured OmpF porin . Dimerization and trimerization were induced by non-polyethyleneoxide-type mild surfactants that are generally used for membrane protein solubilization . The dependence of surfactant concentrations on reassembly was estimated to obtain a minimal concentration required for the reassembly of the protein . Extensive reassembly (to approximately 85% yield) into dimer (a putative assembly intermediate) was observed at a protein concentration of 0.05 mg/ml in 7 mg/ml n-octyl-beta-D-glucopyranoside and 1 mg/ml sodium dodecyl sulfate . This condition will be useful for the studies of the dimer and dimerization of OmpF porin . The role of mixed micelle system on the protein renaturation was discussed. J Protein Chem, 2002 Mar, 21(3), 137 - 43 Aggregation of recombinant bovine granulocyte colony stimulating factor in solution; Bartkowski R et al.; Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE . Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50 degrees C to induce aggregation . The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50 degrees C . Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4 . It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates . The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution . The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE. J Biomol NMR, 2002 Apr, 22(4), 317 - 31 Comparison of protein solution structures refined by molecular dynamics simulation in vacuum, with a generalized Born model, and with explicit water; Xia B et al.; The inclusion of explicit solvent water in molecular dynamics refinement of NMR structures ought to provide the most physically meaningful accounting for the effects of solvent on structure, but is computationally expensive . In order to evaluate the validity of commonly used vacuum refinements and of recently developed continuum solvent model methods, we have used three different methods to refine a set of NMR solution structures of a medium sized protein, Escherichia coli glutaredoxin 2, from starting structures calculated using the program DYANA . The three different refinement protocols used molecular dynamics simulated annealing with the program AMBER in vacuum (VAC), including a generalized Born (GB) solvent model, and a full calculation including explicit solvent water (WAT) . The structures obtained using the three methods of refinements were very similar, a reflection of their generally well-determined nature . However, the structures refined with the generalized Born model were more similar to those from explicit water refinement than those refined in vacuum . Significant improvement was seen in the percentage of backbone dihedral angles in the most favored regions of phi, psi space and in hydrogen bond pattern for structures refined with the GB and WAT models, compared with the structures refined in vacuum . The explicit water calculation took an average of 200 h of CPU time per structure on an SGI cluster, compared to 15-90 h for the GB calculation (depending on the parameters used) and 2 h for the vacuum calculation . The generalized Born solvent model proved to be an excellent compromise between the vacuum and explicit water refinements, giving results comparable to those of the explicit water calculation . Some improvement for phi and psi angle distribution and hydrogen bond pattern can also be achieved by energy minimizing the vacuum structures with the GB model, which takes a much shorter time than MD simulations with the GB model. J Dairy Sci, 2002 Apr, 85(4), 774 - 81 Efficacy of immunization with ferric citrate receptor FecA from Escherichia coli on induced coliform mastitis; Takemura K et al.; The effects of immunization with the ferric citrate receptor FecA on antibody responses and on experimentally induced mastitis following intramammary challenge were investigated . Twenty-one cows were assigned to seven blocks of three cows based on expected parturition . Cows within block were randomly assigned to one of three treatments: 1) FecA immunization, 2) Escherichia coli J5 immunization, and 3) unimmunized controls . Challenge was by infusion of approximately 60 cfu of E . coli 727 into one uninfected mammary gland between 13 and 31 d after parturition . Cows within block were challenged on the same day . Cows immunized with FecA had higher immunoglobulin (Ig)G titers against FecA in serum and in mammary secretions at calving, immediately before challenge, and 7 d after challenge than did cows immunized with E . coli J5 or control cows . Immunization with FecA also increased IgG titers against whole-cell E . coli 727 in serum and in mammary secretions at calving . Serum IgM titers against FecA were higher in FecA immunized cows than in other treatment groups immediately before challenge . Bacterial counts in milk, duration of bacterial isolation in milk, rectal temperature, and milk somatic cell counts following intramammary challenge were similar among treatments . Milk production and dry matter intake did not differ among treatments . The ferric citrate receptor FecA was immunogenic in cows, but immunization had minimal effect on the clinical severity of experimentally induced E . coli mastitis. J Dairy Sci, 2002 Apr, 85(4), 765 - 73 Development of anti-bovine TNF-alpha mAb and ELISA for quantitating TNF-alpha in milk after intramammary injection of endotoxin; Paape MJ et al.; Murine mAb reactive with recombinant bovine tumor necrosis factor-alpha (r-boTNF-alpha) were produced . An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-alpha to sandwich bovine TNF-alpha was developed . Secretion of TNF-alpha in quarter milk increased 1 h after injection of 0.1 mg (four cows) or 0.5 mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5 h later, and returned to control levels in 24 h . There were no differences in body temperature, SCC, TNF-alpha, and blood leukocyte responses between 0.1 and 0.5 mg of LPS . To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1 mg of LPS into the same quarter (two cows) 24 h after the first injection produced a strongly attenuated TNF-alpha response . However, a normal TNF-alpha response was observed when LPS was injected into a contralateral quarter (two cows) 24 h after the first LPS injection . Leukocyte counts in blood decreased and body temperature increased substantially after each injection of LPS . Quarter milk SCC increased 200-fold 8 to 12 h after the LPS injections . It would appear that these changes were not regulated by TNF-alpha secretion because the changes were also similar after the second injection of LPS into the same mammary quarter. Appl Biochem Biotechnol, 2002 Spring, 98-100, 791 - 802 Intracellular release of recombinant green fluorescent protein (gfp(uv)) from Escherichia coli; Penna TC et al.; The recombinant green fluorescent protein (gfp(uv)) was expressed by Escherichia coli DH5-alpha cells transformed with the plasmid pGFPuv . The gfp(uv) was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (-70 degrees C for 15 h), by four freeze (-20 degrees C)/thaw cycles interlaid by sonication . The average content of released gfp(uv) (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (-70 degrees C) and the first, second, third and fourth freeze/thaw cycles, respectively . Superfusion on freezing was observed between -11 degrees C and -14 degrees C, after which it reached -20 degrees C at 0.83 degrees C/min. Int J Hyg Environ Health, 2002 Mar, 205(1-2), 103 - 13 Mapping polycyclic aromatic hydrocarbon and aromatic amine-induced DNA damage in cancer-related genes at the sequence level; Tang MS et al.; Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis . In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair . Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential . Recently, using the E . coli nucleotide excision enzyme complex, UvrABC nucleases in combination with ligation-mediated polymerase chain reaction, we developed a method to map DNA damage in the p53 and ras genes . These studies led us to conclude that targeted DNA damage, in combination with growth selection, contributes greatly in shaping the mutation spectrum in these genes in human cancer . Here we present the rationale and details of this approach, typical experimental results and necessary precautions. Pac Health Dialog, 2001 Mar, 8(1), 110 - 4 Epidemiological applications: a case report of a village epidemic of gastroenteritis; Asuzu MC et al.; This is a case report of an epidemic of gastroenteritis which was investigated and controlled by epidemiological methods only, before laboratory investigations could be done to confirm the original epidemiological conclusions--from contaminated home made ice-cubes . The case and process are reported in order to encourage similar uses of epidemiology by field public health practitioners, especially within the district or primary health care systems and particularly in places where laboratory support are difficult to avail . The case is used also to discuss the equipments and facilities that ought to be part of the support system for every modern field public health practitioner . These should include computers, modern communication facilities and epidemiological support systems, especially senior epidemiologists; as such senior personnel are available to junior colleagues in the other areas of specialist medical practice. J Biol Chem, 2002 Jul 26, 277(30), 26852 - 7 Epub 2002 May 16. Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase; Macol CP et al.; Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme . Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit . In the first case the inactive catalytic subunit had Arg-54 replaced by alanine . The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme . In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine . The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state . Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition . The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions . The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions . Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure . These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme . If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state. J Biol Chem, 2002 Jul 26, 277(30), 27282 - 7 Epub 2002 May 16. Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor; Tam C et al.; Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses . The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase . It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase . RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA . Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183 . Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA . A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition. J Biol Chem, 2002 Aug 2, 277(31), 27733 - 41 Epub 2002 May 16. Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase; Chrestensen CA et al.; The carboxyl-terminal domain (CTD) of the p90 ribosomal S6 kinases (RSKs) is an important regulatory domain in RSK and a model for kinase regulation of FXXFXF(Y) motifs in AGC kinases . Its properties had not been studied . We reconstituted activation of the CTD in Escherichia coli by co-expression with active ERK2 mitogen-activated protein kinase (MAPK) . GST-RSK2-(aa373-740) was phosphorylated in the P-loop (Thr(577)) by MAPK, accompanied by increased phosphorylation on the hydrophobic motif site, Ser(386) . Activated GST-RSK2-(aa373-740) phosphorylates synthetic peptides based on Ser(386) . The peptide RRQLFRGFSFVAK, which was termed CTDtide, was phosphorylated with K(m) and V(max) values of approximately 140 microm and approximately 1 micromol/min/mg, respectively . Residues Leu at p -5 and Arg at p -3 are important for substrate recognition, but a hydrophobic residue at p +4 is not . RSK2 CTD is a much more selective peptide kinase than MAPK-activated protein kinase 2 . CTDtide was used to probe regulation of hemagglutinin-tagged RSK proteins immunopurified from epidermal growth factor-stimulated BHK-21 cells . K100A but not K451A RSK2 phosphorylates CTDtide, indicating a requirement for the CTD . RSK2-(aa1-389) phosphorylates the S6 peptide, and this activity is inactivated by S386A mutation, but RSK2-(aa1-389) does not phosphorylate CTDtide . In contrast, RSK2-(aa373-740) containing only the CTD phosphorylates CTDtide robustly . Thus, CTDtide is phosphorylated by the CTD but not the NH(2)-terminal domain (NTD) . Epidermal growth factor activates the CTD and NTD in parallel . Activity of the CTD for peptide phosphorylation correlates with Thr(577) phosphorylation . CTDtide activity is constrained in full-length RSK2 . Interestingly, mutation of the conserved lysine in the ATP-binding site of the NTD completely eliminates S6 kinase activity, but a similar mutation of the CTD does not completely ablate kinase activity for intramolecular phosphorylation of Ser(386), even though it greatly reduces CTDtide activity . The standard lysine mutation used routinely to study kinase functions in vivo may be unsatisfactory when the substrate is intramolecular or in a tight complex. J Biol Chem, 2002 Jul 26, 277(30), 26987 - 93 Epub 2002 May 16. 1,N(2)-ethenoguanine, a mutagenic DNA adduct, is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase; Saparbaev M et al.; The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro . Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG from defined oligonucleotides containing a single modified base . A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)) . Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity . Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG when it is opposite dG . Using cell-free extracts from genetically modified E . coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N(2)-epsilonG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG . Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N(2)-epsilonG residues in vivo. Bioinformatics, 2002 Apr, 18(4), 507 - 12 Compensation for nucleotide bias in a genome by representation as a discrete channel with noise; Schreiber M et al.; MOTIVATION: Calculation of the information content of motifs in genomes highly biased in nucleotide composition is likely to lead to overestimates of the amount of useful information in the motif . Calculating relative information can compensate for biases, however the resulting information content is the amount seen by an observer and not by a macromolecule binding to the motif . The latter is needed to calculate the discriminatory power of the motif and to compare motifs between species . RESULTS: By treating a biased genome as a discrete channel with noise, in accordance with Shannon Information Theory, we were able to remove both 'Distortion' and 'Noise' from the motif and recover a more instructive biological 'signal.' A Java application, LogoPaint, was developed to remove nucleotide bias distortion and triplet frequency noise from motifs, calculate information content and present the motif as a logo . We demonstrate how this technique can 'unmask' motifs in the translation initiation regions of bacteria that are obscured by strong sequence biases . AVAILABILITY: LogoPaint is available to all users from the authors as an executable JAR file . Source code is available by arrangement. Cell, 2002 May 3, 109(3), 383 - 96 OxyR: a molecular code for redox-related signaling; Kim SO et al.; Redox regulation has been perceived as a simple on-off switch in proteins (corresponding to reduced and oxidized states) . Using the transcription factor OxyR as a model, we have generated, in vitro, several stable, posttranslational modifications of the single regulatory thiol (SH), including S-NO, S-OH, and S-SG, and shown that each occurs in vivo . These modified forms of OxyR are transcriptionally active but differ in structure, cooperative properties, DNA binding affinity, and promoter activities . OxyR can thus process different redox-related signals into distinct transcriptional responses . More generally, our data suggest a code for redox control through which allosteric proteins can subserve either graded (cooperative) or maximal (noncooperative) responses, and through which differential responsivity to redox-related signals can be achieved. J Helminthol, 2002 Jun, 76(2), 165 - 70 Molecular cloning and characterization of a novel protein of Trichinella pseudospiralis excretory-secretory products; Nagano I et al.; A novel excretory-secretory (ES) protein of Trichinella pseudospiralis was produced . A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products . A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass) . A database search revealed that no sequences had a homology to this predicted protein . The recombinant protein was produced in an Escherichia coli expression system . Stage specific expression of this protein was suggested from the following experiments . An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i . muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i . muscle larvae . The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i . muscle larvae . The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i . muscle larvae and adult worms. Arch Virol, 2002 May, 147(5), 917 - 28 In vivo accumulation of Broad bean wilt virus 2 VP37 protein and its ability to bind single-stranded nucleic acid; Qi YJ et al.; The VP37 protein encoded by the RNA2 of Broad bean wilt virus 2 (BBWV2) was overexpressed in Escherichia coli . The protein was purified and a polyclonal antibody specific for the protein was produced . Time course studies by Western blot assays in BBWV2-infected Chenopodium quinoa leaves showed that the VP37 protein was present in cells of the inoculated leaves by 12 h post inoculation and in cells of systemically-infected leaves by 2 days post inoculation . The protein was able to accumulate to a high level in infected leaves at the late infection stage . Gel retardation and UV cross-linking assays demonstrated that the VP37 protein bound preferentially single-stranded (ss) RNA and DNA in a non-sequence-specific manner . The VP37 protein-RNA complex was stable in solutions containing less than 400 mM NaCl, but became fully dissociated in the solutions containing 800 mM NaCl . Sequence analysis of the VP37 protein and its ability to bind ssRNA and ssDNA suggest that the protein may play a role similar to the movement proteins reported for other plant viruses. Appl Microbiol Biotechnol, 2002 May, 58(6), 767 - 71 Epub 2002 Mar 19. Cloning, sequencing and overexpression of a Sinorhizobium meliloti M5N1CS carboxymethyl-cellulase gene; Michaud P et al.; The EndS encoding sequence was isolated from Sinorhizobium meliloti M5N1CS DNA . Comparisons between the deduced amino acid sequence of the mature EndS (337 amino acids, molecular mass 36,418 Da, isoelectric point 4.92) and those of published beta-glycanases showed that this enzyme belongs to family 5 of the glycoside hydrolases . The protein was overproduced in Escherichia coli using a T7 expression system . When the purified overexpressed EndS protein was tested on cellulose-type components, the best substrate was CM-cellulose. Nat Struct Biol, 2002 Jun, 9(6), 419 - 24 The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase; Schiene-Fischer C et al.; Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues . Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases) . APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds . Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D (1)H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed . The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system . Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP . These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides. J Cardiovasc Pharmacol, 2002 Jun, 39(6), 858 - 65 Adenovirus-mediated transfer of ribozyme targeting platelet-derived growth factor A-chain mRNA inhibits growth of vascular smooth muscle cells from spontaneously hypertensive rats; Hu WY et al.; Platelet-derived growth factor (PDGF) is a potent stimulator of growth of vascular smooth muscle cells (VSMCs) . VSMCs from spontaneously hypertensive rats (SHRs) show exaggerated growth and increasingly express PDGF A-chain messenger RNA (mRNA) . To examine adenovirus-mediated transfer of a ribozyme targeting the PDGF A-chain mRNA as a possible gene therapy for vascular proliferative diseases, a recombinant adenovirus vector encoding a ribozyme that targets rat PDGF A-chain mRNA (Ad . ribozyme) was designed and synthesized and its effect on the growth of VSMCs from SHRs was investigated . This vector dose-dependently inhibited DNA synthesis in VMSCs from SHRs, whereas an adenovirus vector encoding the Escherichia coli LacZ gene (Ad . LacZ) did not affect DNA synthesis . Ad . ribozyme significantly suppressed proliferation of VSMCs from SHRs in a dose-dependent manner . Ad . LacZ had no effect . Ad . ribozyme significantly inhibited expression of PDGF A-chain mRNA and PDGF-AA protein in VSMCs from SHRs . Ad . LacZ had no effect . These results demonstrated that adenovirus-mediated transfer of a ribozyme targeting the PDGF A-chain mRNA effectively and specifically inhibited the growth of VSMCs from SHRs with suppression of PDGF A-chain mRNA and PDGF-AA protein expression . Adenovirus-mediated transfer of ribozyme targeting PDGF A-chain mRNA may be a feasible gene therapy for vascular proliferative diseases. Protein Sci, 2002 Jun, 11(6), 1552 - 7 X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation; Estevez M et al.; Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle . Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme . We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site . Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid . To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli . Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate. Protein Sci, 2002 Jun, 11(6), 1424 - 34 Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase; Noonan RC et al.; Analysis of the crystal structures for cytidine deaminase complexed with substrate analog 3-deazacytidine, transition-state analog zebularine 3,4-hydrate, and product uridine establishes significant changes in the magnitude of atomic-scale fluctuations along the (approximate) reaction coordinate of this enzyme . Differences in fluctuations between the substrate analog complex, transition-state analog complex, and product complex are monitored via changes in corresponding crystallographic temperature factors . Previously, we reported that active-site conformational disorder is substantially reduced in the transition-state complex relative to the two ground-state complexes . Here, this result is statistically corroborated by crystallographic data for fluorinated zebularine 3,4-hydrate, a second transition-state analog, and by multiple regression analysis . Multiple regression explains 70% of the total temperature factor variation through a predictive model for the average B-value of an amino acid as a function of the catalytic state of the enzyme (substrate, transition state, product) and five other physical and structural descriptors . Furthermore, correlations of atomic fluctuation magnitudes throughout the body of each complex are quantified through an auto-correlation function . The transition-state analog complex shows the greatest correlations between temperature factor magnitudes for spatially separated atoms, underscoring the strong ability of this reaction-coordinate species to "organize" enzymatic fluctuations . The catalytic significance for decreased atomic-scale motions in the transition state is discussed . A thermodynamic argument indicates that the significant decreases in local enzymatic conformational entropy at the transition state result in enhanced energetic stabilization there. Protein Sci, 2002 Jun, 11(6), 1320 - 9 Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry; Kim MY et al.; Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7) . Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model . Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process . The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX . The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein . Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group . Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX. J Virol, 2002 Jun, 76(12), 5949 - 58 Identification of active-site amino acid residues in the Chiba virus 3C-like protease; Someya Y et al.; The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases . To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease) . Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity . Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139 . His30, Arg89, and Asp138 could not be replaced with any other amino acids . Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage . As for position 88, a positively charged residue was required because the Arg mutant showed significant activity . As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site . Therefore, these are not catalytic residues . On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition . These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis. J Virol, 2002 Jun, 76(12), 5847 - 56 Infectious cDNA clone of the epidemic west nile virus from New York City; Shi PY et al.; We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV) . The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City . It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101 . RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 x 10(9) to 5 x 10(9) PFU/ml . The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides {nt} 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880) . These genetic markers were retained in the recovered progeny virus . Deletion of the 3'-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA . The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses . The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV. J Exp Bot, 2002 Jun, 53(373), 1521 - 4 Cloning and characterization of a phospholipase C from the C(4) plant Digitaria sanguinalis; Coursol S et al.; As a PLC activity was implicated in the light transduction pathway that controls C(4) photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned . The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca(2+) concentration, and with different sensitivity to the PLC inhibitor U-73122. J Biol Chem, 2002 Jul 26, 277(30), 27288 - 93 Epub 2002 May 20. Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling, cross-linking, and mutagenesis; Long JC et al.; The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis . 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin . The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations . This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation . Other substitutions including glutamine, alanine, and leucine were much less detrimental to function . Cross-linking studies with a photoactive cross-linking reagent were carried out . One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation . The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits. Ann N Y Acad Sci, 2002 Apr, 958, 241 - 6 Autoantibodies to IA-2 in type 1 diabetes: measurements with a new enzyme-linked immunosorbent assay; Kawasaki E et al.; The tyrosine phosphatase-like protein IA-2 is an important islet autoantigen in type 1 diabetes . Although the radioligand binding assay with in vitro synthesized (35)S-labeled antigen has been used extensively for measuring autoantibodies to IA-2, disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories . Therefore, we attempted to develop a nonradioactive ELISA for the simple detection of IA-2 autoantibodies . The biotinylated cytoplasmic domain of IA-2 expressed in Escherichia coli was used as an antigen . We evaluated two kinds of ELISA: ELISA with biotin-IA-2 directly captured on streptavidine-coated plates (solid phase) and ELISA with antigen-antibody preincubation in solution in which serum samples were reacted first with biotin-IA-2 and the mixture was transferred to streptavidine-coated plates (liquid phase) . We compared their disease sensitivity and specificity with a conventional radioligand binding assay in 52 patients with recent-onset type 1 diabetes and 138 normal individuals . The radioligand binding assay had 61.5% sensitivity and 99.3% specificity . The liquid-phase ELISA showed relatively higher sensitivity (55.8%) and specificity (99.3%) than the solid-phase ELISA (sensitivity 53.8% and specificity 97.1%) . Furthermore, the mean SD score in IA-2 autoantibody-positive serum samples measured by liquid-phase ELISA was significantly higher than the SD score obtained by solid-phase ELISA (P < 0.0001) . We concluded that this liquid-phase ELISA is suitable for detecting IA-2 autoantibodies in patients with type 1 diabetes with a similar sensitivity and specificity to those of conventional radioligand binding assay. Biol Reprod, 2002 Jun, 66(6), 1869 - 74 DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation; Davis GH et al.; Recent discoveries that high prolificacy in sheep carrying the Booroola gene (FecB) is the result of a mutation in the BMPIB receptor and high prolificacy in Inverdale sheep (FecX(I)) is the result of a mutation in the BMP15 oocyte-derived growth factor gene have allowed direct marker tests to be developed for FecB and FecX(I) . These tests were carried out in seven strains of sheep (Javanese, Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge) in which inheritance patterns have suggested the presence of major genes affecting prolificacy and in the prolific Garole sheep of