Escherichia coli

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 (Gossypium barbadense); Liu J et al.; Terpenoids play an important role in defense against insects and pathogens in cotton . These terpenoids contain phenolic groups . Metabolites in which the phenolic group has been converted to a methoxy group are less toxic to most insects and pathogens and thus may alter resistance . Here is reported the cloning of a gene from Gossypium barbadense that encodes the enzyme that methylates the phenolic group of desoxyhemigossypol (dHG) exclusively at the 6-position, dHG-6-O-methyltransferase (dHG-6-OMT) . Partial peptide sequences from digests of purified dHG-6-OMT were used to design primers for RT-PCR amplification of cDNA fragments from poly(A) mRNA . Fragments were extended to full length using 5' and 3' RACE . The resulting cDNA codes for a 365-residue polypeptide with a calculated molecular weight of 40.6 kDa, in agreement with the molecular mass of purified dHG-6-OMT . When expressed in Escherichia coli, the bacterial lysates showed a high specificity for the methylation of desoxyhemigossypol, differentiating the cloned gene from other pathogen-induced methyltransferases.

Bioconjug Chem, 2002 May-Jun, 13(3), 403 - 7
3-methyladenine-DNA glycosylase II: the crystal structure of an AlkA-hypoxanthine complex suggests the possibility of product inhibition; Teale M et al.; Escherichia coli (E . coli) protein 3-methyladenine-DNA glycosylase II (AlkA) functions primarily by removing alkylation damage from duplex and single stranded DNA . A crystal structure of AlkA was refined to 2.0 A resolution . This structure in turn was used to refine an AlkA-hypoxanthine (substrate) complex structure to 2.4 A resolution . The complex structure shows hypoxanthine located in AlkA's active site stacked between residues W218 and Y239 . The structural analysis of the AlkA and AlkA-hypoxanthine structures indicate that free hypoxanthine binding in the active site may inhibit glycosylase activity.

Biochemistry, 2002 May 21, 41(20), 6561 - 71
Characterization of a viroid-derived RNA promoter for the DNA-dependent RNA polymerase from Escherichia coli; Pelchat M et al.; This paper attributes a novel function, namely, that of transcriptional promoter, to the self-complementary, self-cleaving hammerhead RNA sequences found in RNA derived from the peach latent mosaic viroid (PLMVd) . The features of this RNA promoter, which adopts a hairpin structure that can be utilized by Escherichia coli RNA polymerase (RNAP) for in vitro transcription, that trigger the RNAP driven transcription and are responsible for the specific initiation of synthesis are described . The essential requirement for initiation is a basepaired uridine adjacent to the loop . The presence of a loop composed of at least six nucleotides connected to a relatively unstable stem significantly increases the level of initiation . Finally, we present several insights into the mechanism of the RNAP which reveal that it behaves differently with an RNA template as compared to a DNA one.

Biochemistry, 2002 May 21, 41(20), 6525 - 32
Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli; Vinod MP et al.; The multifunctional PutA flavoprotein from Escherichia coli is a peripherally membrane-bound enzyme that has both proline dehydrogenase (PDH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities . In addition to its enzymatic functions, PutA displays DNA-binding activity and represses proline catabolism by binding to the control region DNA of the put regulon (put intergenic DNA) . Presently, information on structure-function relationships for PutA is derived from primary structure analysis . To gain further insight into the functional organization of PutA, our objective is to dissect PutA into different domains and to characterize them separately . Here, we report the characterization of a bifunctional proline dehydrogenase (PutA(669)) that contains residues 1-669 of the PutA protein . PutA(669) purifies as a dimer and has a PDH specific activity that is 4-fold higher than that of PutA . As anticipated, PutA(669) lacks P5CDH activity . At pH 7.5, an E(m) (E-FAD/E-FADH(-)) of -0.091 V for the two-electron reduction of PutA(669)-bound FAD was determined by potentiometric titrations, which is 15 mV more negative than the E(m) for PutA-bound FAD . The pH behavior of the E(m) for PutA(669)-bound FAD was measured in the pH range 6.5-9.0 at 25 degrees C and exhibited a 0.03 V/pH unit slope . Analysis of the DNA and membrane-binding properties of PutA(669) shows that it binds specifically to the put intergenic control DNA with a binding affinity similar to that of PutA . In contrast, we did not observe functional association of PutA(669) with membrane vesicles . We conclude that PutA(669) has FAD-binding and DNA-binding properties comparable to those of PutA but lacks a membrane-binding domain necessary for stable association with the membrane.

Biochemistry, 2002 May 21, 41(20), 6510 - 6
EPR studies on a stable sulfinyl radical observed in the iron-oxygen-reconstituted Y177F/I263C protein R2 double mutant of ribonucleotide reductase from mouse; Adrait A et al.; Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides . The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity . The radical is located at Y177 in the R2 protein of mouse RNR . The radical is formed concomitantly with the mu-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein . EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2 . The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics . The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein . In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263 . The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2 . The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant . Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

Biochemistry, 2002 May 21, 41(20), 6449 - 59
Structural organization of the fibrin(ogen) alpha C-domain; Tsurupa G et al.; We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure {Weisel and Medved (2001) Ann . N.Y . Acad . Sci . 936, 312-327} . To test this hypothesis, we expressed in E . coli recombinant fragments corresponding to the full-length human alpha C-domain and its NH(2)- and COOH-terminal regions as well as their bovine counterparts, bA alpha 224-568, bA alpha 224-373, and bA alpha 374-568(538), respectively, and tested their folding status by fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC) . All three methods revealed heat-induced unfolding transitions in the full-length bA alpha 224-568 and its two COOH-terminal fragments, indicating that the COOH-terminal portion of the bovine alpha C-domain is folded into a compact cooperative structure . Similar results were obtained by CD and DSC with the full-length and the COOH-terminal h392-610 human fragments . The NH(2)-terminal fragments of both species, b224-373 and h221-392, did not exhibit any sign of a compact structure . However, their heat capacity functions, CD spectra, and temperature dependence of ellipticity at 222 nm were typical for peptides in the extended helical poly(L-proline) type II conformation (PPII), suggesting that they contain this type of regular structure . This is consistent with the presence of proline-rich tandem repeats in the sequence of both bovine and human connector regions . These results indicate that both bovine and human fibrinogen alpha C-domains consist of a compact globular cooperative unit attached to the bulk of the molecule by an extended NH(2)-terminal connector region with a PPII conformation.

Biochemistry, 2002 May 21, 41(20), 6414 - 21
N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation; Feng Z et al.; 4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer . Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis . Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features . To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level . We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP . Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding . We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP . We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated . These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.

Biochemistry, 2002 May 21, 41(20), 6271 - 81
Domain flexibility in ligand-free and inhibitor-bound Escherichia coli adenylate kinase based on a mode-coupling analysis of 15N spin relaxation; Shapiro YE et al.; Adenylate kinase from Escherichia coli (AKeco), consisting of a 23.6-kDa polypeptide chain folded into domains CORE, AMPbd, and LID catalyzes the reaction AMP + ATP <--> 2ADP . The domains AMPbd and LID execute large-amplitude movements during catalysis . Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco is studied with slowly relaxing local structure (SRLS) (15)N relaxation, an approach particularly suited when the global (tau(m)) and the local (tau) motions are likely to be coupled . For AKeco tau(m) = 15.1 ns, whereas for AKeco*AP(5)A tau(m) = 11.6 ns . The CORE domain of AKeco features an average squared order parameter, <S(2)>, of 0.84 and correlation times tau(f) = 5-130 ps . Most of the AKeco*AP(5)A backbone features <S(2)> = 0.90 and tau(f) = 33-193 ps . These data are indicative of relative rigidity . Domains AMPbd and LID of AKeco, and loops beta(1)/alpha(1), alpha(2)/alpha(3), alpha(4)/beta(3), alpha(5)/beta(4), and beta(8)/alpha(7) of AKeco*AP(5)A, feature a novel type of protein flexibility consisting of nanosecond peptide plane reorientation about the C(i-1)(alpha)-C(i)(alpha) axis, with correlation time tau(perpendicular) = 5.6-11.3 ns . The other microdynamic parameters underlying this dynamic model include S(2) = 0.13-0.5, tau(parallel) on the ps time scale, and a diffusion tilt beta(MD) ranging from 12 to 21 degrees . For the ligand-free enzyme the tau(perpendicular) mode was shown to represent segmental domain motion, accompanied by conformational exchange contributions R(ex) < or = 4.4 s(-1) . Loop alpha(4)/beta(3) and alpha(5)/beta(4) dynamics in AKeco*AP(5)A is related to the "energetic counter-balancing of substrate binding" effect apparently driving kinase catalysis . The other flexible AKeco*AP(5)A loops may relate to domain motion toward product release.

Biochemistry, 2002 May 21, 41(20), 6245 - 52
Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin; Griffiths SW et al.; alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors . Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues . In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants . These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism . Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity . The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure . Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues . Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242 . In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.

Anal Biochem, 2002 May 15, 304(2), 231 - 5
Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate; Nakamura M et al.; Biotinylated proteins are widely used as a molecular tool in biotechnological applications . In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin-fluorescein conjugate with a fluorescence image analyzer . Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining . Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method . Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E . coli specifically . The sensitivity and specificity of our method are high, and the procedure is simple . Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin-biotin technology . (c) 2002 Elsevier Science (USA)

Bioelectrochemistry, 2002 May 15, 56(1-2), 195 - 8
Specific natural DNA-bound lipids in post-genome era . The lipid conception of chromatin organization; Struchkov VA et al.; Two pools of DNA-bound lipids were isolated from DNA supramolecular complex (SC-DNA): loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNase I) . The compositions of the two lipid pools from different sources (rat thymus, liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B and T2 phage) were studied . The possible functions of DNA-bound lipids, especially of cardiolipin and cholesterol, at the attachment of DNA loops to the nuclear matrix, in DNA replicon organization, replication and transcription are discussed.

Biochim Biophys Acta, 2002 May 20, 1597(1), 149 - 56
Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods; Felder T et al.; Thymidylate synthase (TS) is a central target for the design of chemotherapeutic agents due to its vital role in DNA synthesis . Structural studies of binary complexes between Escherichia coli TS and various nucleotides suggest the chemotherapeutic agent FdUMP and the natural ligand dUMP bind similarly . We show, however, that FdUMP binding to human TS yields a substantially greater decrease in fluorescence than does dUMP . Because the difference in quenching due to ligand binding was approximately two-fold and this difference was not seen when using ecTS, the intriguing result indicated a significant difference in the mode of FdUMP binding to the human enzyme . We compared the binding affinities of dUMP, FdUMP, and TMP to TS from both species and found no significant differences for the individual ligands . Because binding affinities were not different among the ligands, the method of continuous variation was employed to determine binding stoichiometry . Similar to that found for dUMP binding to human and ecTS, FdUMP displayed single site occupancy with both enzymes . These results show that nucleotide binding differences exist for FdUMP and dUMP binding to the human enzyme . The observed differences are not due to differences in stoichiometry or ligand affinity . Therefore, although the crystal structure of human TS with various nucleotide ligands has not been solved, these results show that the differences observed using fluorescence methods result from as yet unidentified differential interactions between the human enzyme and nucleotide ligands.

Biochim Biophys Acta, 2002 May 20, 1597(1), 97 - 106
Immunoaffinity purification of calpastatin and calpastatin constructs; Wei W et al.; It has been difficult to purify calpastatin without using a step involving heating to 90-100 degrees C . Preparations of calpastatin obtained after heating often contain several polypeptides that have been ascribed to proteolytic degradation . Because calpastatin is highly susceptible to proteolytic degradation and several different calpastatin isoforms can be produced by using different start sites of transcription/translation and/or alternative splicing from the single calpastatin gene, it is not clear whether the different polypeptides observed in purified calpastatin preparations are proteolytic fragments or calpastatin isoforms . It would be useful, therefore, to have a method for purifying calpastatin that does not involve heating . At low ionic strength, calpastatin from skeletal muscle extracts binds quantitatively to an immunoaffinity column made by coupling a monoclonal antibody (MAb) to the C-terminal end of calpastatin (epitope between amino acids 707 and 786) to agarose; the bound calpastatin can be eluted at pH 2.5 . The C-terminal end of the calpastatin polypeptide was used because the known isoforms of calpastatin all contain domain IV . The eluted calpastatin, which retains all its calpain inhibitory activity, consists largely of a 125 kDa polypeptide (70%), and several smaller polypeptides that are labeled with a MAb to calpastatin . Expressed calpastatin constructs representing the full-length XL-IV calpastatin and domains L-IV, II-IV, III-IV, and IV also bind to the immunoaffinity column and can be purified . The immunoaffinity column is especially useful for purifying calpastatin from small tissue samples in a single step.

Biochim Biophys Acta, 2002 May 20, 1597(1), 74 - 80
Substrate recognition by 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus sp . strain 7; Fukuda E et al.; 2-Oxoacid:ferredoxin oxidoreductase (OFOR) catalyzes the coenzyme A-dependent oxidative decarboxylation of 2-oxoacids, at an analogous metabolic position to 2-oxoacid dehydrogenase multienzyme complex . The enzyme from Sulfolobus sp . strain 7, a thermoacidophilic crenarchaeon, is a heterodimer comprising two subunits, a (632 amino acids) and b (305 amino acids) . In contrast to other OFORs, the Sulfolobus enzyme shows a broad specificity for 2-oxoacids such as pyruvate and 2-oxoglutarate . Based on careful multiple alignment of this enzyme family and on the reported three-dimensional structure of the homodimeric pyruvate:ferredoxin oxidoreductase (POR) from Desulfovibrio africanus, we selected five amino acids, T256, R344 and T353 of subunit-a, and K49 and L123 of subunit-b, as candidate 2-oxoacid recognizing residues . To identify the residues determining the 2-oxoacid specificity of the enzyme family, we performed point mutations of these five amino acids, and characterized the resulting mutants . Analyses of the mutants revealed that R344 of subunit-a of the enzyme was essential for the activity, and that K49R and L123N of subunit-b drastically affected the enzyme specificity for pyruvate and 2-oxoglutarate, respectively . Replacement of the five residues resulted in significant changes in both K(m) and V(max), indicating that these amino acids are clearly involved in substrate recognition and catalysis.

Int J Biochem Cell Biol, 2002 Sep, 34(9), 1035 - 50
NADPH-dependent GMP reductase isoenzyme of human (GMPR2) . Expression, purification, and kinetic properties; Deng Y et al.; GMP reductase (EC 1.6.6.8) is the only known metabolic step by which guanine nucleotides can be converted to the pivotal precursor of both adenine and guanine nucleotides . Human GMP reductase has been previously partially purified from erythrocytes and a chromosome 6-linked cDNA has been identified to correspond for encoding human GMP reductase . Here, we reported a distinct cDNA for human GMP reductase isoenzyme isolated from a human fetal brain library, and the GenBank accession number is AF419346 . The deduced protein shows 90% identity with human GMP reductase reported (named GMPR1 compared with GMPR2 of this paper) and 69% with E . coli GMP reductase . Comparison of GMPR2 cDNA sequence with human genome indicates the corresponding gene spans about 6.6kb on chromosome 14, which encodes 348 amino acid residues . Northern hybridization analysis indicates a differential and disproportionate expression of mRNAs for GMPR1 and GMPR2, suggesting the existence of distinct molecular species of GMP reductase in human . The apparent Km of GMPR2 for NADPH and GMP are 26.6 and 17.4 microM, respectively . This is the first report suggesting the existence of two distinct types of human GMP reductase molecular species, which can be used to explain the bimodal saturation curve noted with the purified human erythrocyte GMP reductase.

J Immunol Methods, 2002 May 1, 263(1-2), 133 - 47
Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies; Simmons LC et al.; Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts . In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm . The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs . One E . coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor . As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental . In addition, a limited chimpanzee study suggests that the E . coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.

J Immunol Methods, 2002 May 1, 263(1-2), 97 - 109
Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties; Tanha J et al.; A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s) . Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library . The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction . Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies . The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.

J Periodontal Res, 2002 Apr, 37(2), 101 - 9
Effects of locally-delivered human macrophage products and estrogen on murine inflammatory bone resorption; Crump TB et al.; The objective of this study was to use an in vivo model of periodontitis (mouse calvaria) to quantify the effects of local release of secreted human macrophage products, 17beta-estradiol (E2), and proinflammatory lipopolysaccharide (LPS) on histologic bone resorption . Human THP-1 monocytes (106) were converted to macrophage phenotype by 500 ng/ml phorbol 12-myristate- 13-acetate (PMA) and treated as follows: no stimulation or Escherichia coli LPS (10 microg/ml) alone or in combination with a physiologic dose of E2 (100 pg/ml) for 24 h in RPMI/10% FBS, washed extensively, then incubated for 24 h in serum-free media . Supernatant products were concentrated and incorporated into a 4% (w/v) methylcellulose gel . Separate gels were incorporated with the following: LPS (500 microg/animal) alone, high dose of E2 (10 ng/animal) alone, a combination of LPS + E2, or gel only (controls) . Loaded or control gels were placed into a polylactic acid occlusive dome, inserted subcutaneously over the calvaria of mature ovariectomized ICR Swiss mice (8 mice x 7 groups x 2 times {5/14 days} = 112 animals), then calvaria were evaluated histologically . Macrophage stimulation with LPS alone, but not LPS in combination with E2, produced supernatants which upregulated osteoclast numbers in the suture area compared to gel controls at 5 days (p = 0.009) . The addition of LPS directly to the local delivery gels significantly upregulated osteoclasts in endosteal surfaces compared to gel controls at 5 days (p = 0.024) and at 14 days (p = 0.025) . The addition of E2 to LPS down-regulated resorption to a level not different from gel controls at 14 days . This in vivo model appears effective in studying inflammatory bone resorption, which may be inhibited by E2 directly or through its influence on secreted macrophage products.

