Inflamm Res, 2004 Sep, 53(9), 475 - 83 Diffusible signal to murine alveolar macrophages from lipopolysaccharide- and Escherichia coli-stimulated lung Type II epithelial cells; Farberman MM et al.; OBJECTIVE AND DESIGN: To demonstrate a diffusible intercellular macrophage activation factor secreted by Type II alveolar epithelial cells (AECs) in transwell co-cultures . MATERIALS: T(7), our Type II conditionally immortalized AEC line; MH-S, an alveolar macrophage cell line; Lipopolysaccharide (LPS) or uv-killed Escherichia coli (UVEC) for antigen presentation . METHODS: LPS or UVEC stimulation of T(7) cells in the lower chamber was investigated for ability to activate MH-S cells in the upper chamber, as assayed by nitric oxide production and western blots for inducible nitric oxide synthase-2 . RESULTS: Both transwell and UVEC-conditioned medium experiments showed secretion of an MH-S activation factor by T(7) cells . Many common inflammatory cytokines were ruled out as this immunoactivator . CONCLUSION: Demonstration of a diffusible activation factor produced by Type II AECs supports their potential role as first responders of innate immunity in the lung. Ann Oncol, 2004 Dec, 15(12), 1816 - 24 Phase I trial of intravenous aviscumine (rViscumin) in patients with solid tumors: a study of the European Organization for Research and Treatment of Cancer New Drug Development Group; Schoffski P et al.; BACKGROUND: Aviscumine is an Escherichia coli-derived recombinant type II ribosome-inactivating protein with potent antitumor activity in vitro and in vivo . It is the recombinant counterpart of natural mistletoe lectin-I . The current study was performed to determine the safety profile, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the intravenous (i.v.) administration of aviscumine in cancer patients . Translational research included the evaluation of pharmacokinetics and monitoring of plasma cytokine and anti-aviscumine antibody induction after administration of the drug . PATIENTS AND METHODS: Aviscumine was given twice weekly as a 1 h central i.v . infusion in patients with advanced, refractory progressive, solid malignant tumors who had not been previously exposed to natural mistletoe preparations . They had histologically or cytologically verified disease, were > or =18 years old, had an Eastern Cooperative Oncology Group performance status < or =2 and adequate bone marrow, liver and renal function . DLT was defined as any non-hematological grade 3-4 toxicity (National Cancer Institute Common Toxicity Criteria version 2.0), neutrophil count <500/microl for > or =7 days, febrile neutropenia or thrombocytopenia grade 4 . The MTD was defined as the dose at which >20% of patients experienced DLT during the first treatment cycle . The Continual Reassessment Method was used to determine the number of patients required per dose level . RESULTS: Forty-one fully eligible patients (19 male, 22 female) with a median age of 56 years (range 37-74) were enrolled . Colorectal, ovarian, renal cell and breast cancer were the most common tumor types . Dose levels of aviscumine ranged from 10 to 6400 ng/kg . The median number of cycles was two (range one to eight) . Common clinical toxicities in cycle 1 were fatigue, fever, nausea, vomiting and allergic reactions . Fatigue grade 3 was dose limiting in one of six patients at 4000 ng/kg and reversible grade 3 liver toxicity (elevation in alkaline phosphatase, transaminases and/or gamma-glutamyltransferase) occurred in one of 10 patients at 4800 ng/kg and in two of five patients at 6400 ng/kg . The best response (RECIST criteria) was stable disease in 11 patients, lasting for two to eight cycles . The pharmacokinetic evaluation revealed a short alpha half-life of 13 min and linear kinetics on dose levels > or =1600 ng/kg . Aviscumine stimulated the immune system with a release of cytokines such as interleukin (IL)-1beta, IL-6 and interferon-gamma, and induced immunoglobulin (Ig) G- and/or IgM-anti-aviscumine antibodies of uncertain clinical relevance . CONCLUSIONS: The recommended dose for further clinical trials is 5600 ng/kg twice weekly . Based on the short half-life of the recombinant protein observed in this trial, the exploration of prolonged infusion schedules of aviscumine is warranted. Nucleic Acids Res, 2004, 32(20), 6086 - 95 Print 2004. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages; Sauer B et al.; Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity . DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes . Although these genes from phages P7, phi(w39) and p15B were highly similar to those from P1, those of phage D6 showed significant divergence . Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages . Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox) . Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites . Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination . Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination. Nucleic Acids Res, 2004, 32(20), 6057 - 68 Print 2004. Regulation of 6S RNA biogenesis by switching utilization of both sigma factors and endoribonucleases; Kim KS et al.; In Escherichia coli, 6S RNA functions as a modulator of RNA polymerase sigma70-holoenzyme activity, but its biosynthetic pathway remains uncharacterized . In this study, to further understand the regulatory circuit of 6S RNA biosynthesis for the modulation of Esigma70 activity, we have characterized the biogenesis of 6S RNA . We reveal that there are two different precursors, a long and a short molecule, which are transcribed from the distal P2 and proximal P1 promoter, respectively . Transcription from the P2 promoter is both sigma70- and sigmaS-dependent, whereas, in contrast, P1 transcription is sigma70- but not sigmaS-dependent . Both precursors are processed to generate the 5' end of 6S RNA, and while the long precursor is processed exclusively by RNase E, the short precursor is processed by both RNase G and RNase E . Our data indicate that the switching of the utilization of both sigma factors and endoribonucleases in the biogenesis of 6S RNA would play an essential role in modulating its levels in E.coli. Proc Natl Acad Sci U S A, 2004 Nov 30, 101(48), 16976 - 81 Epub 2004 Nov 30. A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis; Laing WA et al.; Ascorbate is a critical compound in plants and animals . Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants . However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist . We describe a specific L-galactose-1-phosphate (L-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries . The enzyme had a native molecular mass of approximately 65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze L-gal-1-P . The activity had a pH optimum of 7.0, a K(-M(L-gal-1-P) of 20-40 microM and a Ka(Mg2+) of 0.2 mM . The activity was inhibited by Mg2+ at concentrations >2 mM . The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme . The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography-MS . One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase . We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P . The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(L-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme . The data are discussed in terms of the pathway to ascorbate biosynthesis in plants. EMBO J, 2004 Nov 24, 23(23), 4538 - 49 Epub 2004 Nov 24. Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells; Kesty NC et al.; Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries . Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm . We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells . Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells . ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner . Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C . ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts . Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment . We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells . These data demonstrate a role in virulence for ETEC vesicles. Protein Eng Des Sel . 2004 Nov 17; {Epub ahead of print} Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution; Lee PC et al.; Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes . Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to better understand the chain elongation mechanism of this enzyme . Analysis of these mutants revealed three different mechanisms of product chain length specificity . Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases . One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and E . coli C15 farnesyl diphosphate synthase . Finally, one mutant carrying four mutations (V84I, H88R, I177M and M191V) is of interest . Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids. BMC Biochem . 2004 Nov 17;5(1):16. The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis; King SC; BACKGROUND: In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity . A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters . RESULTS: Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration . The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations) . The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for {14C}GABA (4-aminobutyrate) versus {3H}NA (nipecotic acid) . CONCLUSIONS: The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates . A change in the TSR phenotype, called a Delta(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (DeltaGb) that translocation catalysts rely upon to decrease activation energy (Delta G(T)(++) . TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity). Cell Cycle, 2004 Nov, 3(11), 1375 - 7 Epub 2004 Nov 20. Meiotic recombination: an affair of two recombinases; Sehorn MG et al.; In E . coli, homologous recombination is catalyzed by the RecA recombinase . Two RecA-like factors, Rad51 and Dmc1, are found in eukaryotes . Whereas Rad51 is needed for homologous recombination reactions in both mitotic and meiotic cells, the role of Dmc1 is restricted to meiosis . Recent work has shown that, like RecA and Rad51, Dmc1 mediates the homologous DNA pairing strand exchange reaction via a filamentous intermediate assembled on single-stranded DNA . Emerging evidence suggests that the tumor suppressor BRCA2 functions in the assembly of nucleoprotein filaments of Rad51 and Dmc1 . The manner in which Rad51 and Dmc1 functionally cooperate in meiotic recombination remains to be determined. J Bacteriol, 2004 Dec, 186(23), 8156 - 8 Type II isopentenyl diphosphate isomerase from Synechocystis sp . strain PCC 6803; Barkley SJ et al.; Open reading frame sll1556 in the cyanobacterium Synechocystis sp . strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase . The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography . The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1). J Bacteriol, 2004 Dec, 186(23), 8149 - 52 The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 degrees C; Chattopadhyay MK et al.; Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo . In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E . coli cells . Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain . In vitro, like other "chemical chaperones," and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation . These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation. J Bacteriol, 2004 Dec, 186(23), 8105 - 13 Crystal structure of the 65-kilodalton heat shock protein, chaperonin 60.2, of Mycobacterium tuberculosis; Qamra R et al.; Chaperonin 60s are a ubiquitous class of proteins that promote folding and assembly of other cellular polypeptides in an ATP-dependent manner . The oligomeric state of chaperonin 60s has been shown to be crucial to their role as molecular chaperones . Chaperonin 60s are also known to be important stimulators of the immune system . Mycobacterium tuberculosis possesses a duplicate set of chaperonin 60s, both of which have been shown to be potent cytokine stimulators . The M . tuberculosis chaperonin 60s are present in the extracellular milieu at concentrations that are extremely low for the formation of an oligomer . Here we present the crystal structure of one of the chaperonin 60s of M . tuberculosis, also called Hsp65 or chaperonin 60.2, at 3.2-A resolution . We were able to crystallize the protein in its dimeric state . The unusual dimerization of the protein leads to exposure of certain hydrophobic patches on the surface of the protein, and we hypothesize that this might have relevance in binding to immunogenic peptides, as it does in the eukaryotic homologs. J Bacteriol, 2004 Dec, 186(23), 8083 - 8 Structural similarity of YbeD protein from Escherichia coli to allosteric regulatory domains; Kozlov G et al.; Lipoic acid is an essential prosthetic group in several metabolic pathways . The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon . YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region . Here, we report the nuclear magnetic resonance structure of YbeD from E . coli . The structure includes a beta alpha beta beta alpha beta fold with two alpha-helices on one side of a four-strand antiparallel beta-sheet . The beta 2-beta 3 loop shows the highest sequence conservation and is likely functionally important . The beta-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions . YbeD shows striking structural homology to the regulatory domain from d-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system. J Bacteriol, 2004 Dec, 186(23), 8074 - 82 Crystal structure of the PdxY Protein from Escherichia coli; Safo MK et al.; The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-A resolution . PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5' hydroxyl . The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit . The active site is filled with a density that fits that of pyridoxal . In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5'-phosphate . The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY . The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein . A detailed comparison (of functional properties, sequence homology, active site and ATP-binding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily . The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme. J Bacteriol, 2004 Dec, 186(23), 8044 - 57 Phenylphosphate synthase: a new phosphotransferase catalyzing the first step in anaerobic phenol metabolism in Thauera aromatica; Schmeling S et al.; The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation) . In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step . Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied . Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli . The promoter region and operon structure of the phenol gene cluster were investigated . Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue . It catalyzes the exchange of free {(14)C}phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol . Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase . Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H(2)O-->phenylphosphate + MgAMP + orthophosphate . The phosphoryl group in phenylphosphate is derived from the beta-phosphate group of ATP . The free energy of ATP hydrolysis obviously favors the trapping of phenol (K(m), 0.04 mM), even at a low ambient substrate concentration . The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-beta-synthase domains of unknown function but does not show significant overall similarity to known proteins . The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications. J Bacteriol, 2004 Dec, 186(23), 8036 - 43 The chromosomally encoded cation diffusion facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity; Munkelt D et al.; Genomic sequencing of the beta-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family . One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter . The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W . metallidurans or Escherichia coli . The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences . DmeF plays the central role in cobalt homeostasis in W . metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II) . This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup. J Bacteriol, 2004 Dec, 186(23), 8018 - 25 Kinetic analysis of the oxidative conversion of the {4Fe-4S}2+ cluster of FNR to a {2Fe-2S}2+ Cluster; Sutton VR et al.; The ability of FNR to sense and respond to cellular O(2) levels depends on its {4Fe-4S}(2+) cluster . In the presence of O(2), the {4Fe-4S}(2+) cluster is converted to a {2Fe-2S}(2+) cluster, which inactivates FNR as a transcriptional regulator . In this study, we demonstrate that approximately 2 Fe(2+) ions are released from the reaction of O(2) with the {4Fe-4S}(2+) cluster . Fe(2+) release was then used as an assay of reaction progress to investigate the rate of {4Fe-4S}(2+) to {2Fe-2S}(2+) cluster conversion in vitro . We also found that there was no detectable difference in the rate of O(2)-induced cluster conversion for FNR free in solution compared to its DNA-bound form . In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O(2); a comparison of the in vitro and in vivo rates of conversion suggests that O(2)-induced cluster conversion is sufficient to explain FNR inactivation in cells . FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions. J Bacteriol, 2004 Dec, 186(23), 8000 - 9 Two outer membrane proteins are required for maximal type I secretion of the Caulobacter crescentus S-layer protein; Toporowski MC et al.; Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism . Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery . The genes rsaF(a) and rsaF(b) were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C . crescentus genome sequence . The rsaF(a) gene is located several kilobases downstream of the other transporter genes, while rsaF(b) is completely unlinked . An rsaF(a) knockout had approximately 56% secretion compared to wild-type levels, while the rsaF(b) knockout reduced secretion levels to approximately 79% . When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of approximately 9% remained inside the cell, suggesting posttranslational autoregulation . Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion . However, overexpression of rsaF(a) (with normal rsaF(b) levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system . This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system. J Bacteriol, 2004 Dec, 186(23), 7874 - 80 KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum; Panek HR et al.; Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism . Catalases are heme proteins that detoxify H(2)O(2), and many bacteria contain more than one catalase enzyme . Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H(2)O(2) in Escherichia coli . Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity . Four genes encoding putative catalases in the B . japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase . Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H(2)O(2) consumption by whole cells . The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O(2) . By contrast, a B . japonicum ahpCD mutant grew well aerobically and consumed H(2)O(2) at wild-type rates . A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H(2)O(2) . However, cells of the hemA strain were deficient in consumption of high concentrations of H(2)O(2) and were very sensitive to killing by short exposure to H(2)O(2) . In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR . We conclude that aerobic metabolism produces toxic levels of H(2)O(2) in B . japonicum, which is detoxified primarily by KatG . Furthermore, the katG level sufficient for detoxification does not require OxyR. J Bacteriol, 2004 Dec, 186(23), 7858 - 64 Binding of the C-terminal domain of the alpha subunit of RNA polymerase to the phage mu middle promoter; Ma J et al.; The C-terminal domain of the alpha subunit (alpha CTD) of Escherichia coli RNA polymerase is often involved in transcriptional regulation . The alpha CTD typically stimulates transcription via interactions with promoter UP element DNA and transcriptional activators . DNase I footprinting and gel mobility shift assays were used to look for potential interaction of the alpha CTD with the phage Mu middle promoter P(m) and its activator protein Mor . Binding of RNA polymerase to P(m) in the presence of Mor resulted in production of a DNase I footprint downstream of Mor due to open complex formation and generation of a second footprint just upstream of the Mor binding site . Generation of the upstream footprint did not require open complex formation and also occurred in reactions in which the alpha CTD or His-alpha proteins were substituted for RNA polymerase . In gel mobility shift assays, the formation of a supershifted ternary complex demonstrated that Mor and His-alpha bind synergistically to P(m) DNA . Gel shift assays with short DNA fragments demonstrated that only the Mor binding site and a single upstream alpha CTD binding site were required for ternary complex formation . These results suggest that the alpha CTD plays a role in P(m) transcription by binding to P(m) DNA just upstream from Mor and making protein-protein interactions with Mor that stabilize the binding of both proteins. Nucleic Acids Res, 2004, 32(20), 6028 - 37 Print 2004. A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli; Singh NS et al.; The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions . The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation . In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs . The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide . As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed . Thus, we hypothesized that tmRNA may rescue a defect in Pth . Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth(ts)) . Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth(ts) strain . Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli . Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth(ts) strains . We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load. Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16454 - 9 Epub 2004 Nov 23. Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry; Chu F et al.; Among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems . During the signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between Ffh (the protein component of SRP) and FtsY (the SRP receptor) is known to be essential for the efficiency and fidelity of this process . In this work, we studied the Escherichia coli and Thermus aquaticus Ffh.FtsY complexes by using chemical cross-linking and tandem mass spectrometry to identify nine intermolecular cross-linked peptides . This information was used in conjunction with a previously undescribed model-building approach that combines geometric restraint optimization with macromolecular docking . The resulting model of the Ffh.FtsY complex is in good agreement with the crystal structure solved shortly thereafter . Intriguingly, four of the cross-linked pairs involve the M domain of Ffh, which is absent from the crystal structure, providing previously undocumented experimental evidence that the M domain is positioned in close proximity to the Ffh.FtsY interface in the complex. J Clin Endocrinol Metab . 2004 Nov 16; {Epub ahead of print} CYP3A4 IS A VITAMIN D 24- AND 25-HYDROXYLASE: ANALYSIS OF STRUCTURE FUNCTION BY SITE-DIRECTED MUTAGENESIS; Gupta RP et al.; Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25 hydroxylation occurs, and structure function of the enzyme . Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity . CYP3A4, a vitamin D 25-hydroxyl-ase, and CYP1A1 had the highest 24-hydroxylase activity with 1alpha-hydroxyvitamin D2 {1alphaOHD2} as substrate . The ratio of rates of 24 hydroxylation of 1alpha-hydroxyvitamin D3 {1alphaOHD3}, 1alphaOHD2, and vitamin D2 by CYP3A4 was 3.6/2.8/1.0 . Structures of 24-hydroxy-vitamin D2, 1,24(S)-dihydroxyvitamin D2 and 1,24-dihydroxyvitamin D3 were confirmed by HPLC and gas chromatography retention time and mass spectroscopy . In characterized human liver microsomes, 24-hydroxylation of 1alphaOHD2 by CYP3A4 correlated significantly with 6beta-hydroxylation of testosterone, a marker of CYP3A4 activity . 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4 . Rates of 24- and 25-hydroxylation of 1alphaOHD2 and 1alphaOHD3 were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli . Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479 . Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D2, 1alphaOHD2, and 1alphaOHD3. J Biol Chem . 2004 Nov 16; {Epub ahead of print} New genes implicated in the protection of anaerobically grown escherichia coli against nitric oxide; Justino MC et al.; Nitric oxide produced by activated macrophages plays a key role as one of the immune system weapons against pathogens . Since the lifetime of nitric oxide is short in aerobic conditions whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased, like in infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions . In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysR-type (YidZ) . The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated . Furthermore, nitric oxide is shown to cause a significant alteration of the global Escherichia coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulphur cluster assembly systems, DNA repairing enzymes and stress response regulators. Bioorg Med Chem Lett, 2004 Dec 20, 14(24), 5987 - 90 Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA; Maddaford SP et al.; We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site . Binding by the compound was observed using nuclear magnetic resonance and mass spectrometry techniques . We show that the compound binds in the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 37 - 53 Sec-translocase mediated membrane protein biogenesis; Dalbey RE et al.; alpha-Helical transmembrane proteins in bacteria are localized within the plasma membrane . The membrane assembly of these proteins requires protein devices for insertion into the lipid bilayer . In E . coli, membrane proteins require the SRP pathway components Ffh, 4.5S RNA and FtsY for membrane targeting and the SecYEGDF translocase and, in some cases, SecA, for translocation of hydrophilic domains . In addition, YidC, a recently discovered membrane protein, mediates the membrane integration and folding of hydrophobic domains of membrane proteins . In this review, we will describe the current status of the protein targeting and membrane integration pathways. Mol Cell, 2004 Nov 19, 16(4), 575 - 86 20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3; Baugh JM et al.; The molecular basis for coordinated regulation of protein synthesis and degradation is not understood . Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3 . The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components . Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factors in vitro and in vivo, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo . These studies demonstrate the importance of the endoproteolytic activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation. Mol Cell, 2004 Nov 19, 16(4), 537 - 47 Regulation of the pap epigenetic switch by CpxAR: phosphorylated CpxR inhibits transition to the phase ON state by competition with Lrp; Hernday AD et al.; Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON) . Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase . Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P) . CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo . In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo . CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions . These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression. Bioconjug Chem, 2004 Nov-Dec, 15(6), 1454 - 63 Streptavidin in antibody pretargeting . 4 . Site-directed mutation provides evidence that both arginine and lysine residues are involved in kidney localization; Wilbur DS et al.; The in vivo application of the protein streptavidin is limited by its propensity to localize to kidney, particularly when it is used as a carrier of radionuclides in Targeted Radionuclide Therapy . Our previous studies demonstrated that modification of recombinant "core" streptavidin (rSAv) by reaction of lysine residues with succinic anhydride and arginine residues with 1,2-cyclohexanedione dramatically decreases the kidney concentrations over that obtained with wild-type rSAv . In this investigation, we explored the role of lysine and arginine residues in kidney localization further by evaluating site-directed mutants of rSAv . In the five mutants studied, the four lysine residues found in each subunit of rSAv were replaced (independently) with an alanine (K80A, K121A, K132A, K134A), and a specific arginine was replaced with a histidine (R59H) . The rSAv mutants were prepared from a "core" rSAv construct produced by expression in E . coli that had 124 amino acids (residues 13-136) . Another rSAv construct that had 127 amino acids (residues 13-139), used in most of our previous studies, was also included for comparison . As an additional comparison, succinylated rSAv was prepared and evaluated . The rSAv proteins were radioiodinated and injected into athymic mice that were on a biotin-free diet for 5-7 days prior, and biodistribution data were obtained (for most proteins) at 1, 4, 24, and 48 h postinjection . The data obtained show large differences in kidney localizations of the wild-type rSAv and some rSAv mutants . The largest difference in the kidney concentration was noted for the rSAv-K134A mutant (1.90 +/- 0.22%ID/g; 24 h pi) as compared to the wild-type rSAv (31.83 +/-5.26%ID/g) at the same time point . The concentration of rSAv-K134A mutant in kidney was slightly lower than that obtained with succinylated rSAv . At the 24 h time point, the kidney concentrations of the rSAv-R59H mutant (8.95 +/- 2.94%ID/g) and the rSAv-K121A mutant (11.86 +/- 1.61%ID/g) were lower than wild-type rSAv, but the rSAv mutants rSAv-K80A (27.95 +/- 1.82%ID/g) and rSAv-K132A (32.50 +/- 10.09%ID/g) were essentially the same . The data suggests that specific lysine and arginine residues are involved in kidney localization . Possible mechanisms for the observed kidney localization are discussed. J Dairy Sci, 2004 Dec, 87(12), 4150 - 62 Viability of milk neutrophils and severity of bovine coliform mastitis; Mehrzad J et al.; To study the host-pathogen interactions during Escherichia coli mastitis, we first determined whether E . coli infection would change blood and milk polymorphonuclear neutrophil (PMN) chemiluminescence (CL) and viability . We then hypothesized that when E . coli invade the mammary gland, the viable PMN in milk would efficiently phagocytose and destroy E . coli before establishment of infection . We observed that the phagocytosis-dependent and independent CL were closely linked to PMN viability and were crucial to the outcome of mastitis . Maximal PMN influx and colony-forming units in infected quarters appeared at postinfection hours (PIH) 6 to 24 . This further boosted PMN recruitment through bone marrow-blood barrier as well as blood-milk barrier . The survival of recruited PMN in the E . coli-infected quarters was much higher than that of noninfected quarters . Chemiluminescence activity of PMN from the infected quarters significantly increased following E . coli infection, even exceeding that of blood at PIH 6, 12, and 18 to 24; no such increase was observed in noninfected quarters, suggesting that the various responses of milk PMN to stimuli resulted largely from PMN viability . The highest CL intensity and durability was observed in milk PMN from infected quarters at PIH 12 . Whereas an increased viability of PMN in the noninfected quarters was only significant at PIH 6, the viability of PMN in infected quarters was long lasting and significantly higher at PIH 6 to 72 . Importantly, higher preinfection milk PMN viability correlated with bacterial clearance, which was accompanied by faster recovery . Our study strongly supports the hypothesis that boosting milk PMN viability could be a strategy with which to prevent or reduce the severity of coliform mastitis in dairy cows . This strategy might be achieved through strengthening bone marrow functionality. J Biol Chem . 