India, which have been proposed as the ancestor of Australian Booroola Merinos . The FecB mutation was found in the Garole and Javanese sheep but not in Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge sheep . None of the sheep tested had the FecX(I) mutation . These findings present strong evidence to support historical records that the Booroola gene was introduced into Australian flocks from Garole (Bengal) sheep in the late 18th century . It is unknown whether Javanese Thin-tailed sheep acquired the Booroola gene directly from Garole sheep from India or via Merinos from Australia . The DNA mutation test for FecB will enable breeding plans to be developed that allow the most effective use of this gene in Garole and Javanese Thin-tailed sheep and their crosses. J Virol Methods, 2002 Jun, 104(1), 1 - 8 Identification of B cell epitopes of hepatitis C virus RNA dependent RNA polymerase; Hou L et al.; The aim of this study was to identify the B cell epitopes of hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase (RdRp) . The truncated HCV NS5B protein NS5B-dc21 was expressed in Escherichia coli and its antigenicity was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) using 130 HCV-positive human sera and 15 negative sera . Antibodies specific to NS5B-dc21 protein were purified by affinity chromatography using sepharose-4B coupled with the recombinant protein . A 12-mer phage displayed random peptide library was screened four rounds with the purified antibodies . Three epitopes were identified from the phage library, which correspond to amino acids 2444-2452, 2521-2528, and 2915-2925 of HCV RdRp . These epitopes were then expressed in E . coli as fusion proteins with phage M13 pIII protein . ELISA demonstrated that two of these epitopes (P4 and P34, corresponding to amino acids 2443-2452 and amino acids 2512-2528, respectively) have good reactivity and sensitivity . Mutagenesis study of P4 peptide showed that this epitope, which is derived from a phage displayed library, exhibited higher affinity with HCV serum than the corresponding original HCV sequences. Int J Radiat Biol, 2002 May, 78(5), 359 - 74 One-electron oxidation of plasmid DNA by selenium(V) species; Milligan JR et al.; PURPOSE: To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species . MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with 137Cs gamma-rays (662 keV) . The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), as well as auxiliary scavengers such as DMSO or glycerol . In some cases, reducing agents such as ferrocyanide were also present . After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG) . These treatments produced strand breaks in the plasmid . The yields of these strand breaks were quantified by agarose gel electrophoresis . RESULTS: In general, gamma-irradiation produced single-strand breaks (SSB) in plasmid DNA . Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold . The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation . FPG incubation produced significantly larger increases in the SSB yield after gamma-irradiation in the additional presence of selenate and/or biselenite . The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol . This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*- . The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide . The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical . CONCLUSION: By comparing the yields of breaks produced after gamma-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites . By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents . This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation. J Surg Res, 2002 May 15, 104(2), 95 - 100 Control of embryonic lung branching morphogenesis by the Rho activator, cytotoxic necrotizing factor 1; Moore KA et al.; BACKGROUND: Lung development is sensitive to physiological stresses, and its development may be impaired by physical distortion, as in patients with congenital diaphragmatic hernia . Yet, little is known about how mechanical forces can influence lung morphogenesis . Studies with cultured cells suggest that cytoskeletal tension may play a key role in growth control . Since the small GTPase Rho plays an important role in the control of cell tension generation, we carried out studies to test the hypothesis that changes in Rho-mediated cell tension may influence branching morphogenesis . METHODS: Embryonic lung buds from timed pregnant Swiss Webster mice were microdissected on Embryonic Day 12 (E12), and whole organs were cultured in serum-free medium in the presence of the Rho activator cytotoxic necrotizing factor 1 (CNF-1) for 48 h . Serial measurements of the degree of epithelial branch formation and tissue maturation were performed using light microscopy and computerized image analysis . RESULTS: At 48 h, embryonic lungs treated with 2 ng/ml CNF-1 increased their terminal bud count by 236 +/- 18% (P = 0.01) compared with 132 +/- 2% for untreated controls . However, dose-response experiments revealed biphasic behavior: at a higher dose of CNF-1 (200 ng/ml), bud number was actually decreased relative to controls (43 +/- 1%, P < 0.001) . Histological analysis revealed that individual glands appeared to be more highly developed at low-dose CNF-1, whereas the high dose produced gland contraction . CONCLUSIONS: These data support a potential role for Rho and cytoskeletal tension in control of epithelial pattern formation during lung development . (c) 2002 Elsevier Science (USA). J Vet Med A Physiol Pathol Clin Med, 2002 Apr, 49(3), 121 - 4 Reverse 3,3',5'-triiodothyronine suppresses increase in free fatty acids in chickens elicited by dexamethasone or adrenaline; Bobek S et al.; Reverse triiodothyronine (rT3) displays hypometabolic properties and antagonizes the hypermetabolic effect of 3,5,3'-triiodothyronine (T3) . Previous experiments revealed that exogenous rT3 enhanced free fatty acids (FFA) in heat-stressed pullets and in chickens infected with lipopolysaccharide from Escherichia coli . To gain more data concerning the action of rT3, its effect on lipaemia produced by two main stress hormones: glucocorticoids and catecholamines, has been investigated . Synthetic glucocorticoid {dexamethasone (Dex)} and adrenaline (Adr) were used in two experiments . The experiments differed in duration, i.e . 24 h (Dex) or 150 min (Adr), and frequency of rT3 injections, i.e . two (Dex) or single (Adr) injections . The doses of hormones were as follows: rT3: 14 microg 100 g body weight/ injection (subcutaneously): Dex: 5 mg/animal (subcutaneously) and Adr: 1 mg/animal (intramuscularly) . Maximal increases in FFA of 230.5 and 227.5% were noted after 1.5 and 3 h, respectively, in birds treated with Dex . Reverse T3 almost completely suppressed the rise of plasma FFA elicited by Dex . The increase in Dex + rT3-treated fowl was only 30.4% (not significant in comparison to control) . Adr increased FFA by a maximum of 89.1 % and treatment with rT3 (Adr + rT3 group) suppressed this FFA increase to 42.5% . The data obtained demonstrate that rT3 suppresses lipaemia induced by an exogenous glucocorticoid and adrenaline . This suppression was more pronounced in glucocorticoid-treated birds, where Dex produced a higher lipolytic response than Adr. New Microbiol, 2002 Apr, 25(2), 173 - 8 Influence of surface cell structures on physicochemical properties of Escherichia coli; El Ghmari A et al.; The partition of cells in a polyethylene glycol-dextran two phase system was used to compare the relative hydrophobicity of E . coli strains expressing different surface structures . The role of fimbriae and surface antigens on the behavior partition was investigated . The strains expressing PAP fimbriae and/or O-antigen showed a higher surface hydrophobicity than strains which express only type 1 fimbriae and/or R-antigen . No relation was found between K and H antigen and hydrophobicity measurements . The influence of surface structures on electrophoretic mobility has been evaluated . The polysaccharide capsules of AL 213 and AL 499 strains generated a high EPM . For non capsulated E . coli the EPM of rough strains (AL 46, 382) is higher than smooth strain (AL52). Am J Obstet Gynecol, 2002 May, 186(5), 1062 - 8 The fetal maturational and inflammatory responses to different routes of endotoxin infusion in sheep; Newnham JP et al.; OBJECTIVES: In clinical practice, chorioamnionitis has been observed to enhance fetal lung maturation in the short term but may predispose to chronic lung disease thereafter . Using the sheep model, we have previously shown that injection of endotoxin into the amniotic cavity results in inflammatory responses and profoundly enhances newborn lung function after preterm birth . The fetus tolerates intra-amniotic doses of endotoxin considerably greater than those that are lethal if given intramuscularly . This study aimed to explore the mechanisms by which endotoxin matures the lungs by determining whether the route of administration influenced the maturational responses of the fetus . STUDY DESIGN: Date-mated ewes at 118 days of pregnancy were allocated at random to receive endotoxin (Escherichia coli lipopolysaccharide 055;B5) directly into the trachea (n = 7), stomach (n = 6), amniotic cavity (n = 7), or peritoneal cavity (n = 4) of the fetal lamb by surgical implantation of an osmotic pump delivering 1 mg of endotoxin over a 24-hour period . Results were compared with those obtained in saline solution-infused controls (n = 9) . The lambs were delivered by cesarean section at 125 days' gestation (term is 150 days) . RESULTS: Endotoxin infusion into the trachea, stomach, and amniotic cavity each resulted in inflammatory responses in lung fluid and improved postnatal lung function, and effects were similar for each route of administration . These effects occurred with minimal features of systemic inflammation . Intraperitoneal infusion resulted in severe fetal acidosis or death . CONCLUSION: These findings provide further evidence that the lung-maturing effects of intra-amniotic endotoxin are mediated by local factors in the respiratory system rather than by systemic inflammatory responses . Chorioamnionitis may alter lung function and possibly lead to chronic injury without clinical features of systemic inflammation or compromise. J Biol Chem, 2002 Jul 26, 277(30), 27423 - 32 Epub 2002 May 15. Rrn3 phosphorylation is a regulatory checkpoint for ribosome biogenesis; Cavanaugh AH et al.; Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo . The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells . Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I . Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3.rpa43 complex in vivo . Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3 . Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells . These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3.RNA polymerase I complex within the nucleolus. Structure (Camb), 2002 May, 10(5), 701 - 13 Tandem DNA recognition by PhoB, a two-component signal transduction transcriptional activator; Blanco AG et al.; PhoB is a signal transduction response regulator that activates nearly 40 genes in phosphate depletion conditions in E . coli and closely related bacteria . The structure of the PhoB effector domain in complex with its target DNA sequence, or pho box, reveals a novel tandem arrangement in which several monomers bind head to tail to successive 11-base pair direct-repeat sequences, coating one face of a smoothly bent double helix . The protein has a winged helix fold in which the DNA recognition elements comprise helix alpha 3, penetrating the major groove, and a beta hairpin wing interacting with a compressed minor groove via Arg219, tightly sandwiched between the DNA sugar backbones . The transactivation loops protrude laterally in an appropriate orientation to interact with the RNA polymerase sigma(70) subunit, which triggers transcription initiation. Structure (Camb), 2002 May, 10(5), 602 - 3 Tandem DNA binding of E . coli's transactivator PhoB; Volz K; In this issue of Structure, Blanco et al . describe the first structure of a two-component response regulator effector domain bound to its target DNA, showing novel tandem binding to successive direct repeat sequences of pho boxes from the phoA operon promotor. Curr Biol, 2002 May 14, 12(10), 863 - 7 A simple method for genome-wide screening for advantageous insertions of mobile DNAs in Escherichia coli; Edwards RJ et al.; Laboratory evolution in Escherichia coli has revealed that fitness typically increases in experimental populations . These changes are sometimes associated with changes in insertion sequence positions, some of which may themselves cause advantageous phenotypes . We have a novel and general method for identifying genes in Escherichia coli, whose knockout by mobile DNA insertions is beneficial in experimental evolution . Insertion sites in favored clones can be identified by reference to genomic information . We have implemented the method using modified Tn10 transposons bearing kanamycin and chloramphenicol resistance cassettes . Results are consistent across replicated experiments, demonstrating that the insertions are themselves creating selective advantages, rather than hitch-hiking with favorable base substitutions . The successful clones have subsequently been confirmed to have a fitness advantage relative to the progenitor strain . In experiments in shaking culture, we find that advantageous insertions usually fall in operons required in the pathways creating flagella . The method allows a rapid genome-wide screening for advantageous insertions in arbitrary environmental conditions . It allows investigation of the extent to which transient mutations generating environment-dependent selective advantages may help to explain the persistence of mobile DNAs in primarily clonal organisms, such as E . coli. Zhonghua Xue Ye Xue Za Zhi, 2002 Jan, 23(1), 5 - 8 {Construction and expression of a vector containing protein transduction domain and bcr/abl fusion gene}; Liang Y et al.; OBJECTIVE: To construct a vector containing protein transduction domain (PTD) and bcr/abl fusion gene of chronic myelogenous leukemia and express PTD-bcr/abl fusion protein in E . Coli . METHODS: DNA fragment encoding PTD was synthesized and fused to PCR-amplified bcr/abl gene fragment, then inserted into plasmid pET-16b to get the expression vector pEPb containing PTD-bcr/abl fusion gene, which was transfected and expressed in E . Coli LB21 . PTD-bcr/abl fusion protein was purified by affinity chromatography . RESULTS: 523 bp bcr/abl fusion gene was effectively amplified . The PTD-bcr/abl gene sequencing showed the same sequence as scheduled . The fusion peptide was successfully expressed in E . Coli and purified . CONCLUSION: The results may provide a new PTD-bcr/abl fusion peptide for the immunotherapy of CML. Biochem J, 2002 Oct 1, 367(Pt 1), 263 - 9 Origins of the difference in Ca2+ requirement for activation of mu- and m-calpain; Dutt P et al.; The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx . 25 and 325 microM respectively . The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits . As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain . On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d) . In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90% . K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding . Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference. Anticancer Res, 2002 Mar-Apr, 22(2A), 949 - 52 Vitamins B6 and C and mitomycin C-efficiency under irradiation; Svoboda B et al.; The radiation-induced effect of Vitamin B6 (Vit . B6) on mitomycin C (MMC) was investigated by using Escherichia coli bacteria AB 1157 as a model in air-free media as well as in media saturated with nitrous oxide (N20) or air, respectively . In all three types of media Vit.B6 showed cytostatic abilities . The highest synergistic effect of Vit.B6 on MMC was observed in an air-free environment . It decreased 2.8-fold in aerated solution and showed the opposite effect in solutions saturated with N20 . The addition of Vitamin C (Vit.C) to the Vit.B6/MMC-mixture in air-free media reduced the MMC-efficiency by a factor of 3.6, whereas the presence of air led to a MMC enhancement of 1.6-fold . Considerations based on the involvement of the primary transients of water radiolysis were made in order to explain the observed effects. Anticancer Res, 2002 Mar-Apr, 22(2A), 927 - 9 Influence of vitamin B1 on sanazole activity under irradiation . A study in vitro; Heinrich E et al.; The effect of vitamin B1 (thiamine) on sanazole (AK-2123) efficiency was investigated in vitro under irradiation, using E . coli bacteria (AB 1157) as a model . In order to get a deeper insight into the reaction mechanisms, the experiments were performed in media saturated with argon, air or N20 . In the first case vitamin B1 acts as a cytostatic, but in the presence of air or N20 it shows strongly pronounced antioxidant action and leads to an essential increase of sanazole efficiency. Extremophiles, 2002 Apr, 6(2), 151 - 9 Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs; Lee MK et al.; Glutamate dehydrogenases (GDHs) from fresh-water and marine hyperthermophilic Archaea were compared with respect to their responses to different salt concentrations . A gene encoding GDH from the terrestrial hyperthermophilic archaeon Thermococcus waiotapuensis (Twaio) was cloned, sequenced, and expressed at a high level in Escherichia coli . The deduced amino acid sequence, which consists of 418 amino acid residues, revealed a high degree of similarity with GDHs from related marine strains such as Thermococcus litoralis (Tl) and Pyrococcus furiosus (Pfu) . Recombinant Twaio GDH was purified 27-fold to homogeneity . The enzyme is hexameric with a molecular weight of 259,000 . The effects of several salts (KCl, CaCl, MgSO4), temperature, and pH on enzyme activity were determined and compared in three hyperthermophilic GDHs, including T . waiotapuensis, and GDHs from two marine species, T . litoralis and P . furiosus . Kinetic studies suggested a biosynthetic role for the nicotinamide adenine dinucleotide phosphate- (NADP-) specific Twaio GDH in the cell . Interestingly, Twaio GDH revealed no salt responses, whereas the two marine GDHs showed substantial enhancement of activity as well as thermostability at increasing salt concentrations . Because electrostatic interactions between charged amino acid residues are thought to be a key feature of structural integrity and thermostability in hyperthermophilic GDHs, salt availability and its effects on marine enzymes could partially explain a higher thermal stability in marine species than in phyletically related fresh-water species. Extremophiles, 2002 Apr, 6(2), 111 - 22 A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations; Toogood HS et al.; A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli . The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus . It contains one cysteine residue that is highly conserved among aminoacylases . Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85 degrees C in Tris-HCl at pH 8.0 . The recombinant enzyme is thermostable, with a half-life of 25 h at 70 degrees C . Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity . The enzyme was partially inhibited by EDTA and p-hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity . Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity . The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids . preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine. Biopolymers, 2002, 67(4-5), 242 - 6 Secondary and tertiary structure of nucleotide-binding domain of alphasubunit of Na+/K+-ATPase; Hofbauerova K et al.; The nucleotide-binding domain of the alpha subunit of mouse brain Na+/K+-ATPase was expressed and isolated from Escherichia coli cells . A model structure was constructed by comparative modeling with and without docked ATP . This was compared with the secondary structure determination from UV circular dichroism and Raman spectroscopy . Thus, we support the quality of the model and the correct folding of the recombinant protein . ATP binding was followed by Raman difference spectroscopy, and its influence on the secondary structure of the N domain seems to not be significant . Planta, 2002 May, 215(1), 110 - 8 Epub 2002 Feb 02. The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro; Todd CD et al.; Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool . A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound . Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control . At present, regulation of arginase in conifers is not well understood . Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression . We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli . Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte . The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed. Planta, 2002 May, 215(1), 26 - 32 Epub 2002 Feb 01. Immunolocalization of 1- O-sinapoylglucose:malate sinapoyltransferase in Arabidopsis thaliana; Hause B et al.; The serine carboxypeptidase-like protein 1- O-sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the transfer of the sinapoyl moiety of 1- O-sinapoylglucose to malate in the formation of sinapoylmalate in some members of the Brassicaceae . Rabbit polyclonal monospecific antibodies were raised against the recombinant SMT produced in Escherichia coli from the corresponding Arabidopsis thaliana (L.) Heynh . cDNA . Immunoblot analysis of protein from different Arabidopsis tissues showed that the SMT is produced in all plant organs, except in the seeds and young seedlings . The enzyme was most abundant in older seedlings as well as in rosette leaves and the flowering stem of the plant . Minor amounts were found in the cauline leaves, flower buds and siliques . Traces were detected in the root and flowers . Arabidopsis and transgenic tobacco ( Nicotiana tabacum L.) plants expressing the full-length Arabidopsis SMT containing an N-terminal signal peptide showed apparent molecular masses of the protein of 52-55 kDa . The difference of ca . 8 kDa compared to the recombinant protein produced in E . coli was shown to be due to post-translational N-glycosylation of SMT in plants . Immunofluorescent labeling of Arabidopsis leaf sections localized SMT to the central vacuoles of mesophyll and epidermal cells . Comparable leaf sections of an SMT deletion mutant showed no vacuolar immunofluorescent labeling . We conclude that Arabidopsis SMT is synthesized as a precursor protein that is targeted to the endoplasmic reticulum where the signal peptide is removed . The correct N-terminus of the recombinantly produced SMT protein lacking the signal peptide was confirmed by Edman degradation . The protein is probably glycosylated in the Golgi apparatus from where it is subsequently routed to the vacuole. Transpl Int, 2002 May, 15(5), 205 - 11 Epub 2002 Apr 11. Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model . Part I: in vitro assays using porcine aortic endothelial cells; Nagahama M et al.; We assessed whether the adenovirus-mediated gene transfer of triple human complement regulating proteins (hCRPs) to the porcine aortic endothelium (PAE), could possibly exert a synergistic effect to inhibit human complement activation . Adenovirus vectors, encoding E.Coli beta-galactosidase (AxCALacZ), human membrane cofactor protein (MCP) (AxCAMCP), decay-accelerating factor (DAF) (AxCADAF), and CD59 (AxCACD59) were produced by the COS-TPC method . AxCALacZ was transfected to porcine aortic endothelium cells (PAECs) under various multiplicities of infection (MOI) to determine the efficiency of adenovirus-mediated gene transfer by 5-bromo-4-chloro-3-indolyl beta- D-galactopyranoside (X-gal) staining . The mRNA expressions of transfected CRPs were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) . Cellular damage to the PAEC was assessed by an MTT assay . PAEC was most efficiently transfected with the LacZ gene at 10(3) MOI/60-min incubation time (89.1%) . In all samples transfected with the CRP gene, the corresponding mRNAs were detected in the RT-PCR . In the MTT assay, PAECs co-cultured with 20% human serum, showed the highest cellular viability after gene transfer of triple CRPs (117.7%), when compared with those of marker LacZ, single or double CRPs . The adenovirus-mediated multiple gene transfer of CRPs may thus be an efficient method for suppressing complement activation in the porcine-to-human model of hyperacute rejection. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 7166 - 71 The starch-related R1 protein is an alpha -glucan, water dikinase; Ritte G et al.; To determine the enzymatic function of the starch-related R1 protein it was heterologously expressed in Escherichia coli and purified to apparent homogeneity . Incubation of the purified protein with various phosphate donor and acceptor molecules showed that R1 is capable of phosphorylating glucosyl residues of alpha-glucans at both the C-6 and the C-3 positions in a ratio similar to that occurring naturally in starch . Phosphorylation occurs in a dikinase-type reaction in which three substrates, an alpha-polyglucan, ATP, and H(2)O, are converted into three products, an alpha-polyglucan-P, AMP, and orthophosphate . The use of ATP radioactively labeled at either the gamma or beta positions showed that solely the beta phosphate is transferred to the alpha-glucan . The apparent K(m) of the R1 protein for ATP was calculated to be 0.23 microM and for amylopectin 1.7 mg x ml(-1) . The velocity of in vitro phosphorylation strongly depends on the type of the glucan . Glycogen was an extremely poor substrate; however, the efficiency of phosphorylation strongly increased if the glucan chains of glycogen were elongated by phosphorylase . Mg(2+) ions proved to be essential for activity . Incubation of R1 with radioactively labeled ATP in the absence of an alpha-glucan showed that the protein phosphorylates itself with the beta, but not with the gamma phosphate . Autophosphorylation precedes the phosphate transfer to the glucan indicating a ping-pong reaction mechanism. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6889 - 94 ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos; Williams RW et al.; Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression . RNAi is characterized by the conversion of long dsRNA into approximately 21-25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA . Many of the genes required for siRNA production and target mRNA degradation are widely conserved . Notably, members of the Argonaute-like gene family from Arabidopsis, Caenorhabditis elegans, Drosophila, and Neurospora have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway . We show here that mutations in the Drosophila Argonaute1 (AGO1) gene suppress RNAi in embryos . This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro . Furthermore, AGO1 is not required for siRNA production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo . These data suggest that AGO1 functions downstream of siRNA production. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6655 - 60 The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma; Smela ME et al.; A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B(1) (AFB(1)) . Hypotheses have been put forth that AFB(1), in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation . The primary DNA adduct of AFB(1) is 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxyaflatoxin B(1) (AFB(1)-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB(1)-formamidopyrimidine (AFB(1)-FAPY) adduct . AFB(1)-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB(1) exposure, underscoring its high persistence in vivo . The present study reveals two striking properties of this DNA adduct: (i) AFB(1)-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB(1)-N7-Gua, and (ii) one proposed rotamer of AFB(1)-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell . Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6613 - 8 Changing the lactose permease of Escherichia coli into a galactose-specific symporter; Guan L et al.; N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport . Moreover, lactose, melibiose, and beta,d-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by {(14)C}NEM . Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport . Consistently, competitive inhibition of {(14)C}galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected . The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected . In addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition . In contradistinction, none of these replacements has a marked effect on galactose transport . The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the non-galactosyl moiety of D-galactopyranosides . This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides. Plant Physiol, 2002 May, 129(1), 363 - 73 Molecular and biochemical characterization of a cold-regulated phosphoethanolamine N-methyltransferase from wheat; Charron JB et al.; A cDNA that encodes a methyltransferase (MT) was cloned from a cold-acclimated wheat (Triticum aestivum) cDNA library . Molecular analysis indicated that the enzyme WPEAMT (wheat phosphoethanolamine {P-EA} MT) is a bipartite protein with two separate sets of S-adenosyl-L-Met-binding domains, one close to the N-terminal end and the second close to the C-terminal end . The recombinant protein was found to catalyze the three sequential methylations of P-EA to form phosphocholine, a key precursor for the synthesis of phosphatidylcholine and glycine betaine in plants . Deletion and mutation analyses of the two S-adenosyl-L-Met-binding domains indicated that the N-terminal domain could perform the three N-methylation steps transforming P-EA to phosphocholine . This is in contrast to the MT from spinach (Spinacia oleracea), suggesting a different functional evolution for the monocot enzyme . The truncated C-terminal and the N-terminal mutated enzyme were only able to methylate phosphomonomethylethanolamine and phosphodimethylethanolamine, but not P-EA . This may suggest that the C-terminal part is involved in regulating the rate and the equilibrium of the three methylation steps . Northern and western analyses demonstrated that both Wpeamt transcript and the corresponding protein are up-regulated during cold acclimation . This accumulation was associated with an increase in enzyme activity, suggesting that the higher activity is due to de novo protein synthesis . The role of this enzyme during cold acclimation and the development of freezing tolerance are discussed. Plant Physiol, 2002 May, 129(1), 278 - 89 Glucosylation activity and complex formation of two classes of reversibly glycosylated polypeptides; Langeveld SM et al.; Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis . In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch . We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs . Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2 . Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied . After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation . In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose . Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated . Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active . Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes . Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo- and heterodimers . Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes . Taken together, these results suggest the presence of active RGP1 and RGP2 homo- and heteromultimers in wheat endosperm. Plant Physiol, 2002 May, 129(1), 225 - 34 Expression, activation, and biochemical properties of a novel Arabidopsis protein kinase; Gong D et al.; An Arabidopsis SOS2 (salt overly sensitive 2)-like protein kinase gene, PKS6, was expressed in leaves, stems, and siliques, but not detectable in roots of adult plants; its expression in young seedlings was up-regulated by abscisic acid . To determine the biochemical properties of the PKS6 protein, we expressed the PKS6 coding sequence as a glutathione S-transferase fusion protein in Escherichia coli . The bacterially expressed glutathione S-transferase-PKS6 fusion protein was inactive in substrate phosphorylation . We have constructed constitutively active forms of PKS6 by either a deletion of its putative auto-inhibitory FISL motif (i.e . PKS6deltaF) or a substitution of threonine-178 with aspartic acid within the putative activation loop . We found that PKS6deltaF exhibited a strong preference for Mn2+ over Mg2+ as a divalent cation cofactor for kinase activity . PKS6DeltaF displayed substrate specificity against three different peptide substrates and had an optimal pH of approximately 7.5 and temperature optimum of 30 degrees C . The apparent Km values for ATP and the preferred peptide substrate p3 of PKS6deltaF were determined to be 1.7 and 28.5 microM, respectively . These results provide significant insights into the regulation and biochemical properties of the protein kinase PKS6 . In addition, the constitutively active, gain-of-function kinase mutants will be invaluable for future determination of the in planta function of PKS6. Plant Physiol, 2002 May, 129(1), 156 - 68 LeCPK1, a calcium-dependent protein kinase from tomato . Plasma membrane targeting and biochemical characterization; Rutschmann F et al.; The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.) . LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts . The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 microM) . Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439 . Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1 . Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 microM, respectively . LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro . A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo . Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane . Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. Plant Physiol, 2002 May, 129(1), 134 - 44 Isolation and characterization of two germacrene A synthase cDNA clones from chicory; Bouwmeester HJ et al.; Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A . Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins . Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity . Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes . Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels . Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins . On MonoQ, these proteins co-eluted with the two heterologously produced proteins . The K(m) value, pH optimum, and MonoQ elution volume of one of the proteins produced in E . coli were similar to the values reported for the GAS protein that was recently purified from chicory roots . Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene . The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed. Infect Immun, 2002 Jun, 70(6), 3249 - 58 Enhanced delivery of exogenous peptides into the class I antigen processing and presentation pathway; De Haan L et al.; Current immunization strategies, using peptide or protein antigens, generally fail to elicit cytotoxic-T-lymphocyte responses, since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs . In an attempt to circumvent this, we investigated whether the GM1 receptor-binding B subunit of Escherichia coli heat-labile toxin (EtxB) could be used to deliver class I epitopes . When a class I epitope was conjugated to EtxB, it was delivered into the MHC-I presentation pathway in a GM1-binding-dependent fashion and resulted in the appearance of MHC-I-epitope complexes at the cell surface . Importantly, we show that the efficiency of EtxB-mediated epitope delivery could be strikingly enhanced by incorporating, adjacent to the class I epitope, a 10-amino-acid segment from the C terminus of the DNA polymerase (Pol) of herpes simplex virus . The replacement of this 10-amino-acid segment by a heterologous sequence or the introduction of specific amino acid substitutions within this segment either abolished or markedly reduced the efficiency of class I epitope delivery . If the epitope was extended at its C terminus, EtxB-mediated delivery into the class I presentation pathway was found to be completely dependent on proteasome activity . Thus, by combining the GM1-targeting function of EtxB with the 10-amino-acid Pol segment, highly efficient delivery of exogenous epitopes into the endogenous pathway of class I antigen processing and presentation can be achieved. Infect Immun, 2002 Jun, 70(6), 3216 - 26 Phylogenetic analysis and prevalence of urosepsis strains of Escherichia coli bearing pathogenicity island-like domains; Bingen-Bidois M et al.; We characterized 100 Escherichia coli urosepsis isolates from adult patients according to host compromise status by means of ribotyping, PCR phylogenetic grouping, and PCR detection of papG alleles and the virulence-related genes sfa/foc, fyuA, irp-2, aer, hly, cnf-1 and hra . We also tested these strains for copies of pap and hly and their direct physical linkage with other virulence genes in an attempt to look for pathogenicity islands (PAIs) described for the archetypal uropathogenic strains J96, CFT073, and 536 . Most of the isolates belonged to E . coli phylogenetic groups B2 and D and bore papG allele II, aer, and fyuA/irp-2 . papG allele II-bearing strains were more common in noncompromised patients, while papG allele-negative strains were significantly more frequent in compromised patients . Fifteen ribotypes were identified . The three archetypal strains harbored different ribotypes, and only one-third of our urosepsis strains were genetically related to one of the archetypal strains . Three and 18 strains harbored three and two copies of pap, respectively, and 5 strains harbored two copies of hly . papGIII was physically linked to hly, cnf-1, and hra (reported to be PAI II(J96)-like genetic elements) in 14% of the strains . The PAI II(J96)-like domain was inserted within pheR tRNA in 11 strains and near leuX tRNA in 3 strains . Moreover, the colocalized genes cnf-1, hra, and hly were physically linked to papGII in four strains and to no pap gene in three strains . papGII and hly (reported to be PAI I(CFT073)-like genetic elements) were physically linked in 16 strains, pointing to a PAI I(CFT073)-like domain . Three strains contained both a PAI II(J96)-like domain and a PAI I(CFTO73)-like domain . Forty-two strains harbored papGII but not hly, in keeping with the presence of a PAI II(CFT073)-like domain . Only one strain harbored a PAI I(536)-like domain (hly only), and none harbored a PAI I(J96)-like domain (papGI plus hly) or a PAI II(536)-like domain (papGIII plus hly) . This study provides new data on the prevalence and variability of physical genetic linkage between pap and certain virulence-associated genes that are consistent with their colocalization on archetypal PAIs. Infect Immun, 2002 Jun, 70(6), 3187 - 98 Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans; Keller N et al.; The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum . Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process . The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface . Many of the microneme proteins identified so far contain adhesive domains . We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1 . The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively . Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins . The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1 . The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites . Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage . Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant . NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers . Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface . Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans. Infect Immun, 2002 Jun, 70(6), 3111 - 21 Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis; Doherty TM et al.; Immunity against Mycobacterium tuberculosis depends largely on activation of cell-mediated responses, and gamma interferon has been shown to play a crucial role in this process in both humans and animal models . Since the lung is normally the organ in which infection is initiated and is the major site of pathology, immune responses in the lung play a significant role in restricting initial infection with M . tuberculosis . The aim of the present study was to stimulate efficient immunity in the lung by targeting the gut mucosa . Detoxified monophosphoryl lipid A (MPL) has been shown to be a relatively nontoxic adjuvant which efficiently promotes the induction of type 1 responses when it is given by the traditional subcutaneous route . We have therefore compared subcutaneous immunization of mice to oral immunization by using a model subunit vaccine carrying two immunodominant proteins from M . tuberculosis, in combination with MPL-based adjuvants . While less effective when used to prime a response, a heterologous priming and boosting vaccination strategy employing oral boosting induced significant systemic type 1 responses which equaled and surpassed those attained by subcutaneous immunization protocols . Moreover, the increased immune responses observed correlated with the induction of substantial protection against subsequent aerosol infection with virulent M . tuberculosis at levels comparable to, or better than, those obtained by multiple subcutaneous vaccinations . These results demonstrate that booster vaccinations via mucosal surfaces, by combining efficient subunit vaccines with the potent adjuvant MPL, may be an effective method of addressing some of the shortcomings of current vaccination strategies. Infect Immun, 2002 Jun, 70(6), 3012 - 9 The LTR72 mutant of heat-labile enterotoxin of Escherichia coli enhances the ability of peptide antigens to elicit CD4(+) T cells and secrete gamma interferon after coapplication onto bare skin; Beignon AS et al.; Application of antigens with an adjuvant onto bare skin is a needle-free and pain-free immunization procedure that delivers antigens to the immunocompetent cells of the epidermis . We tested here the immunogenicity and adjuvanticity of two mutants of heat-labile enterotoxin (LT) of Escherichia coli, LTK63 and LTR72 . Both mutants were shown to be immunogenic, inducing serum and mucosal antibody responses . The application of LTK63 and LTR72 to bare skin induced significant protection against intraperitoneal challenge with a lethal dose of LT . In addition, both LT mutants enhanced the capacity of peptides TT:830-843 and HA:307-319 (representing T-helper epitopes from tetanus toxin and influenza virus hemagglutinin, respectively) to elicit antigen-specific CD4(+) T cells after coapplication onto bare skin . However, only mutant LTR72 was capable of stimulating the secretion of high levels of gamma interferon . These findings demonstrate that successful skin immunization protocols require the selection of the right adjuvant in order to induce the appropriate type of antigen-specific immune responses in a selective and reliable way . Moreover, the use of adjuvants such the LTK63 and LTR72 mutants, with no or low residual toxicity, holds a lot of promise for the future application of vaccines to the bare skin of humans. Am J Physiol Regul Integr Comp Physiol, 2002 Jun, 282(6), R1680 - 6 Acute stressor exposure facilitates innate immunity more in physically active than in sedentary rats; Fleshner M et al.; Most previous stress-immune research focused on the immunosuppressive effects of stress on acquired immunity . More recently, it has become clear that acute stressor exposure can potentiate innate, as well as suppress acquired, immunity . For example, acute stress improves recovery from bacterial inflammation, a classic in vivo measure of innate immunity . The previous work was done in sedentary organisms . Physical activity status can modulate the impact of stress on immune function . The following studies tested the hypothesis that the effect of stress on inflammation after subcutaneous challenge with bacteria (Escherichia coli) is facilitated by physical activity . The results were that sedentary, stressed rats resolved their inflammation 1-2 days faster and have increased circulating neutrophils compared with their nonstressed, sedentary counterparts . In contrast, physically active, stressed rats resolve their inflammation 3-4 days faster and have increased circulating and inflammatory site neutrophils compared with their nonstressed counterparts . Importantly, the beneficial impact of stress on inflammation recovery and neutrophil migration was greater in the physically active, than sedentary, stressed rats . Thus physical activity status facilitates the positive effect of acute stress on innate immunity. Mol Microbiol, 2002 May, 44(4), 989 - 99 The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference; Kaneshiro ES et al.; Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients . Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth . Recombinant protein technology now makes it possible to avoid some major obstacles . The P . carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism . We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli . Unlike SAM:SMT from other organisms, the P . carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi . Cycloartenol was not a productive substrate . With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively . Thus, the P . carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P . carinii SAM:SMT . These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen. Mol Microbiol, 2002 May, 44(4), 971 - 9 ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase; Jiang Y et al.; Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria . The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death . The ParD protein is a specific ParE antitoxin . In this work, the in vitro activities of these two proteins were examined . The ParE protein was found to inhibit DNA synthesis using an E . coli oriC supercoiled template and a replication-proficient E . coli extract . Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA . The presence of ParD prevented these inhibitory activities of ParE . We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS) . Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex . These results demonstrate that the target of ParE toxin activity in vitro is E . coli gyrase. Physiol Plant, 2002 May, 115(1), 48 - 55 Isolation of mannose 6-phosphate reductase cDNA, changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots; Delavault P et al.; We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite . We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L . attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite . A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E . coli and M6PR activity measurement . Only one M6PR gene was detected in O . ramosa following southern blot analysis . M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e . M6PR expression is constitutive in Orobanche . M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation . Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot . These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development. J Am Chem Soc, 2002 May 22, 124(20), 5702 - 13 Electron-transfer mechanisms through biological redox chains in multicenter enzymes; Jeuken LJ et al.; A new approach for studying intramolecular electron transfer in multicenter enzymes is described . Two fumarate reductases, adsorbed on an electrode in a fully active state, have been studied using square-wave voltammetry as a kinetic method to probe the mechanism of the long-range electron transfer to and from the buried active site . Flavocytochrome c(3) (Fcc(3)), the globular fumarate reductase from Shewanella frigidimarina, and the soluble subcomplex of the membrane-bound fumarate reductase of Escherichia coli (FrdAB) each contain an active site FAD that is redox-connected to the surface by a chain of hemes or Fe-S clusters, respectively . Using square-wave voltammetry with large amplitudes, we have measured the electron-transfer kinetics of the FAD cofactor as a function of overpotential . The results were modeled in terms of the FAD group receiving or donating electrons either via a direct mechanism or one involving hopping via the redox chain . The FrdAB kinetics could be described by both models, while the Fcc(3) data could only be fit on the basis of a direct electron-transfer mechanism . This raises the likelihood that electron transfer can occur via a superexchange mechanism utilizing the heme groups to enhance electronic coupling . Finally, the FrdAB data show, in contrast to Fcc(3), that the maximum ET rate at high overpotential is related to the turnover number for FrdAB measured previously so that electron transfer is the limiting step during catalysis. J Am Chem Soc, 2002 May 22, 124(20), 5652 - 3 A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins; Datta D et al.; Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design . p-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host . DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions. J Agric Food Chem, 2002 May 22, 50(11), 3165 - 72 Cloning and expression of desoxyhemigossypol-6-O-methyltransferase from cotton (