J Nat Toxins, 2002 May, 11(2), 95 - 102
Cloning, expression, and biological activity of recombinant alpha-cinnamomin: toxicity to cranberry and other plant species; Ivanova DG et al.; Elicitins produced by the pathogenic fungi Phytophthora are known to exhibit The elicitin cinnamomin is of nonspecific toxicity to different solanaceous plant species . particular interest for its potential role in the hypersensitive-like cell death and in the biological response of cranberry plants to the fungal pathogen Phytophthora cinnamomi . In order to understand the biochemical steps of the Phytophthora root rot disease in cranberry, we investigated the alpha-cinnamomin-induced plant responses . Toxicity of alpha-cinnamomin, which shows a high degree of sequence homology to the alpha-elicitin group, was tested on Vaccinum macrocarpon, Nicotiana tabacum, Capsicum annuum, Lycopersicon esculentum, Lactuca sativa, and Phaseolus vulgaris plants . Gene corresponding to alpha-cinnamomin gene fused with maltose binding protein gene, was cloned into a pMALTEV expression vector, which was transformed into E . coli cells . Cells containing alpha-cinnamomin clones were cultured and extracted protein was purified on a maltose binding protein affinity column . Biological activity of alpha-cinnamomin fusion protein was examined on propagated plants and cuttings . In cranberry plants treated with cinnamomin necrotic hypersensitive-like response in the proximal areas of the leaf lamina of lower plant leaves was observed after 48-72 hr of incubation . Limited leaf necrosis observed days after application of low amounts of recombinant cinnamomin directly on the leaves of other plants indicates that the recombinant protein might be functioning as a toxin, capable of inducing aging accompanied by plant cell death.

Free Radic Biol Med, 2002 May 15, 32(10), 975 - 81
EC-SOD and the response to inflammatory reactions and aging in mouse lung; Sentman ML et al.; The lung is exposed to high oxygen tension and oxygen free radicals have been implicated in many pathologies of the organ . Extracellular superoxide dismutase occurs in high concentration in the lung and protects against hyperoxia-induced inflammation . We hypothesized that the enzyme might ameliorate other types of inflammation as well as aging-related changes of the organ . Tracheal instillation of endotoxin plus zymosan into extracellular superoxide dismutase knockout and wild-type mice resulted in a marked neutrophilic inflammation and increases in inflammatory cytokines, protein, and lactate dehydrogenase activity in the bronchoalveolar lavage fluid . There were no significant differences between the genotypes . Repeated challenges with ovalbumin caused an allergic inflammation with increases in eosinophils, interleukin-5, protein, and lactate dehydrogenase activity in the bronchoalveolar lavage fluid . Only minimal differences between the genotypes were found . In lungs from 2-year-old mice, marginal increases in inflammatory variables and fibrosis were found in the knockout mice . In conclusion, extracellular superoxide dismutase had a negligible role in the present inflammation and allergy models and for the long-term integrity of the organ.

Trends Pharmacol Sci, 2002 May, 23(5), 199 - 201
A first view of the structure of a type IA topoisomerase with bound DNA; Champoux JJ; The first crystal structure of a type IA topoisomerase with bound DNA has been solved . The structure of Escherichia coli topoisomerase III provides key insights regarding the catalytic mechanism and the conformational changes that accompany DNA binding, and enhances our understanding of how topoisomerases control DNA topology in the cell.

Vet Immunol Immunopathol, 2002 Jul, 86(3-4), 137 - 46
Recruitment of intestinal CD45RA+ and CD45RC+ cells induced by a candidate oral vaccine against porcine post-weaning colibacillosis; Bozic F et al.; To assess the influence of a live attenuated oral vaccine against porcine post-weaning colibacillosis (PWC) induced by enterotoxigenic Escherichia coli (ETEC) on mucosal lymphoid cell CD45 isoforms expression, experimental group of weaned pigs (n=6) was immunized orally with F4ac+ non-ETEC strain (day 0) and challenged with F4ac+ ETEC strain 7 days latter . Non-immunized ETEC-infected pigs (n=6) served as control . All pigs were killed on post-challenge day 7 . The small intestine was excised for isolation of jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) lymphocytes and immunohistochemical studies . The results obtained by immunophenotyping of isolated cells show that the proportion of CD45RA+ and CD45RC+ JLP, but not IPP, cells were higher in the non-ETEC-immunized ETEC-infected pigs versus non-immunized infected . Additionally, while CD45RA+ JLP cells increased only slightly, the expression of CD45RC isoform on the JLP cells was significantly higher (P< or =0.01) in the experimental than in the control group . The results of the quantitative phenotypic analysis of isolated lymphocytes were not confirmed by immunohistochemical in situ staining . The majority of intestinal immune cells was found to express CD45RA antigen in situ, but no differences were observed between the two groups of weaned pigs neither in CD45RA+ nor in CD45RC+ cells . Our overall evidence indicates that the increased expression of CD45RC isoform was in fact induced in a limited number of JLP T cells in the vaccinated pigs . This was accompanied with the impaired protection of the vaccinated pigs from challenge-induced PWC.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 261 - 6
Identification of a novel mycobacterial transcriptional regulator and its involvement in growth rate dependence and stringent control; Kamalakannan V et al.; A novel transcriptional regulator has been identified in the 400-bp upstream region of the guaA gene of Mycobacterium tuberculosis H37Rv that promotes the expression of lacZ gene in Mycobacterium smegmatis mc(2)155 and M . tuberculosis H37Rv but not in Escherichia coli DH5alpha . PCR-mediated deletion mutagenesis and cloning identified a 120-bp fragment upstream from the guaA gene to be the actual regulator . Primer extension analysis mapped the transcription start site to be the first 'G' residue of the translation start codon GTG of the guaA gene . Electrophoretic mobility shift assay showed strong binding of M . smegmatis RNA polymerase holoenzyme to the 400-bp fragment that expresses lacZ in mycobacterial species and a weak binding to the 280-bp fragment that expresses only in E . coli DH5alpha . Both promoter recombinants revealed varied response in the presence of purine nucleotides and exhibited down-regulation when subjected to amino acid starvation.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 243 - 8
Transformation using in vivo and in vitro methylation in Streptomyces griseus; Kwak J et al.; Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer . To efficiently transform S . griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation . Whereas heat treatment of protoplasts or methylation of the E . coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E . coli strains tested in this study highly increased the transformation efficiency . The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E . coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S . griseus . We identified several restriction-modification systems that decrease the transformation efficiency . This research also led us to understand methylation profiles and restriction-modification systems in S . griseus.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 229 - 35
Activity of the Streptomyces coelicolor stress-response sigma factor sigmaH is regulated by an anti-sigma factor; Sevcikova B et al.; The alternative sigma factor sigmaH has been shown to play an important role in stress response and morphological differentiation in Streptomyces coelicolor . Its gene, sigH, is located in an operon with the gene encoding proposed anti-sigma factor UshX, and one of the promoters directing expression of the operon is dependent upon sigH . To clarify the function of S . coelicolor UshX, both the sigmaH and UshX proteins were overproduced in Escherichia coli and purified . In an in vitro transcription assay, sigmaH, after complementation with S . coelicolor core RNA polymerase, was able to recognize the sigH-dependent promoter, sigH-P2 . This transcription was inhibited by UshX, if it was incubated with sigmaH prior to the addition of the core RNA polymerase . When sigmaH and UshX were incubated and electrophoresed through non-denaturing polyacrylamide gels, they formed a specific complex . These results showed that UshX is a specific anti-sigma factor for sigmaH, and the S . coelicolor sigH operon is directly autoregulated.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 209 - 13
Efflux of chloramphenicol by the CmlA1 protein; George AM et al.; The cmlA1 gene cassette contains the cmlA1 gene, that confers resistance to chloramphenicol, as well as a promoter and translational attenuation signals, and expression of cmlA1 is inducible by low concentrations of chloramphenicol . The CmlA1 protein encoded by cmlA1 was localised in the inner membrane . Active efflux of chloramphenicol, additional to the endogenous efflux from Escherichia coli cells, was observed when the cmlA1 gene was present and the production of CmlA1 had been preinduced with subinhibitory concentrations of chloramphenicol . Both endogenous and CmlA1-mediated export of chloramphenicol was driven by the proton-motive force.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 183 - 8
LC-MS analysis of pig intestine sulfatides: interaction with Escherichia coli STb enterotoxin and characterization of molecular species present; Beausoleil HE et al.; STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man . Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb . These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules . However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors . The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue . Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet . The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode . The most abundant ions observed had m/z values of 779, 795, 879 and 907 . For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons . The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.

Biochim Biophys Acta, 2002 Apr 3, 1589(2), 219 - 31
Molecular cloning and characterisation of p15(CDK-BP), a novel CDK-binding protein; Vogel L et al.; The suc1/Cks proteins are well-conserved regulatory components of cyclin-dependent kinases 1 and 2 (CDK1/2) . These small molecular mass proteins form a stable complex with CDK1/2 and are essential for normal regulation of CDKs during the cell division cycle and for degradation of p27(kip1) . Despite the high degree of homology between the nine known CDKs, only CDK1, CDK2 and, to a lesser extent, CDK3 are able to bind to the suc1/Cks proteins . No additional suc1/Cks-related proteins interacting with other CDKs have been reported . We have purified, from starfish oocytes, a 15 kDa protein, p15(CDK-BP), which cross-reacts with anti-Cks antibodies (L . Azzi, L . Meijer, A.C . Ostvold, J . Lew, J.H . Wang, J . Biol . Chem . 269 (1994)) . Following microsequencing of internal peptides and generation of corresponding oligonucleotides we cloned two cDNAs encoding two closely related proteins, p15A and p15B . The predicted protein sequences display distant but distinct homology with the Suc1/Cks proteins, including the genuine starfish Cks homologue protein, p9(CksMg) . P15 transcripts are essentially expressed in oocytes . Recombinant p15B or native p15(CDK-BP) bind a 34 kDa protein cross-reacting with anti-PSTAIRE antibodies, a feature characteristic of CDK-related proteins . In addition p15B interacts tightly with CDK4, CDK6, CDK8 and the yeast CDC28-related kinase Pho85, but not with CDK1, CDK2 or CDK7 . P15 does not appear to alter the catalytic activity of the bound kinases.

Biochim Biophys Acta, 2002 Apr 3, 1589(2), 151 - 9
Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control; Kerkhoff E et al.; Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation . This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins . NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase . We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf . Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells . Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho . With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac . Another feature of cell transformation is the deregulation of cell cycle control . NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase . Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1) . These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.

FEMS Microbiol Lett, 2002 Mar 19, 209(1), 81 - 5
Identification of a CysB-regulated gene involved in glutathione transport in Escherichia coli; Parry J et al.; Growth of Escherichia coli using the tripeptide glutathione as a sulfur source is well documented, but transport of glutathione into E . coli is uncharacterized . We have found that the ybiK gene, at 18.7 min, appears to be involved in the transport of glutathione and have therefore renamed ybiK as spt for sulfur peptide transport . The ybiK/spt gene is the first of what appear to be five cotranscribed genes, three of which show high homology to the peptide transport operon dpp . When the lacZ gene encoding beta-galactosidase was fused to the promoter of ybiK/spt, expression of the ybiK-lacZ fusion was repressed in rich media . This was shown to be due to the presence of exogenous cysteine . The ybiK-lacZ fusion was found to be regulated by cysB, the transcriptional activator for the cysteine regulon . Mutations in the cysB or ybiK genes led to severe growth inhibition when cells were given glutathione as the sole sulfur source . In particular, strains of E . coli containing mutations in both the ybiK and cysA genes were unable to grow when the sole sulfur source provided was glutathione whereas single cysA mutants grew well with glutathione . In contrast, no such defects were seen when L-djenkolic acid or cysteine were used as the sole sulfur source.

Int J Biochem Cell Biol, 2002 Aug, 34(8), 958 - 69
Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily; Riley E et al.; Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines, drugs and xenobiotics . While in most occasions, sulfonation is a detoxication pathway, in the case of certain drugs and carcinogens, it leads to metabolic activation . Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal . In the present study, a novel sulfotransferase isoform (GenBank Accession no . AF360872) was isolated from a rabbit liver cDNA lambdaZAP II library . The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da) . The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species . Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E . coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenol (K(m) and Vmax values of 0.15 microM and 897.5 nmol/min/mg protein, respectively) and dopamine (K(m) and V(max) values of 175.3 microM and 151.1 nmol/min/mg protein, respectively) . Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1 . Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and rectum.

Biochim Biophys Acta, 2002 Apr 29, 1596(2), 283 - 92
Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+; Calderone V et al.; 3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid . Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies . The recombinant human protein, obtained in E . coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed . On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance . A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.

Biochim Biophys Acta, 2002 Apr 29, 1596(2), 212 - 24
Enhanced response to antibody binding in engineered beta-galactosidase enzymatic sensors; Feliu JX et al.; Peptide display on solvent-exposed surfaces of engineered enzymes allows them to respond to anti-peptide antibodies by detectable changes in their enzymatic activity, offering a new principle for biosensor development . In this work, we show that multiple peptide insertion in the vicinity of the Escherichia coli beta-galactosidase active site dramatically increases the enzyme responsiveness to specific anti-peptide antibodies . The modified enzymes HD7872A and HT7278CA, carrying eight and 12 copies respectively of a foot-and-mouth disease peptide per enzyme molecule, show antibody-mediated activation factors higher than those previously observed in the first generation enzymatic sensors, for HT7278CA being close to 400% . The analysis of the signal transduction process with multiple inserted proteins strongly suggests a new, non-exclusive mechanism of enzymatic regulation in which the target proteins might be stabilised by the bound antibody, extending the enzyme half-life and consequently enhancing the signal-background ratio . In addition, the tested sensors are differently responsive to sera from immune farm animals, depending on the antigenic similarity between the B-cell epitopes in the immunising virus and those in the peptide used as sensing element on the enzyme surface . Altogether, these results point out the utility of these enzymatic biosensors for a simple diagnosis of foot-and-mouth disease in an extremely fast homogeneous assay.

J Photochem Photobiol B, 2002 May, 67(1), 39 - 50
Fluorescence investigation of the recombinant cyanobacterial phytochrome (Cph1) and its C-terminally truncated monomeric species (Cph1Delta2): implication for holoprotein assembly, chromophore-apoprotein interaction and photochemistry; Sineshchekov V et al.; Recombinant dimeric full-length Cph1 holophytochrome and its C-terminally-truncated monomeric species {Cph1Delta2, comprising the chromophore-bearing N-terminal sensory module (residues 1 to 514)} from the cyanobacterium Synechocystis expressed in E . coli and reconstituted in vitro with phycocyanobilin (PCB) were investigated with the use of fluorescence spectroscopy and photochemistry in the temperature range from 85 to 293 K . Holoprotein assembly in Cph1 apparently proceeds via intermediate states with the emission maximum at 680-690 nm (I685) and 700 nm (I700) and a half-life time, at room temperature, of < or =5 s . Conversion of the putative I685 into mature Cph1 involves relaxation of the chromophore into a more flexible conformation . Cph1 and Cph1Delta2 were closely similar in their spectroscopic and photochemical characteristics (position of the emission band and its width, character of the temperature dependence of the fluorescence and activation energy of the fluorescence decay, kinetics and extent of the Pr conversion at low and ambient temperatures), suggesting that there is no immediate effect of the C-terminus on the photochemical properties of the chromophore in Cph1 and that chromophore-chromophore interactions in the dimer are not significant . The latter is also supported by the lack of energy transfer from the phycoerythrobilin (PEB) to PCB in the mixed PEB/PCB adduct of Cph1 . At the same time, certain variations in the fluorescence and photochemical parameters of Cph1 with temperature of the sample and intensity of the excitation light and dependence of the emission spectra on excitation wavelength were observed . These variations are interpreted as a manifestation of the Cph1 heterogeneity which may be due to the existence of different conformers of the chromophore and photoproduct formation under excitation light.

Cell, 2002 Apr 19, 109(2), 193 - 203
The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand; Artsimovitch I et al.; The transcriptional regulatory protein RfaH controls expression of several operons that encode extracytoplasmic components in bacteria . Regulation by RfaH occurs during transcript elongation and depends on a 5'-proximal, transcribed nucleic acid sequence called ops that induces transcriptional pausing in vitro and in vivo . We report that RfaH recognizes RNA polymerase transcribing RfaH-regulated operons by interacting with the ops sequence in the exposed nontemplate DNA strand of ops-paused transcription complexes . Although RfaH delays escape from the ops pause, once escape occurs, RfaH enhances elongation by suppressing pausing and rho-dependent termination without apparent involvement of other accessory proteins . This activity predicts a cumulative antitermination model for RfaH's regulation of ops-containing operons in vivo.

Hum Mutat, 2002 Jun, 19(6), 641 - 55
High homocysteine and thrombosis without connective tissue disorders are associated with a novel class of cystathionine beta-synthase (CBS) mutations; Maclean KN et al.; Cystathionine beta-synthase (CBS) is a crucial regulator of plasma levels of the thrombogenic amino acid homocysteine (Hcy) . Homocystinuria due to CBS deficiency confers a dramatically increased risk of thrombosis . Early diagnosis usually occurs after the observation of ectopia lentis, mental retardation, or characteristic skeletal abnormalities . Homocystinurics with this phenotype typically carry mutations in the catalytic region of the protein that abolish CBS activity . We describe a novel class of missense mutations consisting of I435T, P422L, and S466L that are located in the non-catalytic C-terminal region of CBS that yield enzymes that are catalytically active but deficient in their response to S-adenosylmethionine (AdoMet) . The P422L and S466L mutations were found in patients suffering premature thrombosis and homocystinuric levels of Hcy but lacking any of the connective tissue disorders typical of homocystinuria due to CBS deficiency . The P422L and S466L mutants demonstrated a level of CBS activity comparable to that of the AdoMet stimulated wild-type CBS but could not be further induced by the addition of AdoMet . In terms of temperature stability, oligomeric organization, and heme saturation the I435T, P422L, and S466L mutants are indistinguishable from wild-type CBS . Our findings illustrate the importance of AdoMet for the regulation of Hcy metabolism and are consistent with the possibility that the characteristic connective tissue disturbances observed in homocystinuria due to CBS deficiency may not be due to elevated Hcy .