2004 Nov 15; {Epub ahead of print} Crystal structures of the FXIa catalytic domain in complex with ecotin mutants reveal substrate-like interactions; Jin L et al.; Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism and stroke . Although commonly used anticoagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications and there is a need for safer drugs . Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade . Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S.R . (2003) Blood 102, 953-955) . Therefore, FXIa is a potential target for antithrombosis therapy . In order to determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370-607 (rhFXI370-607) . Here we report the first crystal structure of rhFXI370-607 in complex with a substitution mutant of ecotin, a pan-serine protease protein inhibitor secreted by Escherichia coli, to 2.2 resolution . The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa . Subsequently, the sequence from P5 to P2' in ecotin was mutated to the FXIa substrate sequence and the structures of the rhFXI370-607 - ecotin mutant complexes were determined . These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors. J Biol Chem . 2004 Nov 15; {Epub ahead of print} Identification and characterisation of the terminal enzyme of siroheme biosynthesis from arabidopsis thaliana; a plastid-located sirohydrochlorin ferrochelatase containing a 2Fe-2S centre; Raux-Deery E et al.; Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group . Siroheme is synthesised from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation and ferrochelation reactions . In this paper we report on the Arabidopsis thaliana sirohydrochlorin ferrochelatase At-SirB . The complete precursor protein of 225 amino acids, and shorter constructs in which the first 46 or 79 residues had been removed, were shown to complement a defined E . coli sirohydrochlorin ferrochelatase mutant . The mature form of the protein appeared to consist of only 150 amino acids making it much smaller than previously characterised ferrochelatases . Green fluorescent protein tagging revealed that it is located in the chloroplast . The enzyme was easily produced in E . coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmoles/min/mg . Significantly, the protein purified as a brown colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S centre . EPR analysis of the recombinant protein produced a rhombic spectrum with g-values of 2.038, 1.94 and 1.90 and with temperature dependence consistent with a 2Fe-2S centre . Redox titration demonstrated that the Fe-S centre is highly unstable, with an apparent mid-point reduction potential of about -370 mV . This is the first Fe-S centre to be reported in a higher plant ferrochelatase . The implications of the Fe-S centre in an enzyme that is so closely associated with the metabolism of S and Fe are discussed. IUBMB Life, 2004 Aug, 56(8), 501 - 7 Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein; Self WT et al.; The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general . A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii . The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E . coli . The 8.8 kDa gene product contains 81 amino acids . The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite . The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein . The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism. Insect Biochem Mol Biol, 2004 Dec, 34(12), 1289 - 95 Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans); Zhang D et al.; Thrombostasin (TS) is a thrombin inhibitor found in the salivary glands of horn flies (Haematobia irritans) . It is produced as an inactive form with a 76-amino acid propeptide in the N-terminus preceding the mature TS . A minimal recognition sequence by subtilisin-like proprotein convertases, Arg-Xaa-Xaa-Arg, is localized C-terminal to the propeptide . This study demonstrated that a gene cloned from the salivary glands of the horn fly encodes a new convertase, subsequently named horn fly proprotein convertase (HFPC), and that the recombinant HFPC expressed in insect HighFive cell culture specifically cleaves recombinant pro-thrombostasin, produced in E . coli, at the expected site . The relative cleavage efficiency of rHFPC was compared with that of recombinant human furin, a commercially available proprotein convertase . The result indicated that this newly identified proprotein convertase is of importance for the proteolytic maturation of thrombostasin, a protein secreted in horn fly saliva and used by the insect to counteract its host's haemostatic response. Insect Biochem Mol Biol, 2004 Dec, 34(12), 1235 - 46 Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis and an embryonic cell line of the Rocky Mountain wood tick, D . andersoni; Simser JA et al.; Immune-responsive lysozyme encoding cDNAs were identified from two medically important tick species by an expressed sequence tag approach of D . variabilis hemocytes (Dv Lys) and a D . andersoni embryonic derived cell line, DAE100 . Comparative sequence analyses indicated the Dermacentor molecules to be products of orthologous genes and to be most similar to arthropod c-type lysozymes . Northern blotting analyses demonstrated that Dv Lys expression levels were most abundant in tick hemocytes and to a much lesser degree in the midgut while barely detectable in ovary, salivary gland, and Malpighian tubule tissues . Involvement of the Dermacentor c-type lysozymes in innate immunity was demonstrated by Escherichia coli challenges of D . variabilis ticks by injection resulting in a temporal profile of significantly elevated transcript abundances above those of naive controls that was similarly observed of the D . andersoni cells co-cultured with E . coli . In contrast to that reported of the digestive gut lysozyme of the soft tick Ornithodoros moubata, Dv Lys levels were not statistically differentially regulated by blood meal digestion . Additionally, given the differences in tissue distribution, sequence characteristics and phylogenetic placements between the Dermacentor and Ornithodoros lysozymes demonstrates that ticks possess differently adapted c-type lysozymes that are spatially and temporally differentially expressed. J Mol Biol, 2004 Dec 3, 344(4), 1109 - 21 Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles; Lorch M et al.; Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers . We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles . The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated . The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1) . This association rate approaches the diffusion limit of the protein, vesicle reaction . The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase . Protein aggregation competes with vesicle insertion . The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted . This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle . Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers . Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein. J Mol Biol, 2004 Dec 3, 344(4), 951 - 63 Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance; Rajan R et al.; The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks . Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA . In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution . Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A . Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA . Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential . Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding. Biochemistry, 2004 Nov 23, 43(46), 14864 - 72 Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition; Filion ML et al.; The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily . Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond . Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases . In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center . We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors . A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli . Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level . However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg . An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC . The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition . Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity. Biochemistry, 2004 Nov 23, 43(46), 14732 - 43 The exclusion of glycine betaine from anionic biopolymer surface: why glycine betaine is an effective osmoprotectant but also a compatible solute; Felitsky DJ et al.; Paradoxically, glycine betaine (N,N,N-trimethyl glycine; GB) in vivo is both an effective osmoprotectant (efficient at increasing cytoplasmic osmolality and growth rate) and a compatible solute (without deleterious effects on biopolymer function, including stability and activity) . For GB to be an effective osmoprotectant but not greatly affect biopolymer stability, we predict that it must interact very differently with folded protein surface than with that exposed in unfolding . To test this hypothesis, we quantify the preferential interaction of GB with the relatively uncharged surface exposed in unfolding the marginally stable lacI helix-turn-helix (HTH) DNA binding domain using circular dichroism and with the more highly charged surfaces of folded hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) using all-gravimetric vapor pressure osmometry (VPO) and compare these results with results of VPO studies (Hong et al . (2004), Biochemistry, 43, 14744-14758) of the interaction of GB with polyanionic duplex DNA . For these four biopolymer surfaces, we observe that the extent of exclusion of GB per unit of biopolymer surface area increases strongly with increasing fraction of anionic oxygen (protein carboxylate or DNA phosphate) surface . In addition, GB is somewhat more excluded from the surface exposed in unfolding the lacI HTH and from the folded surface of HEWL than expected from their small fraction of anionic surface, consistent with moderate exclusion of GB from polar amide surface, as predicted by the osmophobic model of protein stability (Bolen and Baskakov (2001) J . Mol . Biol . 310, 955-963) . Strong exclusion of GB from anionic surface explains how it can be both an effective osmoprotectant and a compatible solute; analysis of this exclusion yields a lower bound on the hydration of anionic protein carboxylate surface of two layers of water (>or=0.22 H(2)O A(-)(2)). Biochemistry, 2004 Nov 23, 43(46), 14637 - 43 Denaturant-induced unfolding of the acetyl-esterase from Escherichia coli; Del Vecchio P et al.; The stability of acetyl-esterase, Aes, from Escherichia coli against the denaturing action of urea and guanidine hydrochloride, GuHCl, has been investigated by means of circular dichroism and fluorescence measurements . The urea-induced unfolding curves show a single inflection point at 6.2 M urea, whereas the GuHCl-induced curves show two inflection points at 1.4 and 3.1 M GuHCl . The unfolding process is reversible with both urea and GuHCl . These results, together with similar experimental data on the mutant form V20D-Aes, suggest the presence of two domains in the Aes structure, which unfold more or less independently depending on the denaturant used . This is also supported by a 3D model obtained by homology modeling using the structure of brefeldine as a template . The effect of NaCl on the urea-induced unfolding curves of the enzyme has also been investigated. Biochemistry, 2004 Nov 23, 43(46), 14624 - 36 Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA; Hoerter JA et al.; The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation . Recent crystallographic and genetic evidence has called this hypothesis into question . To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E . coli H27 for in vitro physicochemical studies . One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl) . UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link . The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12 . FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit . Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations . This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome. Med Dosw Mikrobiol, 2004, 56(2), 173 - 7 {Using the PCR-RFLP method for comparative analysis of the pathogenic Escherichia coli strains}; Skwark M et al.; The purpose of the study was to find differences and similarities between Escherichia coli strains which utilize or do not utilize disacharide sucrose by PCR-RFLP method . Investigations have been done on chromosomal DNA level using cscA gene associated with conservative sequences . The cscA gene may be find in all of the analysed strains . Genotypic analysis demonstrated presence of the same restriction model consisted of 2 DNA fragments with size of 161 bp and 110 bp in all of E . coli strains . Results of these investigetions have shown that there are no differences between E . coli strains. Pharmazie, 2004 Oct, 59(10), 807 - 11 A new furobenzopyranone and other constituents from Anaphalis lactea; Wang AX et al.; Together with twenty-one known compounds, a new furobenzopyranone was isolated from the whole plant of Anaphalis lactea . Their structures were elucidated by spectroscopic methods MS, IR, UV, NMR, including 2D-NMR techniques . The anti-bacterial activity of compounds 1, 4-6, 14, 15 and the anti-tumor activity of compounds 4-6 were tested. Pharmazie, 2004 Oct, 59(10), 744 - 52 2-(Arylpropionylamino)- and 2-(arylacryloylamino)benzophenones: farnesyltransferase inhibition and antimalarial activity; Fucik K et al.; Structural variation of the 2-acylamino moiety of some benzophenone farnesyltransferase inhibitors led to the para-trifluoromethylphenylpropionyl derivative with relatively low farnesyltransferase inhibition but considerable antimalarial activity and no cytotoxicity. J Chromatogr A, 2004 Oct 22, 1053(1-2), 71 - 8 Capillary liquid chromatographic determination of cellular flavins; Jia L et al.; A capillary LC system was set up and optimized, in which a UV absorbance detector was used and a monolithic silica-ODS column as the separation column . Two on-line concentration techniques, namely, gradient elution mode and in-tube solid-phase ion-pair microextraction (SPIPME), were combined with the capillary LC system, which proved to be beneficial to enhance the concentration sensitivity by enabling the injection of large volumes of samples . The limits of detection at ppb levels for the flavins {riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)} were achieved using the two techniques . For in-tube SPIPME, a monolithic silica-ODS column was employed as the extraction column, on which FAD and FMN were retained b y interaction with an ion-pair reagent, tetrabutylammonium phosphate, resulting in greater than 110-fold enhancement in their concentration sensitivities relative to conventional injection method . The reproducibility and linearity of the two methods were investigated . The two methods were applied to analyze trace amounts of flavins in bacterial Escherichia coli cell extracts and recombinant flavoenzymes. Mol Cell Biochem, 2004 Oct, 265(1-2), 47 - 56 Despite minimal hemodynamic alterations endotoxemia modulates NOS and p38-MAPK phosphorylation via metalloendopeptidases; Gupta A et al.; In the present study, we hypothesized that endotoxemia produces metalloendopeptidase (MEPD)-dependent generation of endothelin-1 (ET-1) and alters NOS expression correlating with p38-mitogen-activated protein kinase (MAPK) phosphorylation in thoracic aorta . Male Sprague-Dawley rats (350-400 g) were subjected to two groups randomly; sham-treated (N = 10) and lipopolysaccharide (LPS)-treated (N = 10) (E . coli LPS 2 mg/kg bolus + 2 mg/kg infusion for 30 min) . The animals in each group were further subdivided into vehicle and MEPD inhibitor phosphoramidon (1 mg/kg bolus, PHOS)-treated groups . LPS produces a significant decrease in mean arterial pressure (MAP) at 2 h post endotoxemia that was blocked by PHOS . PHOS attenuated LPS-induced increase in tumor necrosis factor-alpha (TNF-alpha) concentration at 2- and 24 h post-LPS administration . LPS significantly elevated plasma concentrations of ET-1 at 2- and 24 h post endotoxemia . An upregulated preproET-1 expression following both LPS and MEPD inhibition was observed in thoracic aorta at 2 h post treatment . PHOS effectively blocked conversion of preproET-1 to ET-1 in thoracic aorta locally at 24 h post treatment in endotoxic rats . PHOS inhibited LPS-induced upregulation of inducible NOS (iNOS), downregulation of endothelial NOS (eNOS) and elevation of NO byproducts (NOx) in thoracic aorta . PHOS also blocked LPS-induced upregulated p38-MAPK phosphorylation in thoracic aorta at 24 h post endotoxemia . The data revealed that LPS induces MEPD-sensitive inflammatory response syndrome (SIRS) at 2- and 24 h post endotoxemia . We concluded that inhibition of MEPD not only decreases the levels of ET-1 but also simultaneously downregulates protein expression of iNOS and phosphorylated p38-MAPK while increasing eNOS in thoracic aorta during SIRS in endotoxemia . We suggest that MEPD-dependent ET-1 and NO mechanisms may be involved in endotoxemia-induced altered p38-MAPK phosphorylation. Nat Struct Mol Biol, 2004 Dec, 11(12), 1179 - 85 Epub 2004 Dec. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path; Lee C et al.; The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines . Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial . By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein . In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1) . The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1) . On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR. Mol Cell Biol, 2004 Dec, 24(23), 10390 - 6 3'-End polishing of the kinetoplastid spliced leader RNA is performed by SNIP, a 3'-->5' exonuclease with a Motley assortment of small RNA substrates; Zeiner GM et al.; In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs . The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing . Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei . We named this enzyme SNIP (for snRNA incomplete 3' processing) . The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme . SNIP had a preference for oligo(U) 3' extensions in vitro . RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA . SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking . Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing . We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA. Eur Cytokine Netw, 2004 Jul-Sep, 15(3), 255 - 62 Expression of biologically active mouse ciliary neutrophic factor (CNTF) and soluble CNTFRalpha in Escherichia coli and characterization of their functional specificities; Cognet I et al.; Ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine initially identified in chick embryo . It has been evaluated for the treatment of neurodegenerative diseases . CNTF also acts on non-neuronal cells such as oligodendrocytes, astrocytes, adipocytes and skeletal muscles cells . CNTF has regulatory effects on body weight and is currently in clinical trial for the treatment of diabetes and obesity . CNTF mediates its function by activating a tripartite receptor comprising the CNTF receptor alpha chain (CNTFRalpha), the leukemia inhibitory factor receptor beta chain (LIFRbeta) and gp130 . Human, rat and chicken CNTF have been expressed as recombinant proteins, and most preclinical studies in murine models have been performed using rat recombinant protein . Rat and human CNTF differ in their fine specificities: in addition to CNTFR, rat CNTF has been shown to activate the LIFR (a heterodimer of LIFRbeta and gp130), whereas human CNTF can bind and activate a tripartite receptor comprising the IL-6 receptor alpha chain (IL-6Ralpha) and LIFR . To generate tools designed for mouse models of human diseases; we cloned and expressed in E . coli both mouse CNTF and the CNTFRalpha chain . Recombinant mouse CNTF was active and showed a high level of specificity for mouse CNTFR . It shares the arginine residue with rat CNTF which prevents binding to IL-6Ralpha . It did not activate the LIFR at all concentrations tested . Recombinant mouse CNTF is therefore specific for CNTFR and as such represents a useful tool with which to study CNTF in mouse models . It appears well suited for the comparative evaluation of CNTF and the two additional recently discovered CNTFR ligands, cardiotrophin-like cytokine\cytokine-like factor-1 and neuropoietin. Brain Res, 2004 Dec 17, 1029(2), 148 - 54 Nitric oxide mediates an LPS-induced depression of cytochrome P450 (CYP1A) activity in astrocytes; Nicholson TE et al.; During inflammatory responses in the brain, the expression of cytochrome P450 isoforms in the CNS are modulated and the capacity of the brain to metabolize drugs and to synthesize or degrade certain endogenous chemicals and drugs is diminished . While this response can be attributed in part, to the production and action of cytokines within the brain, it is also likely that other inflammatory mediators play an integral role . This paper investigates a potential role for nitric oxide (NO) in the loss of cytochrome P450 (CYP1A) in the brain during inflammation . Escherichia coli lipopolysaccharide (LPS), a commonly used proinflammatory endotoxin, was incubated with cultured rat astrocytes to provide a model of inflammation in the CNS . CYP1A activity was significantly decreased in cultured astrocytes incubated with LPS for 24 h . This loss in enzyme activity was accompanied by a substantial production of nitric oxide (NO) by these cells . Immunohistochemical examination demonstrated an upregulation of inducible nitric oxide synthase (iNOS) expression following the exposure of astrocytes to LPS . The addition of a selective iNOS blocker (1400W) caused a partial but significant reversal of the LPS-mediated loss in CYP1A . The incubation of astrocytes with the NO-generating compound (DETA NONOate) resulted in a loss of CYP1A . Taken together, these observations suggest that NO plays a pivotal role in the inflammation mediated loss in CYP1A activity in the brain. Arch Biochem Biophys, 2004 Dec 15, 432(2), 136 - 44 Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif; Prosser I et al.; The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant-insect interactions and in a number of biotechnological approaches to pest control . Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D . Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase . Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes . Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D,E), Sc11 is unusual in that this motif occurs as (303)NDTYD . Mutagenesis of this motif to (303)DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity . The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme . Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity . These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought . Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate. Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 726 - 30 Glycogen and related polysaccharides inhibit the laforin dual-specificity protein phosphatase; Wang W et al.; Lafora disease, a progressive myoclonus epilepsy, is an autosomal recessive disease caused in approximately 80% of cases by mutation of the EPM2A gene, which encodes a dual specificity protein phosphatase called laforin . In addition to its phosphatase domain, laforin contains an N-terminal carbohydrate-binding domain (CBD) . Mouse laforin was expressed as an N-terminally polyHis tagged protein in Escherichia coli and purified close to homogeneity . The enzyme was active towards p-nitrophenylphosphate (50-80mmol/min/mg, K(m) 4.5mM) with maximal activity at pH 4.5 . Laforin binds to glycogen, as previously shown, and caused potent inhibition, half maximally at approximately 1mug/ml . Less branched glucose polymers, amylopectin and amylose, were even more potent, with half maximal inhibition at 10 and 100ng/ml, respectively . With all polysaccharides, however, inhibition was incomplete and laforin retained 20-30% of its native activity at high polysaccharide concentrations . Glucose and short oligosaccharides did not affect activity . Substitution of Trp32 in the CBD by Gly, a mutation found in a patient, caused only a 30% decrease in laforin activity but abolished binding to and inhibition by glycogen, indicating that impaired glycogen binding is sufficient to cause Lafora disease. Biochim Biophys Acta, 2004 Nov 1, 1674(3), 319 - 26 The alpha-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions; Mulrooney SB et al.; Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes . It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain . This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system . Alanine was inserted at six positions in the membrane anchor of cyt b5 . Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane . These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions. Z Naturforsch {C}, 2004 Sep-Oct, 59(9-10), 755 - 61 Cloning and expression of Taxus acyltransferase cDNA; Tu J et al.; A new full-length acyltransferase cDNA was obtained from Taxus chinensis by homology-based cloning strategy . The cDNA has an open-reading frame of 1,275 nucleotides, which encodes 425 amino acids with a calculated molecular weight of 47,241 Da and an estimated pI value of 5.93 . The deduced amino acid sequence resembles the sequences of other cloned acyltransferases (56-61% identity; 71-75% similarity) involved directly in taxol biosynthetic pathways . This cDNA was expressed in Escherichia coli using the expression vector pET32a(+) . The expression band corresponds to the calculated mass plus the N-terminal fusion protein derived from the vector. Am Nat, 2004 Nov, 164(5), 651 - 9 Epub 2004 Sep 29. An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity; Alonso-Alvarez C et al.; Carotenoid-based sexual traits are thought to be reliable indicators of male quality because they might be scarce and therefore might indicate the ability of males to gather high-quality food and because they are involved in important physiological functions (as immune enhancers and antioxidants) . We performed an experiment where male and female zebra finches (Taeniopygia guttata) were provided with increasing carotenoid doses in the drinking water during 4 weeks (bill color of this species is a carotenoid-based sexual signal) . Simultaneously, birds were split into two groups: one receiving weekly injections of Escherichia coli lipopolysaccharide in order to activate the immune system, the other being injected with the same volume of phosphate buffered saline . We assessed how carotenoid availability and immune activation affected the amount of circulating plasma carotenoids, the beak color, and the antioxidant defenses (assessed as the resistance of red blood cells to a controlled free radical attack) . Carotenoid availability affected the amount of circulating carotenoids and beak color; both variables reached a plateau at the highest carotenoid doses . Immune activation diverted carotenoids from plasma, and this in turn affected the expression of the sexual trait . Finally, we found a positive correlation between the change in circulating carotenoids and antioxidant defenses . These results support the idea that carotenoids have important physiological properties that ensure the honesty of carotenoid-based sexual traits. Clin Diagn Lab Immunol, 2004 Nov, 11(6), 1085 - 8 Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain; Tachibana H et al.; We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica . To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids . The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E . histolytica trophozoites . Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones . The remaining eight clones exhibited no amino acid change at position 91 or 96 . These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin . Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not . The replacement of Arg96 with Leu did not affect affinity . These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin. Clin Diagn Lab Immunol, 2004 Nov, 11(6), 1016 - 21 Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women; Ferrandiz J et al.; An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection . Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins . Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T . gondii (group 3) were examined for immunoglobulin G (IgG) reactivity . For both antigens, the specificity reached 98% . In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt . In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively . Combination of the readings obtained with both antigens yielded a sensitivity of 91% . A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results . Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections. J Nutr Biochem, 1999 Jun, 10(6), 331 - 7 Can a glutamate-enriched diet counteract glutamine depletion in endotoxemic rats? Chambon-Savanovitch C, Farges MC, Raul F, Blachier F, Davot P, Cynober L, Vasson MP. The study evaluated whether a glutamate-enriched diet would restore glutamine tissue pools and maintain tissue trophicity in endotoxemic rats . For this purpose, young male Sprague-Dawley rats received an intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli at 3 mg/kg body weight . After 24 hours of food deprivation, the rats were enterally refed for 48 hours using Osmolite(R) enriched with glutamate at 4 g/kg/d (LPS-Glu group, n = 7) or glycine isonitrogenous to glutamate (LPS-Gly group, n = 7) . A control group (healthy group, n = 7) had free access to a standard rodent diet . Tissue weights and protein contents were significantly lower in both LPS-treated groups than in the healthy group . No plasma or tissue accumulation of glutamate was observed except in the liver . Glutamine concentrations were increased in the jejunum, liver, and plasma in the LPS-Glu group versus the other two groups (P < 0.05) . Conversely, they were depleted in muscles of the endotoxemic groups versus the healthy group (P < 0.05) . Villus height was significantly greater in the LPS-Glu group than in the LPS-Gly group in the jejunum (P < 0.05), but not in the ileum . In conclusion, a glutamate-enriched diet administered enterally to endotoxemic rats can counteract glutamine depletion in the splanchnic area but not in muscles . In addition, glutamate displayed a trophic effect restricted to the jejunum. Anal Chem, 2004 Nov 15, 76(22), 6521 - 7 Protein microarray system for detecting protein-protein interactions using an anti-His-tag antibody and fluorescence scanning: effects of the heme redox state on protein-protein interactions of heme-regulated phosphodiesterase from Escherichia coli; Sasakura Y et al.; A highly sensitive microarray system for detecting protein-protein interactions has been developed . This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) . To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions . For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate . In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F(c) region of the antibody . Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface . Ec DOS immobilized via anti-(His)6-Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His)6-Tag mAb . Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His)6-Tag mAb as a mediator . Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces. Mol Nutr Food Res, 2004 Dec, 48(7), 515 - 21 A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli; Kuczius T et al.; Subtyping of Shiga toxin (Stx)-encoding genes by conventional polymerase chain reaction (PCR) is time-consuming . We developed a single step real-time fluorescence PCR with melting curve analysis to distinguish rapidly stx1 from its variants, stx1c and stx1d . Melting temperatures (Tm) of 206 Stx-producing Escherichia coli (STEC) identified to harbor stx1 or stx1c were analyzed using a specific hybridization probe over the variable region . 170 of 171 stx1-harboring STEC displayed Tm of 69 degrees C to 70 degrees C, whereas 34 of 35 strains containing stx1c had Tm of 65 degrees C-66 degrees C . This constant and reproducible difference of 4 degrees C demonstrated that melting curve analysis is a reliable technique to differentiate stx1 from stx1c . Two isolates displayed atypical Tm . Sequence analysis showed that one of them was 100% identical to stx1d within a 511 bp DNA stretch . Our data demonstrate that real-time PCR is a rapid and reliable tool to differentiate stx1 from stx1c and stx1d and to detect new stx1 variants . Because stx1-harboring STEC cause diarrhoea and hemolytic-uremic syndrome, whereas those containing stx1c are often shed asymptomatically, a rapid differentiation between stx1 and its variants using the procedure developed here has both clinical implications and a direct significance for the risk assessment analysis of STEC isolated from foods. Mol Nutr Food Res, 2004 Dec, 48(7), 504 - 14 Molecular characteristics of Escherichia coli serogroup O78 strains isolated from diarrheal cases in bovines urge further investigations on their zoonotic potential; Ewers C et al.; We investigated the virulence properties and clonal relationship of 21 Escherichia coli strains of serogroup O78 isolated from diarrhoeic cattle and calves . Isolates were screened for 18 genes representing virulence features of different Escherichia coli pathotypes . None of the strains harboured enterotoxin-genes estIa/Ib, eltIa/Ib, or Shiga toxin (stx) genes, genes involved in adhesion (eae, f5, f41) hemolysin gene hlyA or invasion gene ipaC . With a high prevalence we detected enterotoxin astA (61.9%), genes involved in iron acquisition, like fyuA, irp (each 57.1%) and iucD (81.0%), and the operon sequence of Colicin V plasmids (38.1%) . Some strains possessed toxin genes cdt-IIIB and cnf1/2 (both 14.3%), the invasion gene tia (23.8%), and the serine protease encoding gene espP (23.8%) . Moreover, we could show that E . coli O78 strains under investigation were able to adhere to and invade MDBK-cells with varying efficiencies . The results indicate that the closely related O78 strains, constituting two major PFGE-clusters, harbor various virulence features for bovine intestinal disease but cannot be grouped into one of the common E . coli intestinal pathogenic or other pathotypes according to their virulence gene pattern . Nevertheless, the ability to adhere, invade or harbor toxin genes lets us suggest that O78 strains isolated from diarrheal cases in bovines urges further investigations on the zoonotic potential of these strains. Nature, 2004 Nov 11, 432(7014), 187 - 93 Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks; Singleton MR et al.; RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break . RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site) . The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA . Here we present the crystal structure of RecBCD bound to a DNA substrate . In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits . The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB . Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA . Gating of this tunnel suggests how nuclease activity might be regulated. Protein Sci, 2004 Dec, 13(12), 3214 - 21 Epub 2004 Dec. Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity; Tanaka N et al.; The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system . The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown . In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding . ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate . However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain . In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN) . Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH . The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics . Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate . In addition, these residues are involved in stimulation of ATPase activity in ClpB . Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change. J Biol Chem, 2005 Jan 7, 280(1), 722 - 8 Epub 2004 Nov 10. Crystal Structure of an ATPase-active Form of Rad51 Homolog from Methanococcus voltae: INSIGHTS INTO POTASSIUM DEPENDENCE; Wu Y et al.; Homologous gene recombination is crucial for the repair of DNA . A superfamily of recombinases facilitate a central strand exchange reaction in the repair process . This reaction is initiated by coating single-stranded DNA (ssDNA) with recombinases in the presence of ATP and Mg(2+) co-factors to form helical nucleoprotein filaments with elevated ATPase and strand invasion activities (1) . At the amino acid sequence level, archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA (2) . Unlike the extensively studied Escherichia coli RecA (EcRecA), increasing evidences on yeast and human recombinases imply that their optimal activities are dependent on the presence of a monovalent cation, particularly potassium (3-5) . Here we present the finding that archaeal RadA from Methanococcus voltae (MvRadA) is a stringent potassium-dependent ATPase, and the crystal structure of this protein in complex with the non-hydrolyzable ATP analog adenosine 5'-(beta,gamma-iminotriphosphate), Mg(2+), and K(+) at 2.4 A resolution . Potassium triggered an in situ conformational change in the ssDNA-binding L2 region concerted with incorporation of two potassium ions at the ATPase site in the RadA crystals preformed in K(+)-free medium . Both potassium ions were observed in contact with the gamma-phosphate of the ATP analog, implying a direct role by the monovalent cations in stimulating the ATPase activity . Cross-talk between the ATPase site and the ssDNA-binding L2 region visualized in the MvRadA structure provides an explanation to the co-factor-induced allosteric effect on RecA-like recombinases. J Biol Chem . 2004 Nov 10; {Epub ahead of print} Trypanothione synthesis in Crithidia revisited; Comini MA et al.; In Crithidia fasciculata the biosynthesis of trypanothione {N1,N8bis-(glutathionyl)spermidine; T(SH)2}, a redox mediator unique to, and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), only the first one having been adequately characterized . We here report that the TryS of C . fasciculata, like that of Trypanosoma species, catalyses the entire synthesis of trypanothione, while its GspS appears to be specialised for Gsp synthesis . A gene (Acc . N degrees AY603101) implicated in T(SH)2 synthesis of C . fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus-fusion proteins . The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase and a marginal ATPase activity . The dual specificity of the Cf-TryS preparations was not altered by removal of the tags . Steady-state kinetic analysis of Cf-TryS yielded a pattern that is compatible with a concerted-substitution mechanism, wherein the enzyme forms a ternary complex with Mg++-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine (Gsp) . Limiting Km values for GSH, Mg++-ATP and Gsp were 407, 222 and 480 microM, respectively, and the kcat was 8.7 sec-1 for the TryS reaction . Mutating R553 or R613 to K, L, Q or E resulted in marked reduction or abrogation (R553E) of activity . Limited proteolysis with factor Xa or trypsin resulted in cleavage at R556 that was accompanied by loss of activity . Presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis. J Biol Chem . 