Hum Mutat, 2002 Jun, 19(6), 629 - 40
Propionic acidemia: analysis of mutant propionyl-CoA carboxylase enzymes expressed in Escherichia coli; Chloupkova M et al.; Deficiency of propionyl-CoA carboxylase (PCC) results in propionic acidemia, an autosomal recessive disorder characterized by ketoacidosis sufficiently severe to cause neonatal death . PCC is involved in the catabolism of branched-chain amino acids, odd-chain fatty acids, and cholesterol . The enzyme is a biotin-dependent mitochondrial protein composed of two heterologous subunits arranged into an 800-kDa alpha(6 )beta(6) dodecameric structure . Approximately 60 mutations have been reported in the nuclear genes PCCA and PCCB that encode the two PCC subunits . The vast majority of these mutations have not been examined at the protein level . We present an initial characterization of 13 mutations located in exons 1, 3-7, and 12-14 of PCCB . After expression in E . coli, these recombinant mutant enzymes were analyzed for stability, biotinylation, alpha-beta subunit interaction, and activity . Our results show a functional dichotomy in these PCCB mutations with some mutants (R44P, S106R, G131R, G198D, V205D, I408del, and M442T) capable of varying degrees of assembly but forming catalytically inactive PCC proteins . Other PCCB mutants (R165W, E168K, D178H, P228L, and R410W) that are PCC deficient in patient-derived fibroblasts, were found to be capable of expressing wild-type level PCC activity when assembled in our chaperone-assisted E . coli expression system . This result indicates that these mutations exert their pathogenic effect due to an inability to assemble correctly in patients' cells . This initial screen has identified a range of mutant PCC proteins that are sufficiently stable to be purified and subsequently used for structure-function analysis to further elucidate the complex relationship between genotype and phenotype in propionic acidemia .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 131 - 7
The effect of N-terminal changes on arginyl-tRNA synthetase from Escherichia coli; Liu W et al.; An Asn(2) deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn(2) and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied . The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins . Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in aminoacid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNA(Arg) at all . The mutant deleted Asn(2) and Ile(3) was able to be expressed in Escherichia coli but not stable to be purified . The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme . The data show that the conformation of the mutants are different and the tryptophan residues in the mutants are more exposed than those in the native enzyme . An estimate of the secondary structure of the mutant enzymes from their far ultraviolet CD spectra showed that the chimera mutant contained less alpha-helix, more beta-sheet and slightly higher fraction of random coil, as compared with the native enzyme . The results indicate that an intact N-terminal domain of E.coli arginyl-tRNA synthetase is important to its activity and correct folding.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 248 - 52
{Functional di-domain of neuronal growth inhibitory factor}; Ji QZ et al.; Neuronal growth inhibitory factor (GIF), known also as metallothionein-III (MT-III), was the first validated to be capable of inhibiting growth of neuronal cells in nervous system, its beta-domain being functional . GIF functional di-domain (GIFbeta- beta) was constructed to study the structure and function of GIF . N terminal beta-domain and C terminal beta-domain cDNAs were amplified by PCR, inserted into vector pGEX-4T-1 and expressed in Escherichia coli, as carboxyl terminal extension of glutathione-S-transferase (GST), by IPTG induction . After digestion by thrombin, the fusion protein was isolated by passing through a glutathione-Sepharose 4B affinity chromatography column and was purified by gel fit ration on Sephacryl-S100 . About 60 mg protein per liter of bacterial cell culture was achieved . The results of SDS-PAGE, amino acid composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein showed that the purified protein was the GIFbeta- beta . Circular dichroism (CD) spectroscopy show GIFbeta- beta has characteristic metal-sulfhydryl clusters of metallothionein family . Inhibitory activities detected by the MTT reduction assay are: GIF > GIFbeta-beta > GIF beta-domain.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 199 - 203
{A novel bifunctional in vivo mutation detection system}; Yao ZZ et al.; A shuttle plasmid pMCLacI/neo with two copies of LacI was integrated into mouse genome and a novel system which could detect in vivo mutation of both expression and silence genes was constructed, enabling the comparative analysis of their mutation spectra and mutant frequencies . 486 fertilized eggs from C57BL/6 mice with microinjected pMCLacI/neo plasmid were transferred into oviducts of 18 pseudo-pregnant mice, and 32 alive offsprings were screened and identified by using PCR and Southern blotting . Genomes of 5 mice had pMCLacI/neo plasmid integrated, as verified by Southern blot after the PCR screening . Only one of the two LacI in pMCLacI/neo was in expression state; and this established a model, that the status in vivo of both gene expression and silencing could be simulated . This kind of mice might be used as a novel bifunctional mutation detection system in vivo.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 187 - 92
{Phosphorylation of YLR190w by PAP1 PHO85 kinase complex}; Shi XZ et al.; A yeast two-hybrid screening by using PAP1 was performed to identify targets for PAP1-PHO85 cyclin-CDK complex . N-terminal fragment of protein YLR190w, a yeast gene encoding a 491 amino acids peptide, was identified, and its coding region was amplified by PCR . The interaction of PAP1 and YLR190w was confirmed by both two-hybrid assay and GST pull-down assay in vitro . The PAP1-PHO85 kinase complex obtained from the immunoprecipitates could phosphorylate GST-YLR190w expressed in E.coli, and the phosphorylation of YLR190w was affected by the phosphate concentration, and the phosphorylation sites of YLR190w were Ser/Thr-Promotif, as revealed by protein mutation assay . In another library screen, YAF9, a yeast homolog of human AF9, was isolated using the two-hybrid system with YLR190w as the bait . It was revealed that interaction of YLR190w and YAF9 was affected by phosphate concentration . When all Ser/Thr in Ser/Thr-Pro motif were mutated to Ala, the interaction of YLR190w (mutant) and YAF9 was weakened, and the effect of phosphate concentration was impaired . Ylr190w was not involved in the PHO system by the acid phosphatase activity assay . Deletion of Ylr190w was constructed by homologous recombination and the doubling time of Ylr190w mutant strain as longer than that of wild type.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 181 - 6
{Cloning and expression of human calcyclin binding protein (hCacyBP) gene}; Liu WX et al.; Human calcyclin binding protein (hCacyBP) gene was obtained by the screening of a human cDNA library . The full coding region of CacyBP was cloned into E.coli strain pET28, and then was expressed and purified through affinity chromatography . Rabbit anti-human CacyBP polyclonal antibody was obtained by immunizing rabbit with the purified human CacyBP . Western blots showed that it was expressed extensively in many tissues of mouse . The results of immunohistochemistrial staining showed that the location of CacyBP in BT325 cell line before and after differentiation changed from cytoplasm into nucleus and perinucleus cytoplasm.

Crit Care Med, 2002 May, 30(5), 1091 - 8
n-3 Polyunsaturated fatty acid-enriched diet does not protect from liver injury but attenuates mortality rate in a rat model of systemic endotoxemia; Vollmar B et al.; OBJECTIVE: We investigated the potential of dietary fish oil containing n-3 polyunsaturated fatty acids to attenuate hepatic injury and mortality rate of rats in response to systemic endotoxemia . DESIGN: Prospective, randomized, controlled animal study . SETTING: University laboratory . SUBJECTS: A total of 43 male Sprague Dawley rats . INTERVENTIONS: Rats were fed either fish oil supplement or regular standard lab chow . After 8 wks of feeding, each diet group was subjected to a single exposure of lipopolysaccharide (Escherichia coli, 10 mg/kg intravenously) or saline . Hepatic microvascular response and liver injury were assessed by in vivo analysis of Kupffer cell phagocytic activity, leukocyte-endothelial cell interaction, nutritive sinusoidal perfusion failure, and parenchymal cell apoptosis (intravital fluorescence epi-illumination technique) as well as bile flow, serum liver enzyme activities, and tissue histomorphology . MEASUREMENTS AND MAIN RESULTS: In animals fed a standard diet, livers at 16 hrs after lipopolysaccharide-exposure exhibited depressed Kupffer cell phagocytic activity, enhanced hepatic microvascular leukocyte activation, leukocytic tissue infiltration, sinusoidal perfusion failure, and parenchymal cell apoptosis . Hepatic microvascular injury was further accompanied by reduced bile flow and enhanced liver enzyme release . The fish oil enriched diet did not significantly change the multiple features of endotoxemia-associated liver injury; however, it maintained arterial blood pressure, systemic leukocyte count, and acid base balance and showed a tendency toward improved survival on lipopolysaccharide exposure with a 16 hr-survival rate of 80% (p =.06 vs . survival rate of 40% in animals fed a regular diet) . Moreover, slightly increased serum concentrations of interleukin-10 coincided with enhanced concentrations of interleukin-6 in fish oil fed endotoxemic animals . Healthy, non-lipopolysaccharide-exposed, fish oil fed animals did not differ from those fed with the regular diet, except for dampened Kupffer cell phagocytic activity . CONCLUSIONS: Fish oil feeding does not protect from local endotoxemia-induced hepatic microvascular dysfunction . However, dietary modulation of inflammatory mediator response by macrophages, constituting an appropriate immune response, could add to the survival advantage seen in fish oil-fed animals on exposure to lipopolysaccharide.

Crit Care Med, 2002 May, 30(5), 1071 - 82
Novel phenanthridinone inhibitors of poly (adenosine 5'-diphosphate-ribose) synthetase: potent cytoprotective and antishock agents; Jagtap P et al.; OBJECTIVE: To synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5'-diphosphate {ADP}-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock . DESIGN: Randomized, prospective laboratory study . SETTING: Research laboratory . SUBJECTS: Murine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats . INTERVENTIONS: Macrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis . Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity . Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1-10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor . The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability . Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally) . PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment . Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured . In addition, survival rates and vascular contractile and relaxant responses were recorded . MEASUREMENTS AND MAIN RESULTS: Appropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents . The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM . PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34 . The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized) . The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages . PJ34 was selected for the subsequent in vivo studies . PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse . PJ34 reduced peak plasma concentrations of tumor necrosis factor-alpha, interleukin-1beta, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats . PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut . Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34 . Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment) . CONCLUSIONS: The novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

J Biol Chem, 2002 Jul 12, 277(28), 25703 - 6 Epub 2002 May 02.
Annexin XXI (ANX21) of Giardia lamblia has sequence motifs uniquely sdhared by giardial annexins and is specifically localized in the flagella; Szkodowska A et al.; We have identified a novel annexin, ANX21, in trophozoites of Giardia lamblia . The nucleotide sequence encoding this protein deviated from a published sequence in predicting an additional endonexin fold in the fourth annexin domain . In addition, several motifs exclusively shared by other annexins of G . lamblia in their predicted fourth repeat and predicted to be localized on the opposite (concave) surface of the molecule became apparent . Western blots of trophozoite fractions probed with antiserum against the recombinant protein indicated that this annexin, like the other giardial annexins ANX19 and ANX20, associates with phospholipids in the presence of Ca(2+) . Finally, confocal laser scanning of trophozoites showed that the protein, apart from the median body, was exclusively localized in the eight flagella . Together, these data suggest that ANX21 may function as a Ca(2+)-regulated structural element linking phospholipid bilayer and underlying axoneme.

J Biol Chem, 2002 Jul 12, 277(28), 25527 - 36 Epub 2002 May 02.
Alternative splicing and promoter usage generates an intracellular stromelysin 3 isoform directly translated as an active matrix metalloproteinase; Luo D et al.; Human stromelysin 3 (ST3) is a matrix metalloproteinase (MMP) that has been implicated in cancer progression and in various tissue remodeling processes . Unlike most MMPs, ST3 is characterized by a distinct substrate specificity and a specific regulation and is not directly involved in extracellular matrix degradation . In the present study, we have identified an additional ST3 gene promoter that is accessible to nuclear factors such as C/EBP and retinoic acid receptors . This human specific promoter is inducible and controls the expression of a novel ST3 transcript called the beta-ST3 that is expressed in cultured cells and in placenta . This transcript encodes a 40-kDa ST3 isoform that lacks both the signal peptide common to all secreted MMPs and the prodomain that normally maintains enzyme latency . Consistent with the lack of a signal peptide, the beta-ST3 was found to be intracellular . The relative amount of the extracellular alpha-ST3 isoform was about 20-fold higher than that of the intracellular ST3 isoforms, as estimated by Western blot analysis . Furthermore, recombinant beta-ST3 produced in Escherichia coli exhibits a proteolytic activity against alpha1-proteinase inhibitor, a substrate previously shown to be inactivated by the alpha-ST3 . Therefore, although it was thought that all MMPs were synthesized as inactive zymogens and functioned extracellularly, this is the first MMP isoform reported that is generated by alternative promoter usage and directly translated as an active enzyme . Although the intracellular function of the beta-ST3 remains to be investigated, these data support the idea that the functions of MMPs are not restricted to the extracellular space.

J Biol Chem, 2002 Jul 5, 277(27), 24420 - 6 Epub 2002 May 02.
Use of an in vivo reporter assay to test for transcriptional and translational fidelity in yeast; Shaw RJ et al.; Eukaryotic RNA polymerase II and Escherichia coli RNA polymerase possess an intrinsic ribonuclease activity that is stimulated by the polymerase-binding proteins SII and GreB, respectively . This factor-activated hydrolysis of nascent RNA has been postulated to be involved in transcription elongation as well as removal of incorrect bases misincorporated into RNA . Little is known about the frequency of misincorporation by RNA polymerases in vivo or about the mechanisms involved in improving RNA polymerase accuracy . Here we have developed a luciferase reporter system in an effort to assay for base misincorporation in living Saccharomyces cerevisiae . The assay employs a luciferase open reading frame that contains a premature stop codon . The inactive truncated enzyme would become active if misincorporation by RNA polymerase II took place at the stop triplet . Yeast lacking SII did not display a significant change in reporter activity when compared with wild-type cells . We estimate that under our assay conditions, mRNAs with a misincorporation at the test site could not exceed 1 transcript per 500 cells . The reporter assay was very effective in detecting the previously described process of nonsense suppression (translational read-through) by ribosomes, making it difficult to determine an absolute level of basal (SII-independent) misincorporation by RNA polymerase II . Although these data cannot exclude the possibility that SII is involved in proofreading, they make it unlikely that such a contribution is physiologically significant, especially relative to the high frequency of translational errors.

J Biol Chem, 2002 Jul 12, 277(28), 24995 - 5000 Epub 2002 May 10.
Escherichia coli MoeA and MogA . Function in metal incorporation step of molybdenum cofactor biosynthesis; Nichols J et al.; Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor . Here we show that moeA(-) and mogA(-) cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide . Human sulfite oxidase expressed in E . coli moeA(-) could be activated in vitro in the presence of MoeA and low concentrations of molybdate . Sulfite oxidase purified from the moeA(-) lysate was also activated, although to a lesser extent than observed in the presence of lysate . MogA was incapable of activating sulfite oxidase expressed in E . coli mogA(-) . These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation.

J Biol Chem, 2002 Aug 2, 277(31), 28058 - 64 Epub 2002 May 10.
Transcriptional regulation in constraints-based metabolic models of Escherichia coli; Covert MW et al.; Full genome sequences enable the construction of genome-scale in silico models of complex cellular functions . Genome-scale constraints-based models of Escherichia coli metabolism have been constructed and used to successfully interpret and predict cellular behavior under a range of conditions . These previous models do not account for regulation of gene transcription and thus cannot accurately predict some organism functions . Here we present an in silico model of the central E . coli metabolism that accounts for regulation of gene expression . This model accounts for 149 genes, the products of which include 16 regulatory proteins and 73 enzymes . These enzymes catalyze 113 reactions, 45 of which are controlled by transcriptional regulation . The combined metabolic/regulatory model can predict the ability of mutant E . coli strains to grow on defined media as well as time courses of cell growth, substrate uptake, metabolic by-product secretion, and qualitative gene expression under various conditions, as indicated by comparison with experimental data under a variety of environmental conditions . The in silico model may also be used to interpret dynamic behaviors observed in cell cultures . This combined metabolic/regulatory model is thus an important step toward the goal of synthesizing genome-scale models that accurately represent E . coli behavior.

EMBO J, 2002 May 15, 21(10), 2354 - 63
Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA; Kadokura H et al.; Protein disulfide bond formation in Escherichia coli is catalyzed by the periplasmic protein DsbA . A cytoplasmic membrane protein DsbB maintains DsbA in the oxidized state by transferring electrons from DsbA to quinones in the respiratory chain . Here we show that DsbB activity can be reconstituted by co-expression of N- and C-terminal fragments of the protein, each containing one of its redox-active disulfide bonds . This system has allowed us (i) to demonstrate that the two DsbB redox centers interact directly through a disulfide bond formed between the two DsbB domains and (ii) to identify the specific cysteine residues involved in this covalent interaction . Moreover, we are able to capture an intermediate in the process of electron transfer from one redox center to the other . These results lead us to propose a model that describes how the cysteines cooperate in the early stages of oxidation of DsbA . DsbB appears to adopt a novel mechanism to oxidize DsbA, using its two pairs of cysteines in a coordinated reaction to accept electrons from the active cysteines in DsbA.

EMBO J, 2002 May 15, 21(10), 2312 - 22
A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids; Kokoeva MV et al.; Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively . Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein . We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes . Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers . Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems . The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.

Am J Physiol Endocrinol Metab, 2002 Jun, 282(6), E1276 - 85
Euglycemic hyperinsulinemia augments the cytokine and endocrine responses to endotoxin in humans; Soop M et al.; Type 2 diabetes is associated with biochemical evidence of low-grade inflammation, and experimental studies have suggested that both insulin and glucose affect inflammatory responses . To determine the effect of in vivo changes in glucose availability and plasma insulin concentrations in humans, we administered 20 U/kg Escherichia coli lipopolysaccharide (LPS) or saline (control) to 14 subjects during a euglycemic hyperinsulinemic clamp (n = 6) or an infusion of sterile saline (n = 8) . Parallel in vitro studies on human whole blood were undertaken to determine whether there was a direct effect of glucose, insulin, and leptin on proinflammatory cytokine production . Infusion of glucose and insulin significantly amplified and/or prolonged the cardiovascular, plasma interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and counterregulatory hormone responses to LPS, whereas the effects on fever, plasma norepinephrine concentrations, and oxygen consumption were unaffected . In vitro studies showed no modulation of LPS-stimulated IL-6 or TNF-alpha production by glucose, insulin, or leptin at physiologically relevant concentrations . Hyperinsulinemia indirectly enhances key components of the systemic inflammatory and stress responses in this human model of infection.

Phys Rev E Stat Nonlin Soft Matter Phys . 2002 Apr;65(4 Pt 1):041905 . Epub 2002 Mar 25.
Analysis of symbolic sequences using the Jensen-Shannon divergence; Grosse I et al.; We study statistical properties of the Jensen-Shannon divergence D, which quantifies the difference between probability distributions, and which has been widely applied to analyses of symbolic sequences . We present three interpretations of D in the framework of statistical physics, information theory, and mathematical statistics, and obtain approximations of the mean, the variance, and the probability distribution of D in random, uncorrelated sequences . We present a segmentation method based on D that is able to segment a nonstationary symbolic sequence into stationary subsequences, and apply this method to DNA sequences, which are known to be nonstationary on a wide range of different length scales.

Biomacromolecules, 2002 May-Jun, 3(3), 411 - 4
A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand; Uzawa H et al.; A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method . The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film . Then, the film was transferred onto a QCM cell surface modified with alkanethiols . Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell . Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins . On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.

Structure (Camb), 2002 Mar, 10(3), 329 - 42
Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis; Ramon-Maiques S et al.; N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis . The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices . In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate . The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.

Structure (Camb), 2002 Mar, 10(3), 283 - 4
Chloride channel function: partial charges and dipolar rods; Mancia F et al.; The regulated flow of ions across biological membranes is a process fundamental to all living organisms . The crystal structures of representative chloride channels recently published in Nature, together with the previously determined structures of a potassium channel, provide a solid basis for understanding the chemical principles that govern selective ion flow.

Life Sci, 2001 Dec 7, 70(3), 269 - 78
Effects of six diterpenes on macrophage eicosanoid biosynthesis; de las Heras B et al.; Six diterpenes (three clerodanes, two abietanes and one rosane) were tested for interactions with the cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism and for effects of nitric oxide production . Two abietane diterpenes, aethiopinone and 11,12-dihydroxy-6-oxo-8,11,13-abietatriene and the rosane lagascatriol showed a remarkable effect on COX-1 pathway of PGE2 release in calcium ionophore A23187-stimulated peritoneal macrophages . Only the two latter diterpenes showed inhibition on COX-2 pathway of PGE2 release in E . coli LPS-stimulated peritoneal macrophages . In addition, all compounds assayed were inhibitors of LTC4 release with IC50 < or = 10 microM . Clerodane diterpenes were inactive in COX assay . None of the diterpenes assayed, except 11,12-dihydroxy-6-oxo-8,11,13-abietatriene, affected NO production . The results obtained suggest that the cellular mechanisms of action of some of these substances may involve inhibition of cyclooxygenase/lipoxygenase pathways and nitric oxide production.

Clin Chem Lab Med, 2002 Mar, 40(3), 293 - 7
Pancreatic phospholipase A2 activity in acute pancreatitis: a prognostic marker for early identification of patients at risk; Aufenanger J et al.; Remarkably elevated levels of phospholipase A2 (PLA2) are measurable in human blood samples in cases of acute pancreatitis . The source of the enzyme was first thought to be exclusively the pancreas, but now it is generally accepted that two isoenzymes--the pancreatic PLA2, group I, and the extrapancreatic PLA2, group II--contribute to the raised activity . In contrast to the group II-PLA2, the pancreatic PLA2 is heat-resistant for 1 hour at 60 degrees C . The catalytically inactive proenzyme of the pancreatic PLA2 can be activated by trypsin . The aim of our study was to evaluate the diagnostic value of PLA2 isoenzyme activity measurements to identify patients with severe complications in acute pancreatitis . Blood samples from patients suffering from acute pancreatitis were analyzed for catalytically active pancreatic PLA2 on day 1 and 2 of hospitalization with a modified radiometric Escherichia coli-based PLA2 assay . In 10 of 41 patients clearly elevated values of catalytically active, heat-resistant pancreatic PLA2 (7.2 to 81.2 U/l) were observed . This group of patients was characterized by severe complications (necrotizing pancreatitis, shock, sepsis, respiratory problems) of which two patients subsequently died . Patients with low or undetectable activity (<7 U/l) of pancreatic PLA2 recovered rapidly . According to these results the presence of catalytically active pancreatic PLA2 in serum is associated with severe complications of acute pancreatitis . In contrast to total serum-PLA2, the catalytic concentration of pancreatic PLA2 can serve as a prognostic marker in acute pancreatitis.

Biosci Biotechnol Biochem, 2002 Mar, 66(3), 622 - 7
Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase; Fujii T et al.; The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified . We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA . This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E . coli . The complementation experiment of proC32 in E . coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA . Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro . These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA . Biotransformation of L-Lys to L-PA using lat-expressing E . coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation . It is noteworthy that the ee-value of the produced pipecolic acid was 100%.

Biosci Biotechnol Biochem, 2002 Mar, 66(3), 558 - 65
Cloning and expression of Bombyx mori silk gland elongation factor 1gamma in Escherichia coli; Kamiie K et al.; Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of alpha-, beta-, gamma-, and delta-subunits . EF-1alpha GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP . EF-1betagammadelta catalyzes the exchange of EF-1alpha-bound GDP for exogenous GTP and stimulates the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosomes . EF-1gamma cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library . The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively . The silk gland EF-1gamma shares 67.3% amino acid identity with Artemia salina EF-lgamma . The N-terminal domain (amino acid residues 1-211) of silk gland EF-lgamma is 29.3% identical to maize glutathione S-transferase . We demonstrated that silk gland EF-lgamma bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1gamma may have the capacity to bind to glutathione.

J Biol Chem, 2002 Jul 19, 277(29), 26171 - 6 Epub 2002 May 09.
Rad52 protein has a second stimulatory role in DNA strand exchange that complements replication protein-A function; New JH et al.; Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae . We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange . This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concentration) of replication protein-A (RPA) are used . Under these conditions, Rad52 protein is needed for extensive DNA strand exchange . Interestingly, in this new role, Rad52 protein neither acts simply as a single strand DNA-binding protein per se nor, in contrast to its previously identified stimulatory roles, does it require physical interaction with RPA because it can be substituted by the Escherichia coli single strand DNA-binding protein . We propose that Rad52 protein acts by stabilizing the Rad51 presynaptic filament.

J Biol Chem, 2002 Jul 19, 277(29), 26157 - 62 Epub 2002 May 09.
The phase property of membrane phospholipids is affected by the functionality of signal peptides from the Escherichia coli ribose-binding protein; Ahn T et al.; We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers . The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition . The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species . The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid . The wild-type and revertant signal peptides predominantly formed alpha-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy . In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer . However, the alpha-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer . An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not . The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid . We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.

J Biol Chem, 2002 Jul 26, 277(30), 27360 - 6 Epub 2002 May 09.
The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding; Walther M et al.; The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes . This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process . To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics . We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding . Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme . These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.

J Biol Chem, 2002 Jul 26, 277(30), 26769 - 78 Epub 2002 May 09.
Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases . Variation from the SYK dogma; Creuzenet C et al.; Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified . FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily . This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis . The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis . A Glu(589) residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM . Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant . Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad . Mutations of residues specific for the short homologues (Asp(70), Asp(149)-Lys(150), Cys(103)) abolished the activity of FlaA1 . Among them, C103M prevented dimerization but did not significantly affect the secondary structure . The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases . Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding . None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.

J Bacteriol, 2002 Jun, 184(11), 3142 - 5
Behavior of sister copies of mini-F plasmid after synchronized plasmid replication in Escherichia coli cells; Onogi T et al.; To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F . Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.

J Bacteriol, 2002 Jun, 184(11), 3106 - 13
CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus; Santamarta I et al.; The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography . In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies . The partially purified CcaR protein from S . clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region . In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene . The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies . ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S . clavuligerus . These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR . Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S . clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator . These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.

J Bacteriol, 2002 Jun, 184(11), 3069 - 77
The N terminus of the Escherichia coli transcription activator MalT is the domain of interaction with MalY; Schlegel A et al.; The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins . The system is positively regulated by MalT, its transcriptional activator . MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins . MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity . By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK . This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain . Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors . The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them . Some of them became nearly completely resistant to Aes while still being sensitive to MalY . These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.

J Bacteriol, 2002 Jun, 184(11), 3044 - 52
Glycerol-3-phosphate-induced catabolite repression in Escherichia coli; Eppler T et al.; The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression . For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA(Glc), as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present . We followed the level of EIIA(Glc) phosphorylation after the addition of glycerol or G3P . In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA(Glc) . Isopropyl-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA(Glc) . A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression . We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds . Further metabolism of these compounds is not necessary for repression . Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol . Some of the prominently repressed proteins were identified by peptide mass fingerprinting . Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.

J Bacteriol, 2002 Jun, 184(11), 3017 - 26
Characterization of the class 3 integron and the site-specific recombination system it determines; Collis CM et al.; Integrons capture gene cassettes by using a site-specific recombination mechanism . As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined . The configuration of the three potentially definitive features of integrons, the intI3 gene, the adjacent attI3 recombination site, and the P(c) promoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5' conserved segment) of class 1 integrons . The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encoding tniR gene on the other, suggesting that they are part of a transposable element related to Tn402 but with the integron module in the opposite orientation . The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, the attI3 site in the integron, and different cassette-associated 59-be (59-base element) sites . Both integration of circularized cassettes into the attI3 site and excision of integrated cassettes were also catalyzed by IntI3 . The attI3 site was localized to a short region adjacent to the intI3 gene . Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.

J Bacteriol, 2002 Jun, 184(11), 2994 - 9
Sugar transport through maltoporin of Escherichia coli: role of the greasy slide; Van Gelder P et al.; The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding . To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction . The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis . Crystallographic analysis of the W74A mutant showed no perturbation of the structure . All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues . Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel . Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition . The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.

J Bacteriol, 2002 Jun, 184(11), 2951 - 62
Targeting of (D)MinC/MinD and (D)MinC/DicB complexes to septal rings in Escherichia coli suggests a multistep mechanism for MinC-mediated destruction of nascent FtsZ rings; Johnson JE et al.; The MinC protein is an important determinant of septal ring positioning in Escherichia coli . The N-terminal domain ((Z)MinC) suppresses septal ring formation by interfering with FtsZ polymerization, whereas the C-terminal domain ((D)MinC) is required for dimerization as well as for interaction with the MinD protein . MinD oscillates between the membrane of both cell halves in a MinE-dependent fashion . MinC oscillates along with MinD such that the time-integrated concentration of (Z)MinC at the membrane is minimal, and hence the stability of FtsZ polymers is maximal, at the cell center . MinC is cytoplasmic and fails to block FtsZ assembly in the absence of MinD, indicating that recruitment of MinC by MinD to the membrane enhances (Z)MinC function . Here, we present evidence that the binding of (D)MinC to MinD endows the MinC/MinD complex with a more specific affinity for a septal ring-associated target in vivo . Thus, MinD does not merely attract MinC to the membrane but also aids MinC in specifically binding to, or in close proximity to, the substrate of its (Z)MinC domain . MinC-mediated division inhibition can also be activated in a MinD-independent fashion by the DicB protein of cryptic prophage Kim . DicB shows little homology to MinD, and how it stimulates MinC function has been unclear . Similar to the results obtained with MinD, we find that DicB interacts directly with (D)MinC, that the (D)MinC/DicB complex has a high affinity for some septal ring target(s), and that MinC/DicB interferes with the assembly and/or integrity of FtsZ rings in vivo . The results suggest a multistep mechanism for the activation of MinC-mediated division inhibition by either MinD or DicB and further expand the number of properties that can be ascribed to the Min proteins.

J Bacteriol, 2002 Jun, 184(11), 2898 - 905
Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli; Vulic M et al.; During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells . In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies . The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low . Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion . The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium . Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.

J Bacteriol, 2002 Jun, 184(11), 2889 - 97
Identification of a lycopene beta-cyclase required for bacteriorhodopsin biogenesis in the archaeon Halobacterium salinarum; Peck RF et al.; Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor . As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H . salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor . The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi . To test crtY function, we constructed H . salinarum strains with in-frame deletions in the gene . In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein) . Heterologous expression of H . salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production . These results indicate that H . salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis . Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.

J Bacteriol, 2002 Jun, 184(11), 2863 - 9
The PtlE protein of Bordetella pertussis has peptidoglycanase activity required for Ptl-mediated pertussis toxin secretion; Rambow-Larsen AA et al.; Pertussis toxin of Bordetella pertussis is secreted by a type IV secretion system comprised of the products of the nine ptl (pertussis toxin liberation) genes . These proteins are believed to form a complex spanning both the inner and outer membranes and passing through the peptidoglycan layer . Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules . Assembled pertussis toxin and the secretion component proteins PtlC through PtlH are too large to diffuse through intact peptidoglycan . Therefore, we hypothesized that the Ptl system contains a peptidoglycanase activity . The PtlE protein was found to exhibit a sequence match to the active site of glycohydrolase enzymes . An N-terminally polyhistidine-tagged PtlE fusion protein, constructed and expressed in Escherichia coli and in B . pertussis, exhibited peptidoglycanase activity on activity gels . A fusion protein with alanine substitutions at the putative active site residues (aspartic acid at position 53 and glutamic acid at position 62) lacked peptidoglycanase activity . B . pertussis strains with the amino acid substitutions were deficient for pertussis toxin secretion . Based on these results, we concluded that PtlE is a peptidoglycanase responsible for the local removal or rearrangement of the peptidoglycan layer during Ptl secretion complex assembly.

Am J Physiol Heart Circ Physiol, 2002 Jun, 282(6), H2316 - 23
Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart; Nemoto S et al.; The precise molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined . Toll-like receptor-4 (TLR-4) engages lipopolysaccharide (LPS) and activates signaling pathways leading to the expression of proinflammatory cytokines implicated in myocardial dysfunction . We determined whether TLR-4 was necessary for LPS-induced myocardial dysfunction in vivo . The effects of LPS on left ventricular (LV) function were studied in mice with defective TLR-4 signaling (C3H/HeJ, TLR-4 deficient) and wild-type mice (C3HeB/FeJ) . Mice (n = 5/group) were injected with LPS or diluent, and LV function was examined by using two-dimensional echocardiography and conductance catheters . LPS significantly decreased all indexes of LV function in wild-type mice when compared with controls; LV function was not depressed in the LPS-treated TLR-4-deficient mice relative to controls . LPS increased myocardial nitric oxide synthase-2 expression and cGMP only in wild-type mice . This study suggests that TLR-4 mediates the LV dysfunction that occurs in LPS-induced shock . Therefore, TLR-4 might be a therapeutic target for attenuating the effects of LPS on the heart.

Am J Physiol Lung Cell Mol Physiol, 2002 Jun, 282(6), L1245 - 52
Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice; Maus UA et al.; Intratracheal instillation of the monocyte chemoattractant JE/monocyte chemoattractant protein (MCP)-1 in mice was recently shown to cause increased alveolar monocyte accumulation in the absence of lung inflammation, whereas combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute lung inflammation with early alveolar neutrophil and delayed alveolar monocyte influx . We evaluated the role of resident alveolar macrophages (rAM) in these leukocyte recruitment events and related phenomena of lung inflammation . Depletion of rAM by pretreatment of mice with liposomal clodronate did not affect the JE/MCP-1-driven alveolar monocyte accumulation, despite the observation that rAM constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by flow cytometry and immunohistochemistry . In contrast, depletion of rAM largely suppressed alveolar cytokine release as well as neutrophil and monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment . Despite this strongly attenuated alveolar inflammatory response, increased lung permeability was still observed in rAM-depleted mice undergoing JE/MCP-1/LPS challenge . Lung leakage was abrogated by codepletion of circulating neutrophils or administration of anti-CD18 . Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs but largely contribute to the alveolar cytokine response and enhanced early neutrophil and delayed monocyte influx under inflammatory conditions (JE/MCP-1/LPS deposition) . Loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils via beta(2)-integrin engagement.

J Org Chem, 2002 May 17, 67(10), 3499 - 501
A novel water-soluble Hantzsch 1,4-dihydropyridine compound that functions in biological processes through NADH regeneration; Sambongi Y et al.; A novel Hantzsch 1,4-dihydropyridine derivative could function in an organic solvent-free solution, and thus it could function in biological systems . These functions can be accomplished through regeneration of the reduced form of nicotinamine adenine dinucleotide (NADH), an essential compound for living organisms . The results obtained here demonstrate the usefulness of a water-soluble Hantzsch 1,4-dihydropyridine derivative and its wide applicability as a chemical energy source, which drives various biological processes efficiently.

Crit Rev Microbiol, 2002, 28(1), 43 - 60
Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1): a new toxin with an old twist; Menard LP et al.; Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) is a small protein that was first detected more than a decade ago in an enteroaggregative E . coli (EAEC) strain isolated from the stools of a diarrheic child . The EAST1 gene, astA, is not solely present in EAEC, but also in other categories of diarrheagenic E . coli . Strains expressing EAST1 have been shown to induce diarrhea principally in humans, although they have also been associated with piglets and calves . EAST1 toxin has been proposed as a virulence factor implicated in the mechanism of pathogenesis of EAEC and could play a role in the pathogenicity of other enteropathogens as well . This toxin is often compared to E . coli STa enterotoxin because they share some physical and mechanistic similarities . This review summarizes the various observations on EAST1 since its discovery.