2004 Nov 9; {Epub ahead of print} A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2; Jiang B et al.; Trastuzumab, a humanized antibody to HER-2, has been shown to be effective in the treatment of breast cancer in which HER-2 overexpression and metastasis occurs . In our search for an effective mimic epitope of HER-2 binding with trastuzumab and in order to develop HER-2 peptide vaccine, we screened a phage display 12-mer peptide library with trastuzumab as the target . A mimetic peptide (mimotope) H98 (LLGPYELWELSH) that could specifically recognize trastuzumab was isolated . The DNA encoding peptide H98 was cloned and expressed as the fusion protein GST-H98 in Escherichia . coli BL21 . The purified GST-H98 could specifically bind to trastuzumab and block the binding of trastuzumab to HER-2 protein . Moreover, H98 could significantly block the function of trastuzumab inhibiting growth of cancer cell . Mice that were immunized with GST-H98 made specific antibody to H98 as well as to HER-2 . In addition, T-cell proliferation occurred in mice immunized with GST-H98 . Although no sequence homology was found between H98 and HER-2, through the use of structure analysis we were able to determine that peptide H98 contributed to a conformational epitope of HER-2 . Furthermore, we determined that the last two amino acids at the C-terminus, and the third together with the fourth amino acid at the N-terminus of peptide H98 are critical to the binding of H98 to trastuzumab . As a result, we conclude that the peptide H98 has potential for being developed as a HER-2 vaccine for biotherapy of cancer with HER-2 overexpression. Biochim Biophys Acta, 2004 Nov 18, 1675 1-3, 174 - 83 Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen; Hiemori M et al.; One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide) . However, the 23-kDa peptide has never been characterized . In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean . Several proteins were detected by immunoblotting with the mAb . All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor . Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons . Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean . The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety . This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen. Biochim Biophys Acta, 2004 Nov 18, 1675 1-3, 155 - 64 Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity; Pevzner IB et al.; Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant . Earlier we have cloned the full-length gene of MLIII precursor {A.G . Tonevitsky, I.I . Agapov, I.B . Pevzner, N.V . Maluchenko, M.M . Mojsenovich, U . Pfueller, M.P . Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press} . Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit . We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit . The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively . The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity . The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence . The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed. Vet Res, 2004 Nov-Dec, 35(6), 651 - 9 Serum amyloid A and TNF alpha in serum and milk during experimental endotoxin mastitis; Lehtolainen T et al.; A cross-over study was conducted to investigate the effect of intramammarily infused lipopolysaccharide (LPS) on the acute phase reaction in early (EL) and in late (LL) lactation . Nine cows received intramammary injections of 100 microg of Escherichia coli 0111:B4 LPS during EL and LL . The severity of each cows systemic and local signs and change in milk appearance were recorded and scored throughout the experiment . Systemic and local signs were found to be more serious in EL cows . Tumor necrosis factor alpha (TNF alpha) was detected in milk but not in serum . Serum amyloid A (SAA) concentrations increased both in serum and in milk . The milk TNF alpha concentrations peaked at 8 h post-challenge (PC) . SAA concentrations started to increase at 8 h PC, and peak concentrations were seen at 32 and 48 h PC in milk and serum, respectively . The milk TNF alpha and SAA seemed to be correlated, being on average higher in EL . Serum SAA concentration was not correlated with milk TNF alpha or SAA, nor with the severity of local or systemic signs, but was correlated with changes in milk appearance. Genome Biol . 2004;5(11):R87 . Epub 2004 Nov 01. Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli; Peter BJ et al.; BACKGROUND: The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli . In turn, supercoiling influences local DNA structure and can affect gene expression . We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure . RESULTS: We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant . Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased . These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells . Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions . CONCLUSIONS: The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome . We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell. Genome Biol . 2004;5(11):R86 . Epub 2004 Oct 27. Spatial patterns of transcriptional activity in the chromosome of Escherichia coli; Jeong KS et al.; BACKGROUND: Although genes on the chromosome are organized in a fixed order, the spatial correlations in transcription have not been systematically evaluated . We used a combination of genomic and signal processing techniques to investigate the properties of transcription in the genome of Escherichia coli K12 as a function of the position of genes on the chromosome . RESULTS: Spectral analysis of transcriptional series revealed the existence of statistically significant patterns in the spatial series of transcriptional activity . These patterns could be classified into three categories: short-range, of up to 16 kilobases (kb); medium-range, over 100-125 kb; and long-range, over 600-800 kb . We show that the significant similarities in gene activities extend beyond the length of an operon and that local patterns of coexpression are dependent on DNA supercoiling . Unlike short-range patterns, the formation of medium and long-range transcriptional patterns does not strictly depend on the level of DNA supercoiling . The long-range patterns appear to correlate with the patterns of distribution of DNA gyrase on the bacterial chromosome . CONCLUSIONS: Localization of structural components in the transcriptional signal revealed an asymmetry in the distribution of transcriptional patterns along the bacterial chromosome . The demonstration that spatial patterns of transcription could be modulated pharmacologically and genetically, along with the identification of molecular correlates of transcriptional patterns, offer for the first time strong evidence of physiologically determined higher-order organization of transcription in the bacterial chromosome. J Am Chem Soc, 2004 Nov 17, 126(45), 14879 - 89 Catalytic mechanism of yeast cytosine deaminase: an ONIOM computational study; Sklenak S et al.; The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method . Cytosine deamination proceeds via a sequential mechanism involving the protonation of N(3), the nucleophilic attack of C(4) by the zinc-coordinated hydroxide, and the cleavage of the C(4)-N(4) bond . The last step is the rate determining step for the generation of the zinc bound uracil . Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C(4)-O(Zn) cleavage, and the regeneration of the Zn-coordinated water . The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction. Nucleic Acids Res, 2004 Nov 08, 32(19), 5962 - 71 Print 2004. Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia; Pollard LM et al.; Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood . Replication of (GAA*TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability . There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template . DNA replication was also significantly slower in this orientation . To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets) . Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions . The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment . Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions . Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication. Nucleic Acids Res, 2004 Nov 08, 32(19), 5935 - 44 Print 2004. DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus; Ceci P et al.; Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress . The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively . Gel mobility and atomic force microscopy imaging showed that at pH 6.3, both wild type and DpsDelta8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes . Conversely, DpsDelta18 does not self-aggregate and binds DNA without causing condensation . At pH 8.2, DpsDelta8 and DpsDelta18 neither self-aggregate nor cause DNA condensation, a behavior also displayed by wild-type Dps at pH 8.7 . Thus, Dps self-aggregation and Dps-driven DNA condensation are parallel phenomena that reflect the properties of the N-terminus . DNA protection against the toxic action of Fe(II) and H2O2 is not affected by the N-terminal deletions either in vitro or in vivo, in accordance with the different structural basis of this property. Nucleic Acids Res . 2004 Nov 08;32(19):e155. A method for cloning and sequencing long palindromic DNA junctions; Rattray AJ; DNA sequences containing long adjacent inverted repeats (palindromes) are inherently unstable and are associated with many types of chromosomal rearrangements . The instability associated with palindromic sequences also creates difficulties in their molecular analysis: long palindromes (>250 bp/arm) are highly unstable in Escherichia coli, and cannot be directly PCR amplified or sequenced due to their propensity to form intra-strand hairpins . Here, we show that DNA molecules containing long palindromes (>900 bp/arm) can be transformed and stably maintained in Saccharomyces cerevisiae cells lacking a functional SAE2 gene . Treatment of the palindrome-containing DNA with sodium bisulfite at high temperature results in deamination of cytosine, converting it to uracil and thus reducing the propensity to form intra-strand hairpins . The bisulfite-treated DNA can then be PCR amplified, cloned and sequenced, allowing determination of the nucleotide sequence of the junctions . Our data demonstrates that long palindromes with either no spacer (perfect) or a 2 bp spacer can be stably maintained, recovered and