Life Sci, 2002 Mar 22, 70(18), 2125 - 38
Activation of lung vagal sensory receptors by circulatory endotoxin in rats; Lai CJ et al.; Although endotoxin is known to induce various pulmonary responses that are linked to the function of lung vagal sensory receptors, its effects on these pulmonary receptors are still not clear . This study investigated the effects of circulatory endotoxin on the afferent activity of lung vagal sensory receptors in rats . We recorded afferent activity arising from vagal pulmonary C fibers (CFs), rapidly adapting receptors (RARs), tonic pulmonary stretch receptors (T-PSRs), and phasic pulmonary stretch receptors (P-PSRs) in 64 anesthetized, paralyzed, and artificially ventilated rats . Intravenous injection of endotoxin (50 mg/kg; lipopolysaccharide) stimulated 7 of the 8 CFs, 8 of the 8 RARs, and 4 of the 8 T-PSRs studied, while having no effect on the 8 P-PSRs tested . The stimulation started 3-16 min after endotoxin injection and lasted until the end of the 90-min observation period . The evoked discharge of either CFs or RARs was not in phase with the ventilatory cycle, whereas that of T-PSRs showed a respiratory modulation . Injection of a saline vehicle caused no significant change in the discharge of these receptors . Additionally, endotoxin significantly produced an increase in total lung resistance, and decreases in dynamic lung compliance and arterial blood pressure . Our results demonstrate that a majority of lung vagal sensory receptors are activated following intravenous injection of endotoxin, and support the notion that these pulmonary receptors may function as an important afferent system during endotoxemia.

Proteins, 2002 Jun 1, 47(4), 513 - 20
Flexibility in monomeric Cu,Zn superoxide dismutase detected by limited proteolysis and molecular dynamics simulation; Falconi M et al.; Limited proteolysis by trypsin of monomeric Cu,Zn superoxide dismutase from Escherichia coli induces a specific cleavage of the polypeptide chain at the level of Lys60 located in the S-S subloop of loop 6,5 where, when compared to the eukaryotic enzyme, a seven-residues insertion, completely exposed to the solvent, is observed . This result suggests that this subloop is disordered and flexible, thus enabling binding and adaptation to the active site of the proteolytic enzyme . Indeed, molecular dynamics simulation indicates that the S-S subloop undergoes high fluctuations and that its high flexibility coupled to an high solvent accessibility can explain the specific bond selection of the protease . As a matter of fact, of the possible 14 solvent accessible proteolytic sites only the Lys60 flexible site is cleaved . High flexibility and solvent exposure are confirmed by the short water residence time for the residues corresponding to the cleavage site evaluated by molecular dynamics simulation . These experiments demonstrate that molecular dynamics simulation and limited proteolysis are complementary and unambiguous tools to identify flexible sites in proteins .

Nucleic Acids Res . 2002 May 15;30(10):e42.
Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution; Naimuddin M et al.; We developed a method to produce, identify and analyze DNA fragments for the purpose of taxonomic classification . Genome profiling (GP) is a strategy that identifies genomic DNA fragments common to closely related species without prior knowledge of the DNA sequence . Random PCR, one of the key technologies of GP, is used to produce fragments and may be used even when there are mutations at the priming site . These fragments can then be distinguished based on the information of mobility and melting pattern when subjected to temperature gradient gel electrophoresis (TGGE) . Corresponding fragments among several species, designated as commonly conserved genetic fragments (CCGFs), likely have the same genetic origin or correspond to the same gene . The criteria for identification of CCGFs has been defined and presented here . To assess this prediction, some of the fragments were sequenced and were confirmed to be CCGFs . We show that genome profiles bearing evolutionarily conserved CCGFs can be used to classify organisms and trace evolutionary pathways, among other profound applications.

Nucleic Acids Res . 2002 May 15;30(10):e41.
A reliable and efficient method for deleting operational sequences in PACs and BACs; Nistala R et al.; P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice . They carry large fragments of genomic DNA (> or =100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene . Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression . Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number . Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression . Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them . We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method . Specifically, we generated and employed a Lox511 'floxed' CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.

Nucleic Acids Res, 2002 May 15, 30(10), 2172 - 82
Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability; Godthelp BC et al.; The eukaryotic Rad51 protein is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination . Five paralogs of Rad51 have been identified in vertebrates, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are also implicated in recombination and genome stability . Here, we identify a mammalian cell mutant in Rad51C . We show that the Chinese hamster cell mutant, CL-V4B, has a defect in Rad51C . Sequencing of the hamster Rad51C cDNA revealed a 132 bp deletion corresponding to an alternatively spliced transcript with lack of exon 5 . CL-V4B was hypersensitive to the interstrand cross-linking agents mitomycin C (MMC) and cisplatinum, the alkylating agent methyl methanesulfonate and the topoisomerase I inhibitor campthotecin and showed impaired Rad51 foci formation in response to DNA damage . The defect in Rad51C also resulted in an increase of spontaneous and MMC-induced chromosomal aberrations as well as a lack of induction of sister chromatid exchanges . However, centrosome formation was not affected . Intriguingly, a reduced level of sister chromatid cohesion was found in CL-V4B cells . These results reveal a role for Rad51C that is unique among the Rad51 paralogs.

Nucleic Acids Res, 2002 May 15, 30(10), 2103 - 13
Identification of essential domains for Escherichia coli tRNA(leu) aminoacylation and amino acid editing using minimalist RNA molecules; Larkin DC et al.; Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) molecules . Each has a distinct anticodon and varied nucleotides in other regions of the tRNA . Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful . Herein, we describe the smallest tRNA(leu) analog that has been aminoacylated to a significant extent to date . A series of tRNA(leu) analogs with various domains and combinations of domains deleted was constructed . The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem-loop and variable arm stem-loop, but neither the D-arm nor T-arm, were deleted . Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA . The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase . The anticodon and variable stem-loop domains were also dispensable for hydrolysis of the charged tRNA(leu) mimics . These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.

J Biol Chem, 2002 Jul 19, 277(29), 26356 - 63 Epub 2002 May 08.
Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo; Min G et al.; Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus . Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells . In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo . Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding . N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region . In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist . Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold . In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice . Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment . In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed . In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR . These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and activation of CAR.

Plant J, 2002 May, 30(3), 373 - 83
Regulation of Arabidopsis thaliana Em genes: role of ABI5; Carles C et al.; In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated . Germination tests on ABA showed that the most affected mutant had a weak abi phenotype . Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5-5 . In addition to reducing the final level of Em transcripts in the dry seed, the abi5-5 mutation causes a delay in the accumulation of AtEm1 during seed development . An additional characteristic of the abi5-5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol . The abi5-5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene . The wild type and mutant recombinant proteins were produced in E . coli and were assayed for DNA-binding activity on their target promoters by electrophoretic mobility shift assay (EMSA) . The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting . Among the ABRE-type sequences selected on both Em promoters, the G-box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.

Insect Mol Biol, 2002 Jun, 11(3), 233 - 9
Sequence analysis and expression of a virus-like particle protein, VLP2, from the parasitic wasp Venturia canescens; Reineke A et al.; Endoparasitoid wasps produce maternal protein secretions, which are transported into the body of insect hosts at oviposition to regulate host physiology for successful development of their offspring . Venturia canescens calyx fluid contains so-called virus-like particles (VLPs) that are essential for immune evasion of the developing parasitoid inside the host . VLPs consist of four major proteins . In this paper, we describe the isolation and molecular cloning of a gene (vlp2) that is a constituent of VLPs and discuss its possible role in VLP structure and function.

Plant J, 2002 Apr, 30(2), 177 - 87
Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor; Kim YK et al.; Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi . However, the molecular basis of this requirement is unknown . We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact . We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases . chip6 expressed in E . coli catalyses glucosyl transfer from UDP-glucose to cholesterol . Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation . The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase.

Plant Mol Biol, 2002 Mar-Apr, 48(5-6), 751 - 64
Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis; Skibbe DS et al.; Cytoplasmic male sterility is a maternally transmitted inability to produce viable pollen . Male sterility occurs in Texas (T) cytoplasm maize as a consequence of the premature degeneration of the tapetal cell layer during microspore development . This sterility can be overcome by the combined action of two nuclear restorer genes, rf1 and rf2a . The rf2a gene encodes a mitochondrial aldehyde dehydrogenase (ALDH) that is capable of oxidizing a variety of aldehydes . Six additional ALDH genes were cloned from maize and Arabidopsis . In vivo complementation assays and in vitro enzyme analyses demonstrated that all six genes encode functional ALDHs . Some of these ALDHs are predicted to accumulate in the mitochondria, others in the cytosol . The intron/exon boundaries of these genes are highly conserved across maize and Arabidopsis and between mitochondrial and cytosolic ALDHs . Although animal, fungal, and plant genomes each encode both mitochondrial and cytosolic ALDHs, it appears that either the gene duplications that generated the mitochondrial and the cytosolic ALDHs occurred independently within each lineage or that homogenizing gene conversion-like events have occurred independently within each lineage . All studied plant genomes contain two confirmed or predicted mitochondrial ALDHs . It appears that these mitochondrial ALDH genes arose via independent duplications after the divergence of monocots and dicots or that independent gene conversion-like events have homogenized the mitochondrial ALDH genes in the monocot and dicot lineages . A computation approach was used to identify amino acid residues likely to be responsible for functional differences between mitochondrial and cytosolic ALDHs.

J Chromatogr A, 2002 Mar 8, 949(1-2), 173 - 84
Global internal standard technology for comparative proteomics; Chakraborty A et al.; The work described in this paper tests the efficacy of a global isotope labeling (global internal standard technology, GIST) strategy for quantification in proteomics . Using GIST, overexpression of beta-galactosidase in Escherichia coli was identified and quantified . The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting tryptic peptides, mixing the differentially labeled control and experimental digests, fractionation of the peptide mixture by reversed-phase chromatography, and isotope ratio analysis by mass spectrometry . N-Acetoxysuccinimide and N-acetoxy-{2H3}succinimide were used to differentially derivatize primary amino groups in peptides from experimental and control samples, respectively . The relative concentration of isotopically labeled peptides was determined by isotope ratio analysis with both matrix-assisted laser desorption ionization mass spectrometry and tandem mass spectrometry (MS-MS) . Peptide masses and sequences obtained by MS-MS were used to identify proteins . MS-MS was found to be uniquely suited for isobaric peptide quantification.

J Chromatogr A, 2002 Mar 8, 949(1-2), 153 - 62
Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates; Wang S et al.; This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates . Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange . Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper . Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry . The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced . Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above . Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.

Free Radic Res, 2002 Jan, 36(1), 73 - 8
Thiol-linked peroxidase activity of human sensitive to apoptosis gene (SAG) protein; Kim SY et al.; SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined . Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT) . However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide . The peroxidase activity was completely abolished by the reaction of SAG with N-ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins . These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide . In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t-butylhydroperoxide (t-BOOH) and fatty acid hydroperoxide-selective peroxidase activity.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 481 - 3
Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase; Yamamoto H et al.; The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis . Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 476 - 80
High-level expression and characterization of fully active recombinant conger eel galectins in Eschericia coli; Ogawa T et al.; An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli . Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield . Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency . Purification of recombinant proteins were done by only two chromatographical steps from E . coli extract . The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 434 - 8
Cloning and expression of an alpha-L-arabinofuranosidase gene (stxIV) from Streptomyces thermoviolaceus OPC-520, and characterization of the enzyme; Tsujibo H et al.; The gene encoding alpha-L-arabinofuranosidase (STX-IV), located upstream of the previously reported stxI gene, was cloned and sequenced . The gene is divergently transcribed from the stxI gene, and the two genes are separated by 661 nucleotides . The stxIV gene consists of a 1,092-bp open reading frame encoding 363 amino acids . The deduced amino acid sequence of the gene showed that STX-IV was an enzyme consisting of only a catalytic domain, and that the enzyme had significant similarity with alpha-L-arabinofuranosidases belonging to family 62 of glycosyl hydrolases . The stxIV gene was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity . Arabinoxylan and oat spelt xylan were good substrates for STX-IV, however, the enzyme showed a low activity with p-nitrophenyl alpha-L-arabinofuranoside . The optimum pH and temperature were 5.0 and 60 degrees C, respectively.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 285 - 92
Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae; Kimura T et al.; The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji . The structural part of xynF3 was found to be 1468 bp . The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids . Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids . The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi . The xynF3 gene was successfully overexpressed in A . oryzae and the XynF3 was purified . The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000 . This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence . The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C . It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate . Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter . The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 277 - 84
Molecular cloning, functional expression, and mutagenesis of cDNA encoding rye (Secale cereale) seed chitinase-c; Ohnuma T et al.; We cloned a complete cDNA encoding rye seed chitinase-c, designated RSC-c, by rapid amplification of cDNA end and PCR procedures . The cDNA of RSC-c consists of 1,018 nucleotides and includes an open reading frame encoding a polypeptide of 266 amino acid residues . A recombinant RSC-c was produced by expression in Escherichia coli Origami(DE3) and purified . rRSC-c had almost the same chitinase activity toward glycolchitin and antifungal activity against Trichoderma sp . as the authentic RSC-c did . RSC-c mutants were subsequently constructed and characterized with respect to their chitinase and antifungal activities . Mutation of Glu67 to Gln completely abolished the chitinase activity and diminished the antifungal activity . Considerable decreases in both activities were observed in the mutations of Trp72 and Ser120 to Ala, and Glu89 to Gln . The roles of these residues in the catalytic event of RSC-c are discussed.

Immunobiology, 2002 Mar, 205(1), 17 - 34
Effects of pentoxifylline on inflammatory cytokine expression and acute pleuropneumonia in swine; Myers MJ et al.; Pentoxifylline, a methylxanthine derivative and nonspecific type 4 phosphodiesterase inhibitor, has been used to improve survival of animals with sepsis and to attenuate lung injury in acute lung inflammation . The purpose of this study was to examine whether pentoxifylline would inhibit the expression of inflammatory cytokines, particularly tumor necrosis factor alpha (TNF), and thereby decrease the pathophysiology of acute porcine pleuropneumonia . E . coli lipopolysaccharide (LPS) and bacterial extracts of A . pleuropneumoniae--induced elevations in TNF mRNA which were fully abrogated by addition of pentoxifylline in both alveolar macrophage and neutrophil cultures . A 30% reduction in the level of LPS-induced interleukin (IL)-1beta mRNA levels also was achieved in macrophages . Pentoxifylline did not affect either IL-1alpha or IL-8 expression in vitro . Pentoxifylline therapy in vivo significantly reduced the number of band neutrophils in swine but did not reduce the pathology associated with pleuropneumonia, including changes in serum zinc, iron, or haptoglobin . Neither did it alter TNF, IL-1, IL-6, or IL-8 expression . Measurement of pentoxifylline and its metabolites in pig sera suggested that efficacious doses of pentoxifylline were probably not achieved in vivo . However, subcutaneous doses of pentoxifylline higher than 25 mg/kg produced transient diarrhea, vomiting, and tremors . These results suggest that pentoxifylline is an effective pharmacological tool for the dissection of cytokine regulation in vitro, but inhibitory concentrations may not be achievable for in vivo pharmacological use in swine.

Mycopathologia, 2002, 153(3), 157 - 62
Collagenase production in an antarctic strain of Arthrobotrys tortor Jarowaja; Tosi S et al.; This paper describes the results of a comparative screening between the nematophagous Antarctic fungus Arthrobotrys tortor and other species of that genus for the production of extracellular collagenases . The nematode species used in this study was Caenorhabditis elegans, feeding on Escherichia coli cultures . Determination of collagenase activity was made using insoluble collagen from bovine Achilles tendon and determining the amount of solubilized hydroxyproline produced . The results show that the total amount of collagenase produced by the Antarctic strain of A . tortor was about threefold higher than that observed for the other species . In the Antarctic strain, collagenase was shown to be a constitutive enzyme.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6731 - 6 Epub 2002 May 07.
Energetics of glycerol conduction through aquaglyceroporin GlpF; Jensen MO et al.; Aquaglyceroporin GlpF selectively conducts water and linear polyalcohols, such as glycerol, across the inner membrane of Escherichia coli . We report steered molecular dynamics simulations of glycerol conduction through GlpF, in which external forces accelerate the transchannel conduction in a manner that preserves the intrinsic conduction mechanism . The simulations reveal channel-glycerol hydrogen bonding interactions and the stereoselectivity of the channel . Employing Jarzynski's identity between free energy and irreversible work, we reconstruct the potential of mean force along the conduction pathway through a time series analysis of molecular dynamics trajectories . This potential locates binding sites and barriers inside the channel; it also reveals a low energy periplasmic vestibule suited for efficient uptake of glycerol from the environment.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6679 - 83 Epub 2002 May 07.
The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H; Mihara H et al.; Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from L-cysteine and L-selenocysteine, respectively, to form L-alanine . These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin . Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism . We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA, suggesting that the sulfur atom released from L-cysteine by the action of IscS is incorporated into mnm(5)s(2)U . In contrast, neither CSD nor CsdB was essential for production of mnm(5)s(2)U and 5-methylaminomethyl-2-selenouridine . The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H . Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.

J Exp Bot, 2002 May, 53(372), 1305 - 19
Regulation and function of ascorbate peroxidase isoenzymes; Shigeoka S et al.; Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS) . Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level . Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle . Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants . APX is also found in eukaryotic algae . The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes . Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons . Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses . Transgenic plants expressing E . coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions . It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.

Biochim Biophys Acta, 2002 Feb 15, 1553(3), 296 - 301
pH dependent inactivation of solubilized F1F0 ATP synthase by dicyclohexylcarbodiimide: pK(a) of detergent unmasked aspartyl-61 in Escherichia coli subunit c; Valiyaveetil F et al.; The pH dependence of the reaction of dicyclohexylcarbodiimide with the essential aspartyl-61 residue in subunit c of Escherichia coli ATP synthase was compared in membranes and in a detergent dispersed preparation of the enzyme . The rate of reaction was estimated by measuring the inactivation of ATPase activity . The reaction with the detergent dispersed form of the enzyme proved to be pH sensitive with the essential aspartyl group titrating with a pK(a)=8 . However, when measured with E . coli membranes, the reaction proved to be pH insensitive . The results suggest that the reacting aspartyl-61 residues are shielded from the bulk aqueous solvent when in the membrane, but then become aqueous-accessible following detergent solubilization.

Biochim Biophys Acta, 2002 Feb 15, 1553(3), 268 - 78
Interaction of purified NDH-1 from Escherichia coli with ubiquinone analogues; David P et al.; The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I . The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation . All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis . In addition, some minor contaminants were observed and identified . The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized . Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors . The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors . Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.

Biochim Biophys Acta, 2002 Feb 15, 1553(3), 188 - 211
The molecular mechanism of ATP synthesis by F1F0-ATP synthase; Senior AE et al.; ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production . Earlier mutagenesis studies had gone some way to describing the mechanism . More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor . Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined . In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.

FEBS Lett, 2002 May 8, 518(1-3), 173 - 6
PspE (phage-shock protein E) of Escherichia coli is a rhodanese; Adams H et al.; The psp (phage-shock protein) operon of Escherichia coli is induced when the bacteria are infected by filamentous phage and under several other stress conditions . The physiological role of the individual Psp proteins is still not known . We demonstrate here that the last gene of the operon, pspE, encodes a thiosulfate:cyanide sulfurtransferase (EC 2.8.1.1; rhodanese) . Kinetic analysis revealed that catalysis occurs via a double displacement mechanism as described for other rhodaneses . The K(m)s for SSO3(2-) and CN- were 4.6 and 27 mM, respectively.

FEBS Lett, 2002 May 8, 518(1-3), 93 - 6
One functional subunit is sufficient for catalytic activity and substrate specificity of Escherichia coli endoribonuclease III artificial heterodimers; Conrad C et al.; To study the intersubunit communication required for the activity of the normally homodimeric enzyme endoribonuclease III of Escherichia coli we have constructed and analysed an artificial heterodimer . This heterodimer is composed of one wild-type and one catalytically inactive subunit . The inactive subunit has one amino acid exchanged (E117K, rnc70 mutant) which abolishes cleavage activity but still allows substrate binding of a rnc70-homodimer . Our results show that one functional active site is sufficient for cleavage activity of the heterodimer.

FEBS Lett, 2002 May 8, 518(1-3), 10 - 6
PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity; McEwan AG et al.; PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M . and Kaplan, S . (2000) Biochemistry 39, 2052-2062; Oh, J.-I . and Kaplan, S . (2000) EMBO J . 19, 4237-4247) . It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence . In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase . Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper . PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site . Following removal of Ni2+ and formation of renatured metal-free rPrrC (apo-PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a lambda(max) of 360 nm . The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre . The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive . The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC . Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role.

Mutat Res, 2002 May 22, 502(1-2), 39 - 46
Instability of repeated DNAs during transformation in Escherichia coli; Hashem VI et al.; Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA . Changes in triplet repeat length during growth following transformation in E . coli have been used as a measure of repeat instability . However, very little is known about the molecular and biological changes that may occur on transformation . Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells . To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E . coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E . coli . Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic . The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation . The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent . Thus, the process of transformation was required to observe the instability . Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation . The magnitude of the instability is dependent on the nature and length of the repeat . Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements . Moreover, different E . coli genetic backgrounds show different levels of instability during transformation.

Mutat Res, 2002 May 22, 502(1-2), 25 - 37
Genetic assays for measuring rates of (CAG).(CTG) repeat instability in Escherichia coli; Hashem VI et al.; Genetic selection assays were developed to measure rates of deletion of one or more (CAG).(CTG) repeats, or an entire repeat tract, in Escherichia coli . In-frame insertions of >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype . When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 10(-2) revertants per cell per generation . The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly . When (CTG)n comprised the leading template strand the Cm(r) mutation rates were 100-1000 lower than for the opposite orientation . As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined . The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand . Replication errors made with the opposite repeat orientation were apparently not recognized . For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain . In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5 x 10(-9) to 1 x 10(-7) per cell per generation . These assays allow careful quantitation of triplet repeat instability in E . coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.

Biochem Pharmacol, 2002 Apr 15, 63(8), 1575 - 8
Verapamil decreases glucuronidase activity in the gut; Lotsch J et al.; The present investigation addressed the role of verapamil for oral pharmacokinetics of morphine-6-beta-glucuronide (M6G) . Male Sprague-Dawley rats received 62.5 mg kg(-1) M6G-dihydrate orally w/wo pre-treatment with 70 mg kg(-1) verapamil . Intravenous M6G (3.9 mg kg(-1) ) and oral morphine (52.7 mg kg(-1) morphine-hydrochloride) were also employed . Oral bioavailability of M6G and the fraction of M6G deglucuronidated to morphine were estimated from areas under the plasma-concentration vs . time curves (AUC) of morphine and its glucuronides . As initial results pointed towards inhibition of glucuronidases by verapamil, its capability to specifically inhibit E . coli and/or rat intestinal beta-glucuronidase was assessed using altered cleavage of the model substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) . Oral bioavailability of M6G was 2.1%; 13% of oral M6G was deglucuronidated to morphine . Co-administration of verapamil did not increase the AUC of M6G . AUCs of morphine and morphine-3-glucuronide were smaller in the verapamil group than in controls . Verapamil co-administration decreased the fraction of M6G deglucuronidated to morphine to 4.6% . In vitro experiments provided evidence that verapamil inhibits beta-glucuronidase from E . coli with an IC(50) of 30 microM, whereas no inhibition of the rat beta-glucuronidase from small intestine was seen . In conclusion, verapamil decreased intestinal deglucuronidation of M6G by inhibiting E . coli beta-glucuronidase . This indicates that verapamil is not suited as P-gp inhibitor in experiments involving glucuronides . An increase in the intestinal absorption of M6G due to P-gp-inhibition was not observed at the verapamil dose studied.

Emerg Infect Dis, 2002 May, 8(5), 508 - 13
Typical and atypical enteropathogenic Escherichia coli; Trabulsi LR et al.; Typical and atypical enteropathogenic Escherichia coli (EPEC) strains differ in several characteristics . Typical EPEC, a leading cause of infantile diarrhea in developing countries, is rare in industrialized countries, where atypical EPEC seems to be a more important cause of diarrhea . For typical EPEC, the only reservoir is humans; for atypical EPEC, both animals and humans can be reservoirs . Typical and atypical EPEC also differ in genetic characteristics, serotypes, and virulence properties . Atypical EPEC is more closely related to Shiga toxin-producing E . coli (STEC), and like STEC these strains appear to be emerging pathogens.

Biochem J, 2002 Aug 15, 366(Pt 1), 245 - 53
Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins; Gianazza E et al.; Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS . For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed . The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M) . A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M) . A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments . Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40 . A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances . Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.

J Am Chem Soc, 2002 May 15, 124(19), 5268 - 9
Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B; Kato Y et al.; A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus . The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit . Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.

Analyst, 2002 Mar, 127(3), 373 - 7
Effect of static magnetic field on growth of Escherichia coli and relative response model of series piezoelectric quartz crystal; Zhang S et al.; The effect of magnetic field on the growth of bacteria was studied with the series piezoelectric quartz crystal (SPQC) sensing technique . The growth situations of Escherichia coli (E . coli) in the absence and presence of different intensities of static magnetic fields were examined and analyzed . The results showed that the growth of E . coli was inhibited due to the presence of magnetic fields . By fitting frequency shift (deltaD) versus time curves according to the frequency shift response equation of SPQC, the relationships between three kinetic growth parameters, i.e., the asymptote A, the maximum specific growth rate mu(m) and lag time lambda, and magnetic field intensity were established . Based on these results, a new response model containing the magnetic field intensity was derived as: delta(f) = 167.7 (7.25 - 7.11B)/{1 + exp{4 x 2.46e(-3.97B)/(7.25 -7.1 IB)} x (4.42 + 16.46B - t) + 2}} The kinetic parameters of bacterial growth obtained from this model are close to those obtained from the logistics popular growth model, in which the concentration of the bacteria was determined by the traditional pour plate count method.

Acta Biochim Pol, 2001, 48(4), 985 - 94
Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers; Kolasa IK et al.; The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro . Previously, this promoter was used as a control one in studies on the effect of DNA bending by An x Tn sequences on transcription initiation and shown to be fully functional in E . coli (Lozinski et al., 1991, Nucleic Acids Res . 19, 2947; Lozinski & Wierzchowski, 1996, Acta Biochim . Polon . 43, 265) . The data now obtained demonstrate that the mechanism of Pa-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters . Measurements of the dissociation rate constant of Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 4, being bound to the RPo in the course of renaturation of the melted DNA region . This number was found constant in the temperature range of 25-37 degrees C, which indicates that under these conditions the complex remaines fully open . This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim . Polon . 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

Acta Biochim Pol, 2001, 48(4), 851 - 65
GTP-binding properties of the membrane-bound form of porcine liver annexin VI; Kirilenko A et al.; Annexin VI (AnxVI) of molecular mass 68-70 kDa belongs to a multigenic family of ubiquitous Ca2+- and phospholipid-binding proteins . In this report, we describe the GTP-binding properties of porcine liver AnxVI, determined with a fluorescent GTP analogue, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) . The optimal binding of TNP-GTP to AnxVI was observed in the presence of Ca2+ and asolectin liposomes, as evidenced by a 5.5-fold increase of TNP-GTP fluorescence and a concomitant blue shift (by 17 nm) of its maximal emission wavelength . Titration of AnxVI with TNP-GTP resulted in the determination of the dissociation constant (Kd) and binding stoichiometry that amounted to 1.3 microM and 1:1 TNP-GTP/AnxVI, mole/mole, respectively . In addition, the intrinsic fluorescence of the membrane-bound form of AnxVI was quenched by TNP-GTP and this was accompanied by fluorescence resonance energy transfer (FRET) from AnxVI Trp residues to TNP-GTP . This indicates that the GTP-binding site within the AnxVI molecule is probably located in the vicinity of a Trp-containing domain of the protein . By controlled proteolysis of human recombinant AnxVI, followed by purification of the proteolytic fragments by affinity chromatography on GTP-agarose, we isolated a 35 kDa fragment corresponding to the N-terminal half of AnxVI containing Trp192 . On the basis of these results, we suggest that AnxVI is a GTP-binding protein and the binding of the nucleotide may have a regulatory impact on the interaction of annexin with membranes, e.g . formation of ion channels by the protein.

J Immunol, 2002 May 15, 168(10), 4889 - 96
Human V gamma 2V delta 2 T cells augment migration-inhibitory factor secretion and counteract the inhibitory effect of glucocorticoids on IL-1 beta and TNF-alpha production; Wang L et al.; In immune cells, proinflammatory cytokine gene expression is regulated by glucocorticoids, whereas migration-inhibitory factor (MIF), a pleiotropic cytokine, has the unique property of counteracting the inhibitory effect of glucocorticoids on TNF-alpha and IL-1beta secretion . A few lines of evidence suggest that gammadelta T cells play an important role in immunoregulation . However, it is unknown whether human gammadelta T cells participate in regulating MIF secretion, and how gammadelta T cells, glucocorticoids, and cytokines converge to give a unified physiological response . In this study, we demonstrate that human Vgamma2Vdelta2 T cells augment MIF secretion . Remarkably, these Vgamma2Vdelta2 T cells, functioning similarly to MIF in part, counteracted inhibition of dexamethasone on production of IL-1beta and TNF-alpha . SCID mice reconstituted with human PBMC that were mock depleted of Vdelta2 T cells and repeatedly infected with lethal dose of Escherichia coli had shorter survival time than those reconstituted with PBMC that were depleted of Vdelta2 T cells . Thus, human Vgamma2Vdelta2 T cells are likely to play broad-spectrum roles in immunoregulation and immunopathology by influencing MIF secretion and the immunomodulatory function of glucocorticoids.

J Biol Chem, 2002 Jul 26, 277(30), 27353 - 9 Epub 2002 May 06.
Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU; Hoff KG et al.; Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli . Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown . We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU . Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif . Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members . A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66 . Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20 . Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

J Biol Chem, 2002 Jul 19, 277(29), 25920 - 8 Epub 2002 May 06.
Mutational analysis of the transcription factor IIIB-DNA target of Ty3 retroelement integration; Yieh L et al.; The Ty3 retrovirus-like element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III . The requirements for transcription factor (TF) IIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined . Ty3 integrates at low but detectable frequencies in the presence of TFIIIB subunits Brf1 and TATA-binding protein . Integration increases in the presence of the third subunit, Bdp1 . TFIIIC is not essential, but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site . In the current study, recombinant wild type TATA-binding protein, wild type and mutant Brf1, and Bdp1 proteins and highly purified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration . The amino-terminal half of Brf1, which contains a TFIIB-like repeat, contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting . Each half of Bdp1 split at amino acid 352 enhanced integration . In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared with the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo . Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration . Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for polymerase III recruitment but defective in promoter opening were competent for Ty3 integration irrespective of the state of DNA supercoiling . These results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription.

J Biol Chem, 2002 Jul 19, 277(29), 26486 - 95 Epub 2002 May 06.
Mutating conserved residues in the ribonuclease H domain of Ty3 reverse transcriptase affects specialized cleavage events; Lener D et al.; The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts . However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1 . In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT . The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae . Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg(2+)- and Mn(2+)-dependent RNase H function . Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg(2+), whereas in the presence of Mn(2+) these mutants displayed a lack of turnover . Despite this, mutations at all positions were lethal for transposition . To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme . Mutants retaining RNase H activity on a heteropolymeric RNA.DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

Am J Epidemiol, 2002 May 15, 155(10), 941 - 8
Population-based trends in pediatric hemolytic uremic syndrome in California, 1994-1999: substantial underreporting and public health implications; Cummings KC et al.; This paper describes the epidemiology of childhood hemolytic uremic syndrome (HUS) in California, for which hospitalization data were used, and the proportion of cases reported to public health authorities . HUS discharge data for children < or =17 years of age were extracted from the population-based California Patient Discharge Data Set for 1994-1999 and were linked electronically with HUS reports to public health authorities . Incidence rates per 100,000 children were calculated . The authors identified 585 HUS hospitalizations; 369 were incident cases . The average HUS incidence rate was 0.67 (95% confidence interval: 0.61, 0.74); rates rose modestly from 1994 (0.59, 95% confidence interval: 0.44, 0.78) to 1997 (0.80, 95% confidence interval: 0.63, 10.0) and decreased modestly thereafter (0.59, 95% confidence interval: 0.45, 0.77) . Rates were highest for northern California children < or =5 years of age (1.85, 95% confidence interval: 1.55, 2.19) . The hospital case-fatality rate was 2.7% (95% confidence interval: 1.1, 4.4); the median charge was $39,500 per child . Only 43.9% of HUS cases in the California Patient Discharge Data Set were reported to public health authorities . Despite heightened efforts to control Shiga toxin-producing Escherichia coli exposures (the predominant cause of childhood HUS in the United States), HUS incidence rates remained relatively stable in California . Reporting HUS cases to public health authorities is important for disease control.

Mol Microbiol, 2002 May, 44(3), 783 - 92
Primosome assembly requirement for replication restart in the Escherichia coli holDG10 replication mutant; Flores MJ et al.; In this report, we study the role of pre-primosome proteins in a strain in which the frequency of replication arrest is increased because of a mutation in a replication protein . The holDG10 mutant was used, in which replication restart involves replication fork reversal . As expected, PriA primosome assembly function is essential for growth of the holDG10 mutant . The priA300 mutation, which inactivates only the helicase function of PriA in vitro, and priB inactivation strongly impair viability . In contrast, priC inactivation has no effect . Therefore, PriB is more important than PriC for PriA-dependent replication fork restart in vivo . The gain of function mutation dnaC809 restores the viability of holDG10 priA and holDG10 priB mutants only to some extent . The dnaC809 820 double mutation restores full viability to the holDG10 mutant lacking either PriA or PriB . Similarly to the holDG10 single mutant, the holDG10 priA dnaC809 820 strain is depend-ent on RecBC for viability, indicating that facilitating primosome assembly using the dnaC809 820 mutation does not allow bypass of replication fork reversal.

Mol Microbiol, 2002 May, 44(3), 709 - 19
A NarX-Tar chimera mediates repellent chemotaxis to nitrate and nitrite; Ward SM et al.; Membrane receptors communicate between the external world and the cell interior . In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator . Here, we show that a chimeric protein that joins the ligand-binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite . Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself . We conclude that the NarX-Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts . This observation implies that ligand-dependent activation of a sensor kinase and repellent-initiated activation of receptor-coupled CheA kinase involve a similar transmembrane signal.

Biochemistry, 2002 May 14, 41(19), 6100 - 6
Translesion synthesis by human DNA polymerase kappa on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene); Suzuki N et al.; Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts . These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation . Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene) adducts was explored . Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C . Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed . Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions . (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%) . Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers . Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions . The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs . Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE . Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.

Biochemistry, 2002 May 14, 41(19), 6032 - 44
Stopped-flow studies of the kinetics of single-stranded DNA binding and wrapping around the Escherichia coli SSB tetramer; Kozlov AG et al.; We have examined the kinetic mechanism for binding of the homotetrameric Escherichia coliSSB protein to single-stranded oligodeoxynucleotides {(dT)(70) and (dT)(35)} under conditions that favor the formation of a fully wrapped ssDNA complex in which all four subunits interact with DNA . Under these conditions, a so-called (SSB)(65) complex is formed in which either one molecule of (dT)(70) or two molecules of (dT)(35) bind per tetramer . Stopped-flow studies monitoring quenching of the intrinsic SSB Trp fluorescence were used to examine the initial binding step . To examine the kinetics of ssDNA wrapping, we used a single-stranded oligodeoxythymidylate, (dT)(66), that was labeled on its 3'-end with a fluorescent donor (Cy3) and on its 5'-end with a fluorescent acceptor (Cy5) . Formation of the fully wrapped structure was accompanied by extensive fluorescence resonance energy transfer (FRET) from Cy3 to Cy5 since the two ends of (dT)(66) are in close proximity in the fully wrapped complex . Our results indicate that initial ssDNA binding to the tetramer is very rapid, with a bimolecular rate constant, k(1,app), of nearly 10(9) M(-1) s(-1) in the limit of low salt concentration (<0.2 M NaCl, pH 8.1, 25.0 degrees C), whereas the rate of dissociation is very low at all salt concentrations that were examined (20 mM to 2 M NaCl or NaBr) . However, the rate of initial binding and the rate of formation of the fully wrapped complex are identical, indicating that the rate of wrapping of the ssDNA around the SSB tetramer is very rapid, with a lower limit rate of 700 s(-1) . The implications of this rapid binding and wrapping reaction are discussed.

J Gastrointest Surg, 2002 Mar-Apr, 6(2), 159 - 66; discussion 166
Specific targeting of tumor vasculature by diphtheria toxin-vascular endothelial growth factor fusion protein reduces angiogenesis and growth of pancreatic cancer; Hotz HG et al.; Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), a mediator of neoangiogenesis . The aim of this study was to specifically target and damage the vasculature of pancreatic cancer (PaCa) by fusing VEGF to diphtheria toxin (DT), which inhibits protein synthesis of target cells . DT-VEGF fusion protein was produced in vector pGEX-KG and expressed in E . coli SG12036 . Human PaCa cell lines (HPAF-2 and AsPC-1) and human endothelial cells (HUVEC) were exposed to DT-VEGF (10 ng/ml - 10,000 ng/ml) . Proliferation was assessed after 3 days . One mm(3) fragments of subcutaneous PaCa donor tumors were implanted into the pancreas of nude mice that received either DT-VEGF (200 microg/kg, every other day) or phosphate-buffered saline intraperitoneally for 14 weeks . Tumor volume, metastatic spread, and animal weight were determined at autopsy . Microvessel density was analyzed in CD31-stained tumor sections . Proliferation of PaCa cells was inhibited at high concentrations of DT-VEGF (>1000 ng/ml) . DT-VEGF decreased the growth of HUVEC at 10 ng/ml . In vivo, DT-VEGF reduced tumor volume (HPAF-2, 76%; AsPC-1, 53%), microvessel density (HPAF-2, 54%; AsPC-1, 62%), and tumor spread (HPAF-2, 89%; AsPC-1, 50%) . Survival was increased (HPAF-2, 7/8 vs . 4/8 animals; AsPC-1, 6/8 vs . 1/8 animals) . Weight was not influenced by DT-VEGF . The DT-VEGF effect is due to its toxic action on the tumor vasculature rather than to direct inhibition of PaCa cell growth . DT-VEGF therapy was not associated with systemic side effects.

Bioorg Med Chem Lett, 2002 May 20, 12(10), 1391 - 3
A unique unnatural base pair between a C analogue, pseudoisocytosine, and an A analogue, 6-methoxypurine, in replication; Hirao I et al.; Pseudoisocytidine, a C-nucleoside analogue of cytosine, has two possible isomers of the H1- and H3-forms . Enzymatic incorporation experiments confirmed the existence of the two isomers in solution, and the 2'-deoxyribonucleoside triphosphate of pseudoisocytosine (PIC) was incorporated into DNA opposite both guanine and 6-methoxypurine (M) by the Klenow fragment of Escherichia coli DNA polymerase I . In addition to the PIC*M pairing in replication, M also functioned as an A analogue and T was efficiently incorporated opposite M . Thus, the PIC*M pair is regarded as a base pair between a C analogue and an A analogue, and can mediate the interconversion between the G*C and A*T base pairs . The combination of PIC and M could be used as a G*C<-->A*T transition mutagen.

J Control Release, 2002 May 17, 81(1-2), 201 - 17
Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations; Reschel T et al.; Biophysical properties of polycation/DNA complexes designed for gene delivery were studied with respect to the conditions of their preparation, chemical structure and molecular weight of the polycations involved . The polycations used included a variety of cationic polymers and copolymers containing primary and tertiary amino or quaternary ammonium groups . It was found that the molecular weight and the size of these polyelectrolyte complexes (PECs) increase with increasing temperature and pH of the buffer . By decreasing the molecular weight of polycations used for PEC formation, the complexes become unstable towards coagulation in aqueous solution at lower pH . The self-assembly of DNA with low-molecular-weight polycations in water provides PECs with the lowest molecular weight, smallest size and the lowest density but their stability in NaCl solutions is very poor . Despite the complexity of the multistep transfection process, a direct correlation between the transfection efficiency in vitro and the stability of the complexes in NaCl solutions and coagulation in 0.15 M NaCl solution was found . DNA complexes with polycations containing primary amino groups showed the best stability in saline solutions and also the best transfection activity . PECs formed by polycations with quaternary ammonium groups were the least resistant to destruction by the added salt and provided the lowest activity in transfection assays . The highest transfection activity was found for DNA complexes formed with a statistical copolymer containing primary and tertiary amines.

J Virol, 2002 Jun, 76(11), 5829 - 34
Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice; Mori Y et al.; Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences . The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.

J Virol, 2002 Jun, 76(11), 5605 - 11
Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses; Pijlman GP et al.; The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied . Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production . These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid) . Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events . Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells . This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences . The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.

J Virol, 2002 Jun, 76(11), 5557 - 64
Overcoming the phage replication threshold: a mathematical model with implications for phage therapy; Kasman LM et al.; Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur . Such a replication threshold density would have broad implications for the therapeutic use of phage . In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution . This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml . In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time . Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations . In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.

J Virol, 2002 Jun, 76(11), 5503 - 14
Analysis of an Autographa californica multicapsid nucleopolyhedrovirus lef-6-null virus: LEF-6 is not essential for viral replication but appears to accelerate late gene transcription; Lin G et al.; The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays . In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle . Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor . To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli . Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAc(lef6KO)) was able to propagate in cell culture, although virus yields were substantially reduced . Thus, the lef-6 gene is not essential for viral replication in Sf9 cells . Two "repair" AcMNPV BACmids (vAc(lef6KO-REP-P) and vAc(lef6KO-REP-ie1P)) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc(lef6KO) BACmid . Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus . The lef-6-null BACmid (vAc(lef6KO)) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection . The lef-6 deletion did not appear to affect viral DNA replication . Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid . This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus . Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription . Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

RNA, 2002 Apr, 8(4), 534 - 41
Lead(II) as a probe for investigating RNA structure in vivo; Lindell M et al.; In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe . Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent . Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50-100 mM lead(II) acetate for 3-7 min, resulting in partial cleavage of RNA in vivo . Cleavage positions were mapped by reverse transcription analysis of total extracted RNA . Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA . All three RNAs had previously been analyzed in vitro, and secondary structure models were available . The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work . These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.

Allergy, 2002 Jan, 57(1), 40 - 3
Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens; Ledesma A et al.; In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli . We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8) . Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen . Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens . Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other . Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

Adv Biochem Eng Biotechnol, 2002, 74, 171 - 81
Fed-batch cultures of Escherichia coli cells with oxygen-dependent nar promoter systems; Chang HN et al.; The recombinant proteins produced from Escherichia coli as a host cell need to be made at as low a cost as possible because of the end of the monopoly following expiry of the patent on early pharmaceutical proteins, and thus expanding applications to non-pharmaceutical large-scale products . We review in this article how the various promoters used in recombinant E . coli could affect its protein products, especially with emphasis on relatively new oxygen-dependent nar promoters for beta-galactosidase production . Several studies carried out in the authors' laboratory show that the nar promoter does not require any chemicals except 1% nitrate and oxygen for protein production . And according to recent work with the modified strains it is possible to produce the enzyme (beta-galactosidase) even without the nitrate ions at 45% of its total protein content when its cell density reached OD = 176.

Crit Care Med, 2002 Mar, 30(3), 508 - 17
L-arginine supplementation in hyperdynamic endotoxemic pigs: effect on nitric oxide synthesis by the different organs; Bruins MJ et al.; OBJECTIVES: Under septic conditions, the protective role of nitric oxide in the organs may become compromised at a time of increased demand as a result of decreased availability of L-arginine . It remains unknown whether supplementation with L-arginine, as a substrate, can modulate organ nitric oxide production . DESIGN: Controlled study with laboratory animals . SETTING: University research laboratory . SUBJECTS: Female crossbred pigs . INTERVENTION: Pigs were challenged with Escherichia coli endotoxin (intravenously) and received intravenous fluid resuscitation for 24 hrs to reproduce a model of long-lasting hyperdynamic endotoxemia . Pigs were infused with either L-arginine or L-alanine intravenously during endotoxin and via the intragastric route after cessation of endotoxin infusion . The effects of L-arginine supplementation on nitric oxide synthesis and the relationships with arginine metabolism were determined with a stable isotope infusion protocol . Also, organ nitrite plus nitrate fluxes were measured . Implantation of multiple catheters enabled in vivo measurements across the hindquarter muscle, the portal-drained viscera, the liver, and the kidneys . MEASUREMENTS AND RESULTS: The isotope conversion method showed that L-arginine intervention significantly increased nitric oxide production by the portal-drained viscera, liver, and kidneys, resulting in elevated whole-body nitric oxide synthesis under endotoxemic and postendotoxemic conditions . Organ nitrite plus nitrate fluxes only tended to increase because of high variance among data . CONCLUSIONS: In this endotoxemia model, supplemental use of L-arginine favored nitric oxide synthesis in various organs.

J Med Microbiol, 2002 May, 51(5), 385 - 91
Inhibition of cell proliferation and induction of apoptosis by Helicobacter pylori through increased phosphorylated p53, p21 and Bax expression in endothelial cells; Kurosawa A et al.; Microcirculation plays a crucial role in mucosal physiological function as well as repair of gastric mucosal damage . Endothelial cell damage is known to disturb microcirculation and suppress angiogenesis . Therefore, the direct effect of Helicobacter pylori on endothelial cells in vitro was investigated with H . pylori water extract . The effect of H . pylori water extract on cell proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) was evaluated . The ratio of BrdU-positive HUVECs in both cagA/vacA-positive and -negative H . pylori water extract-treated groups was significantly lower at 24 h than that in the control group, but Escherichia coli water extract did not affect the proliferation of these endothelial cells . Apoptosis was induced by H . pylori water extracts after incubation for 24 h in a cagA/vacA-independent manner . In the mitochondrial permeability transition assay, tetramethylrhodamine methyl ester was accumulated in mitochondria of HUVECs . Western blot analysis showed no difference in the level of total p53 protein in H . pylori water extract-treated and non-treated cells, but the level of phosphorylated p53 protein was increased in the treated cells at 15 and 60 min after addition of the extract . Reverse transcription (RT)-PCR products for p21 and Bax were elevated in the H . pylori water extract-treated cells . p21 levels began to increase 0.5-1 h after addition of the extract, whereas Bax increased in the period 0.5-2 h . H . pylori induced a disturbance of cell proliferation and apoptosis in the vascular endothelial cells which may contribute to gastric mucosal injury and to delayed healing of gastric lesions.

J Neurotrauma, 2002 Apr, 19(4), 439 - 48
Fiberoptic intraparenchymal brain pressure monitoring with the Camino V420 monitor: reflections on our experience in 163 severely head-injured patients; Poca MA et al.; To assess the safety and accuracy of the Camino intraparenchymal sensor, we prospectively evaluated hemorrhagic complications, zero-drift, infection, and system malfunction in 163 patients monitored after a severe head injury . Mean duration of intracranial pressure (ICP) monitoring was 5 +/- 2.2 days (range: 12 h to 11 days) . Of the 141 patients with a control CT scan, four showed a 1-2-cc collection of blood at the catheter's end . When removed, the sensors underread the true ICP value (negative zero-drift) in 80 of the 126 sensors evaluated (63.5%) . Fourteen sensors showed no zero-drift, and 32 sensors overread the true ICP value (positive zero-drift) (median: -1 mm Hg; interquartile range: -4 to +1 mm Hg) . No significant relationship was found between zero-drift, the surgeon who implanted the sensor, intracranial hypertension, or duration of ICP monitoring . No clinical infections could be attributed to the devices . Sixteen patients (9.8%) required more than one ICP sensor due to malfunctioning of the system . In conclusion, continuous ICP monitoring using the Camino intraparenchymal sensor has a low complication rate . However, this sensor may underread the real ICP values in a high number of patients . The lack of correlation between duration of ICP monitoring and zero-drift suggests that, contrary to the recommendations of other reports, the intraparenchymatous Camino sensor can provide reliable readings after the fifth day of use.

J Pediatr Hematol Oncol, 2002 Mar-Apr, 24(3), 175 - 81
A pharmacoeconomic analysis of pegaspargase versus native Escherichia coli L-asparaginase for the treatment of children with standard-risk, acute lymphoblastic leukemia: the Children's Cancer Group study (CCG-1962); Kurre HA et al.; PURPOSE: The purpose of this pharmacoeconomic analysis was to compare pegaspargase . a newer chemotherapeutic agent used for treating acute lymphoblastic leukemia, with native Escherichia coli L-asparaginase in induction, delayed intensification 1 and delayed intensification 2 . MATERIALS AND METHODS: A subset of patients with newly diagnosed, standard-risk, acute lymphoblastic leukemia enrolled in the Children's Cancer Group (CCG) study CCG-1962 at seven participating institutions gave consent and was enrolled in our pharmacoeconomic analysis study . Societal (transportation, lodging, missed workdays, food, babysitter) and payer (frequency of encounters) cost data were collected from diaries (n = 27) . Additional payer costs, such as drug costs, cost per clinic visit, and cost per inpatient day stay were collected from patients in CCG-1962 and participating institutions . We considered costs of therapy, including higher pegaspargase costs when comparing regimens of pegaspargase versus native E . coli L-asparaginase in induction, delayed intensification 1, and delayed intensification 2 . RESULTS: Our results showed that the costs of the two therapies were similar from the payer perspective, with pegaspargase costing 1.8% more than E . coli L-asparaginase . The difference between groups also was small (<1%) from the societal perspective . Inpatient stay accounted for 88% of pegaspargase payer costs and 91% of the native E . coli L-asparaginase costs . CONCLUSION: We recommend that pegaspargase not be withheld from treatment protocols solely because of its higher pharmacy costs.

Cell Calcium, 2002 Jan, 31(1), 13 - 25
Monoclonal antibodies recognizing epitopes of calretinins: dependence on Ca2+-binding status and differences in antigen accessibility in colon cancer cells; Zimmermann L et al.; Monoclonal antibodies are very helpful tools to investigate the localization and sometimes even the function of specific proteins in cells and tissues . By generating monoclonal antibodies against calretinin-22k (CR-22k), a C-terminally truncated isoform of calretinin (CR) as a result of alternative splicing of the CR mRNA, we envisaged that screening multiple monoclonal antibodies would allow the identification of CR-22k as well as CR . Both proteins share the first 178 amino acids, but have different C-termini . All three antibodies 10C10, 6B3 and 2H4 recognize recombinant CR-22k and the specificity to also recognize CR was demonstrated in brain extracts of different species and human tumour cells, which express CR . All monoclonal antibodies did not crossreact with the closely related protein calbindin D-28k . Antibody binding was depending on the Ca2+-binding status of both forms of calretinin . Generally, the Ca2+-bound form was better recognized than the Ca2+-free form . Carboxy- and amino-terminally truncated CR proteins were expressed in E . coli in order to characterize the epitopes recognized by the three antibodies . Additionally, tryptic and cyanogen bromide fragments were produced to further narrow down the sequences recognized by the three antibodies . 10C10 recognizes an epitope consisting of the linker region between EF-hand domains I and II and the N-terminal part of EF-hand II, while the others (6B3, 2H4) bind to a region including the linker between EF-hand domains III and IV . These antibodies are valuable tools to further investigate the distribution and eventually the specific function of these two proteins in the nervous tissue and under pathological conditions, e.g . in colon tumours and mesotheliomas.

Mol Membr Biol, 2002 Jan-Mar, 19(1), 51 - 8
P-glycoprotein misfolds in Escherichia coli: evidence against alternating-topology models of the transport cycle; Linton KJ et al.; P-glycoprotein (P-gp) is a drug transporter which pumps toxic hydrophobic compounds out of cells, conferring mutidrug resistance . P-gp is predicted to consist of 12 transmembrane alpha-helices and there is a strong body of experimental support for this model . However, a number of studies, including those on P-gp expressed in E . coli, have reported topologies with fewer than 12 transmembrane alpha-helices, leading to the hypothesis that the transmembrane topology of the protein changes during function . It is well established that P-gp undergoes conformational changes during its transport cycle and it has been recently shown that these changes are large in magnitude and could, potentially, reflect a changing transmembrane topology . One therefore, reassessed the transmembrane topology of P-gp expressed in E . coli and compared it directly with the topology of the protein expressed in mammalian cells . It was clear that the transmembrane topology of the protein was different in the different cell types and that the misfolding of P-gp in E . coli was due to the misrecognition of multiple P-gp sequences as topogenic signals . Thus, the alternative transmembrane topologies reported for P-gp in E . coli are artefacts of the heterologous expression system used, and models based on such data in which the transmembrane topology changes during drug transport are unlikely to be correct . Instead, the large conformational changes observed during the transport cycle are more likely due to changes in alpha-helix packing.

Can J Vet Res, 2002 Apr, 66(2), 93 - 8
In vitro expression of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 8 mRNA by bovine macrophages following exposure to Porphyromonas levii; Walter MR et al.; The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by evaluating the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 8 (IL-8) . Bovine macrophages detect the presence of bacteria, such as P . levii, and respond by upregulating transcription of the genes for pro-inflammatory cytokines TNF-alpha and IL-1beta in addition to the neutrophil chemoattractant IL-8 . Monocytes were isolated from blood obtained from Holstein steers . These cells were cultured and differentiated into macrophages over 7 d, followed by exposure to P . levii, Escherichia coli lipopolysaccharide (LPS), or tissue culture medium for 1, 1.5, 2, 4, 6, 8,12, or 24 h . Total RNA was extracted, and reverse transcriptase polymerase chain reaction was conducted to examine the presence of TNF-alpha, IL-1beta, or IL-8 mRNA . Products were visualized on agarose gels to determine the presence or absence of cytokine mRNA amplified DNA . Bovine macrophages, when exposed to P . levii or E . coli LPS, produced mRNA for TNF-alpha, IL-1beta, and IL-8 . Expression of all 3 cytokines was higher in the P . levii and LPS-exposed macrophages at all time points examined, compared with tissue culture medium-treated cells . Expression of these cytokines by macrophages is likely directly involved in orchestration of the immune response, and particularly in neutrophil recruitment to affected tissues in acute interdigital phlegmon.

Indian J Biochem Biophys, 2001 Dec, 38(6), 353 - 60
UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies; Nayar S et al.; UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor . The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0 . There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation . Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here . These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition . The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure . Thermodynamical parameters associated with some transitions have been quantitated . The results have been discussed with the X-ray crystallographic structure of the enzyme.

Mol Biotechnol, 2002 May, 21(1), 39 - 41
Flexprep: scale-flexible rapid plasmid preparation for analysis of recombinant clones; Aranishi F; A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones, this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation . It can process up to 20 mL of E . coli cells carrying 3-10 kbp plasmid vectors in < 10 min . Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.

Microbiology, 2002 May, 148(Pt 5), 1571 - 9
Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis; Hobson RJ et al.; The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes . To identify such genes, a promoter-probe library was constructed in which fragments of M . tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector . Mycobacterium bovis Bacille Calmette-Guerin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions . From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite . Subsequent sequence analyses identified 26 of these clones as containing potential promoters . After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in M . tuberculosis during macrophage infection, by real-time RT-PCR . Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.

Microbiology, 2002 May, 148(Pt 5), 1553 - 9
The regulation of Enzyme IIA(Glc) expression controls adenylate cyclase activity in Escherichia coli; Krin E et al.; During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions . In this study, the possible effect of H-NS on the cAMP level was investigated . In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity . This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter . This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.

Microbiology, 2002 May, 148(Pt 5), 1421 - 5
cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor; Bralley P et al.; In Escherichia coli the poly(A) tails of messenger and rRNAs are a major determinant of RNA stability . These tails are formed primarily by poly(A) polymerase I (PAP I) in wild-type strains or by polynucleotide phosphorylase (PNPase) in PAP I-deficient strains . In Streptomyces coelicolor it has been shown that mycelial RNAs display biochemical characteristics consistent with the presence of poly(A) tails . To confirm the occurrence of polyadenylation, rRNA and mRNA transcripts from S . coelicolor were isolated by oligo(dT)-dependent RT-PCR followed by cDNA cloning . One of the clones obtained was polyadenylated at a site corresponding to the mature 3' terminus of 16S rRNA, while two 23S rRNA cDNA clones were polyadenylated at precursor processing sites . Other clones identified polyadenylation sites internal to the coding regions of both 16S and 23S rRNAs, and redD and actII-orf4 mRNAs . While most rRNA cDNA clones displayed adenosine homopolymer tails, the poly(A) tails of three rRNAs and all the redD and actII-orf4 clones consisted of a variety of heteropolymers . These results suggest that the enzyme primarily responsible for polyadenylation in S . coelicolor is PNPase rather than a PAP I homologue.

Microbiology, 2002 May, 148(Pt 5), 1335 - 47
Repression of chsB expression reveals the functional importance of class IV chitin synthase gene chsD in hyphal growth and conidiation of Aspergillus nidulans; Ichinomiya M et al.; The functions of two previously identified chitin synthase genes in Aspergillus nidulans, chsB and chsD, were analysed . First, a conditional chsB mutant was constructed in which the expression of chsB is under the control of a repressible promoter, the alcA promoter, of A . nidulans . Under repressing conditions, the mutant grew slowly and produced highly branched hyphae, supporting the idea that chsB is involved in normal hyphal growth . The involvement of chsB in conidiation was also demonstrated . Next, double mutants of chsB and chsD were constructed, in which chsB was placed under the control of the alcA promoter and chsD was replaced with the argB gene of A . nidulans . These double mutants grew more slowly than the chsB single mutant under high-osmolarity conditions . The hyphae of the double mutant appeared to be more disorganized than those of the chsB single mutant . It was also found that ChsD was functionally implicated in conidiation when the expression of chsB was limited . These results indicate the importance of the ChsD function in the absence of chsB expression . The roles of ChsB and ChsD in hyphal growth and in conidiation were supported by the analysis of the spatial expression patterns of chsB and chsD, using lacZ of Escherichia coli as a reporter gene.

Annu Rev Biophys Biomol Struct, 2002, 31, 207 - 33 Epub 2001 Oct 25.
The alpha-helix and the organization and gating of channels; Spencer RH et al.; The structures of an increasing number of channels and other alpha-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family . In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins . In the case of channels, alpha-helices create the sealed barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes . The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle . The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity . Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.

Neurosci Lett, 2002 May 17, 324(2), 145 - 8
Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line; Omori N et al.; In microglia, it is difficult to introduce exogenous genes of interest even by recombinant adenovirus vectors (Ad) which can infect with high efficiency only to the cells expressing coxackievirus and adenovirus receptors (CAR) . We found a lack of CAR expression in primary cultured murine microglia (PCMG) and its immortalized cell line MG5 by reverse transcription-polymerase chain reaction . In order to improve the efficiency of gene transfer, we generated a novel Ad (Ad-RGD) by an incorporation of the Arg-Gly-Asp motif (RGD) containing peptide in the HI loop of the viral fiber knob domain, which enables the virus to contact target cells through alpha V integrins which are known to be ubiquitously expressed on the surface of mammalian cells . Ad-RGD showed a remarkable improvement (13-18-fold) in the delivery of Escherichia coli LacZ gene in MG5 cells and a moderate increase in PCMG cells under the treatment with granulocyte-macrophage colony stimulating factor . These results suggest that Ad-RGD may be a potent tool for the delivery of genes to microglia activated by optimum stimulation, and thus analyzing the function of microglia with utilization of MG5 and PCMG cells.

Biochem J, 2002 May 15, 364(Pt 1), 227 - 34
Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme; Lee MH et al.; We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE) . Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition . Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature . The activities of these mutants were examined by measuring their binding affinities (K(app)(i)) and association rates (k(on)) against TACE . Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants . On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE . In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays . Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha . Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition . Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.

Biochem J, 2002 May 15, 364(Pt 1), 129 - 36
Isoaspartyl dipeptidase activity of plant-type asparaginases; Hejazi M et al.; Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp . PCC (Pasteur culture collection) 6803 and Anabaena sp . PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E . coli with either an N-terminal or a C-terminal His tag, and purified . Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG . The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides . As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation . The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.

Biopolymers, 2001-2002, 61(3), 201 - 13
Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes; Heyduk T et al.; Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions . This feature makes FRET a powerful tool to study complicated biological assemblies . In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase . The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase . FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase . FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase . Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated .

Biopolymers, 2001-2002, 61(3), 145 - 58
The stretched DNA geometry of recombination and repair nucleoprotein filaments; Singleton SF et al.; The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks . In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes . To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods . Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions . Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands . All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA .

J Ind Microbiol Biotechnol, 2002 Apr, 28(4), 239 - 43
Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S . clavuligerus NRRL3585; Loke P et al.; Malate synthases (MS) from Streptomyces coelicolor A3(2) and S . clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli . The fusion GST-MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS . With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study . Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS . The average purified specific activities of S . coelicolor and S . clavuligerus MS were 26199 and 11821 nmol/mg min, respectively . Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar Km value for acetyl-CoA, but S . coelicolor MS had a Km value for glyoxylate that is approximately sixfold higher than S . clavuligerus MS.

Heredity, 2002 May, 88(5), 366 - 70
Highly repeated DNA sequences in three species of the genus Pteropus (Megachiroptera, Mammalia); Barragan MJ et al.; Bat genomes are characterised by an A-T richness and by a small C-value compared with other mammalian groups . It has been suggested that the small C-value is mainly due to the lack of repetitive DNA sequences . However, little information about repetitive DNA sequences in this mammalian group is available at the molecular level . Here we describe a PstI family of repetitive DNA sequences present in three species of the genus Pteropus . These repetitive sequences are 54.97% G-C rich, organised in tandem and with a unit length of 744 bp . Methylation analysis indicates that some of the CCGG target sites present in these repetitive DNA sequences have methylated cytosines and that there are small differences in the methylation pattern between species . Several features of this family of repetitive sequences suggest that they evolved by concerted evolution.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 44 - 7
{Characterization of intertype specific epitopes on adenoviruses hexon}; Zhang T et al.; BACKGROUND: To characterize the intertype epitopes on human adenovirus (HAdV) hexon . METHODS: Based on computerized analysis on adenoviruses sequence of genomic alignment, antigenicity prediction and 3-D structure characteristics of hexon subunit, several peptides of hexon of adenoviruses were chosen to be synthesized or recombinant proteins of the hexon were expressed in E . coli by use of PGEX-5X . To identify the existence of intertype epitopes, the antisera raised with synthetic peptides or purified recombinant proteins were analyzed with Western blot and immunofluorescent assay . RESULTS: The results of Western blot indicated that both peptide and recombinant antibodies showed specific reactivities with hexons of HADv-3, 4, 7 individually . Meanwhile, typical stain of immunofluorescence was found on HeLa cells infected with these HAdV by incubation with peptide as well as recombinant antibodies . Also, antibodies raised against peptide recognized the recombinant hexon protein in which a corresponding region of peptides was covered . CONCLUSIONS: Most of the predicted intertype epitopes of HAdV hexon wer e exclusively found in synthetic peptides and recombinant proteins . These intertype epitopes showed to be continuous and sequential which could be employed for development of antibodies of diagnostic use.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 40 - 3
{Detection of the expression of alpha3-integrin on hantavirus permissive cells}; Dong J et al.; BACKGROUND: To express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin . METHODS: The human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E . coli . The gene expression was confirmed by Western blot assay . Rabbit was inoculated with purified antigen to stimulate the antibody generation . The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis . The separated cell clones were confirmed by immunofluorescence and Western blot assay . RESULTS: The alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity . High titer antibody was generated . However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells . CONCLUSIONS: The results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 20 - 2
{Expression of thermal stable, soluble hepatitis E virus recombinant antigen}; Zhang M et al.; BACKGROUND: To obtain thermal stable, soluble, biologically active hepatitis E virus recombinant antigen using thioredoxin fusion expression system . METHODS: HEV ORF2 gene fragment (6964-7126 nt) was inserted into thioredoxin fusion expression vector pThioHisA . The recombinant plasmid was transformed into E . coli BL21 strain . After induction with IPTG, cells were lysed and the supernatant was subjected to 80 degree treatment for 10 minutes . After centrifugation, the supernatant was tested by ELISA . RESULTS: SDS-PAGE analysis showed the thioredoxin . HEV fusion protein was highly expressed and was thermally stable, soluble . HEV specific ELISA confirmed this fusion protein possessing HEV specific antigenicity . CONCLUSIONS: Using thioredoxin fusion expression system, a soluble, thermal stable, biologically active HEV recombinant antigen was successfully expressed.

Clin Diagn Lab Immunol, 2002 May, 9(3), 731 - 5
Activated T lymphocytes disappear from circulation during endotoxemia in humans; Krabbe KS et al.; Seventeen volunteers received an intravenous bolus of endotoxin (2 ng/kg of body weight) . Endotoxin-induced lymphopenia was constituted mainly by cells with an immature phenotype (CD45RA(+) CD45RO(-)) that were less likely to undergo apoptosis (CD28(+)), whereas cells with the highest rates of disappearance were characterized by an activated phenotype (CD45RA(-) CD45RO(+)) as well as a phenotype linked to apoptosis (CD95(+) CD28(-)) . In conclusion, endotoxin-induced lymphopenia reflects the disappearance from the circulation of activated lymphocytes prone to undergo apoptosis.

Clin Diagn Lab Immunol, 2002 May, 9(3), 698 - 703
Importance of M-protein C terminus as substrate antigen for serodetection of equine arteritis virus infection; Jeronimo C et al.; Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group . In a previous paper (A . Kheyar, S . Martin, G . St.-Laurent, P . J . Timoney, W . H . McCollum, and D . Archambault, Clin . Diagn . Lab . Immunol . 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV . In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombinant glutathione S-transferase-M fusion protein . Various deletion mutant clones, which covered the entire sequence of the M protein, were then generated by inverse PCR and expressed in Escherichia coli to examine, by a Western blot assay, the antigenic reactivity of the clone-derived truncated M proteins with sera from horses either experimentally or naturally infected with EAV . Deletion of the hydrophobic N-terminal 87 aa did not abolish immune reactivity of the protein with serum antibodies to EAV, thereby demonstrating the antigenicity of the C-terminal region (aa 88 to 162) of the M protein . Further truncations of the M-protein C-terminal domain defined particular linear epitope-containing amino acid sequence regions . However, only the M-protein C-terminal region was readily recognized by all EAV-specific horse antisera tested in this study . Based on these findings, only the M-protein C-terminal polypeptide composed of aa 88 to 162 is necessary to identify horse serum antibodies specific to the EAV M protein . Thus, this polypeptide might be useful for serodetection of EAV-infected animals.

Clin Diagn Lab Immunol, 2002 May, 9(3), 687 - 92
Polyclonal antibodies to glutathione S-transferase--verotoxin subunit a fusion proteins neutralize verotoxins; Leung PH et al.; The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins . The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields . Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively . The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains . The antibodies had significantly high neutralizing activities against their homologous toxins . The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% +/- 7.9% and 3.6% +/- 2.3%, respectively, and those of VT2 were 1.7% +/- 2.3% and 82.5% +/- 13.9%, respectively . VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant . To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins . The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.

FEMS Immunol Med Microbiol, 2002 Mar 25, 33(1), 23 - 6
Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells; Miyazaki J et al.; Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis . In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E . coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line . We found all isolates adherent to T-24 cells within 15 min of infection . In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive . About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates . The amplitude of invasiveness was also independent of the adhesins . In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.

Biochim Biophys Acta, 2002 Mar 15, 1570(2), 81 - 8
Reduction of streptavidin RYDS-mediated renal adhesion by site-directed mutagenesis; Murray S et al.; Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention . This retention is mediated by an integrin-binding motif-RYDS-that shares homology to the more common RGDS . We generated a c-Strep molecule constituting amino acids 13-139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES . RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E . coli and purified on a 2-imminobiotin matrix . Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers . The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay . RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin . Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.

Mol Microbiol, 2002 Jan, 43(2), 521 - 36
Analysis of the kefA2 mutation suggests that KefA is a cation-specific channel involved in osmotic adaptation in Escherichia coli; McLaggan D et al.; Mechanosensitive channels play an essential role in the regulation of turgor pressure in bacteria . In Escherichia coli, there are multiple mechanosensitive channels that have been characterized genetically: MscL, YggB and KefA . In this report, we describe the cloning of the kefA gene, the organization of the KefA protein and the phenotype of a missense mutation, kefA, which affects the KefA mechanosensitive channel . The altered function of the channel is manifest through increased sensitivity to K+ during growth at low osmolarity and complete inhibition of growth in media containing high K+ concentrations (0.6 M) in the presence of betaine or proline . Growth in high Na+ medium (0.6 M NaCl plus 20 mM K+) is normal . Analysis of the cytoplasmic pools shows that the mutant cannot regulate the K+ content of the cytoplasm when grown in high K+ medium . However, regulation of pools of amino acids is essentially normal and the mutant can accumulate high pools of proline during growth inhibition . The mutant shows increased sensitivity to acid hypo-osmotic shock (transition from neutral to acid pH combined with a reduction in osmolarity) . The data are consistent with abnormal regulation of KefA in the presence of high K+ concentrations and either betaine or proline.

Mol Microbiol, 2002 Jan, 43(2), 487 - 95
Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli; Guzman EC et al.; Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min . This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure . If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes . This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme . The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain . We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.

Mol Microbiol, 2002 Jan, 43(2), 355 - 70
Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli; Martin RG et al.; Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator-DNA-RNA polymerase (RNP) ternary complex . Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator-DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently . We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA . In addition, we find that the ternary MarA-DNA-RNP and SoxS-DNA-RNP complexes are more stable than the corresponding Rob-DNA-RNP complex, although the binary Rob-DNA complex is often more stable than the corresponding MarA- or SoxS-DNA complexes . These results may help to explain certain puzzling aspects of the MarA/SoxS/Rob system . We suggest that activator-RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.

Mol Microbiol, 2002 Jan, 43(2), 323 - 33
Structure of the Lrp-regulated serA promoter of Escherichia coli K-12; Yang L et al.; Expression of the Escherichia coli serA gene is activated in vivo by the product of the lrp gene, leucine-responsive regulatory protein (Lrp), an effect partially reversed by L-leucine . We show here that serA is transcribed from two promoters, P1 45 bp upstream of the translation start site, and P2 92 bp further upstream . Lrp binds to a long AT-rich sequence from -158 to -82 from the start of the coding region, i.e . upstream of P1 and overlapping P2 . It activates transcription from P1 and represses expression from P2 . A second regulator, cAMP/CRP, activates P2, an effect that is largely inhibited by Lrp, such that catabolite repressor protein (Crp) and Lrp are rival activators of serA transcription.

Mol Microbiol, 2002 Jan, 43(2), 269 - 79
Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase; Makinoshima H et al.; Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation . The cell separation was found to result from the difference in buoyant density but not the size difference . The cell density increases upon transition from exponential growth to stationary phase . Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities . Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting . These findings together suggest that the growth phase-coupled transition of E . coli phenotype is discontinuous.

Eur J Biochem, 2002 May, 269(9), 2414 - 20
Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities; Herbette S et al.; This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX) . Both purified recombinant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione . Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate . Moreover, thioredoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective Vmax and Km values . We therefore conclude that these two plant GPX-like proteins are antioxidant enzymes showing PHGPX and TPX activities.

Eur J Biochem, 2002 May, 269(9), 2403 - 13
Systematic search for zinc-binding proteins in Escherichia coli; Katayama A et al.; A systematic search for Escherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E . coli proteins fractionated by two methods of two-dimensional gel electrophoresis . A total of 30-40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing . In addition to five known zinc-binding proteins, nine zinc-binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ) . Together with about 20 known zinc-binding proteins, the total number of zinc-binding proteins in E . coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions) . The specificity and affinity of zinc-binding were analysed for some of the zinc-binding proteins.

Eur J Biochem, 2002 May, 269(9), 2347 - 52
Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis; Justin AM et al.; In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns) . In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol . Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously . Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP . Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis . All of the PtdIns metabolizing activities require free manganese ions . EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.

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