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| J Biol Chem, 2001 Dec 14, 276(50), 46770 - 8 Epub 2001 Sep 27. Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase; Shimada N et al.; Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration . We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure . Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor . However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms . mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases. J Biol Chem, 2001 Nov 30, 276(48), 44481 - 7 Epub 2001 Sep 27. Mimic of photocycle by a protein folding reaction in photoactive yellow protein; Lee BC et al.; The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore . Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP . This implies a direct link between transient protein unfolding and photosensory signal transduction . We utilize this link to study general issues in protein folding . Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear . We propose transition state movement to explain this phenomenon . The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore . This provides a clear example of kinetic control in a protein unfolding reaction . These results demonstrate the power of PYP as a light-triggered model system to study protein folding. FEBS Lett, 2001 Sep 21, 505(3), 426 - 30 Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site; Kramer RA et al.; Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser(99) and His(212) as active site residues . The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed . Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants . Our results support the involvement of a nucleophilic water molecule that is activated by the Asp(210)/His(212) catalytic dyad . Activity is also strongly dependent on Asp(83) and Asp(85) . Both may function in binding of the water molecule and/or oxyanion stabilization . The proposed mechanism implies a novel proteolytic catalytic site. Plant J, 2001 Sep, 27(5), 373 - 82 Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranes; Tan BC et al.; Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges . ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway . Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E . coli . An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids . Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain . The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes . Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence . A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting . Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane . The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea . Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane. J Pept Res, 2001 Sep, 58(3), 221 - 8 Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants; Duenas-Carrera S et al.; Two variants of the hepatitis C virus (HCV) E2 envelope protein, lacking the C-terminal domain and comprising amino acids 458-650 (E2A) and 382-605 (E2C), respectively, were efficiently produced in BL21 (DE3) Escherichia coli cells . E2A and E2C were used to immunize mice . The E2C variant induced the maximal mean antibody titer . Anti-E2C mouse sera reacted mainly with E2 synthetic peptides covering the 70 amino acid N-terminal region of the E2 protein . Moreover, a panel of anti-HCV positive human sera recognized only the E2C protein (28.2%) and the synthetic peptide covering the HVR-1 of the E2 protein (23.1%) . These data indicate the existence of an immunologically relevant region in the HVR-1 of the HCV E2 protein. J Mol Biol, 2001 Sep 28, 312(4), 833 - 47 Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein; Greenfield NJ et al.; Tropomyosin is an alpha-helical coiled-coil protein that aligns head-to-tail along the length of the actin filament and regulates its function . The solution structure of the functionally important N terminus of a short 247-residue non-muscle tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short alpha-tropomyosin and the last 18 residues of the GCN4 leucine zipper . A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli . Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances . Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H(N)-H(alpha)) scalar coupling were analyzed using AutoStructure . This is the first application of this new program to determine the three-dimensional structure of a symmetric homodimer and a structure not previously reported . Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical . Heteronuclear (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible . The (13)C' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield . Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha-helical backbone and side-chains at the coiled-coil interface broadened or shifted with T(M) values approximately 20 degrees C lower than the loss of alpha-helix measured by circular dichroism, suggesting the presence of a folding intermediate . The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations . The conformational flexibility of the N termini of short tropomyosins may be important for their binding specificity . J Mol Biol, 2001 Sep 28, 312(4), 625 - 35 Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli; Chen S et al.; Lrp is a global regulatory protein in Escherichia coli that activates expression of more than a dozen operons and represses expression of another dozen . For some operons, exogenous leucine reduces the extent of Lrp action, for others it potentiates the effect of Lrp, and for yet other operons it has no effect . In an effort to understand how leucine affects Lrp-mediated expression, we examined Lrp self-association and the effect of leucine on self-association using light scattering, chemical cross-linking, and analytical ultracentrifugation . The following results were obtained . (i) Lrp self-associates to a hexadecamer and octamer with the predominant species being hexadecamer at microM concentrations . (ii) Lrp undergoes a leucine-induced dissociation of hexadecamer to octamer . (iii) A mutant Lrp lacking 11 amino acid residues at the C terminus does not form higher-order oligomers, suggesting that the C terminus is involved in subunit association . (iv) At nM concentrations, Lrp dissociates to a dimer . It is proposed that leucine regulates the equilibrium between Lrp oligomers and thus Lrp occupancy of sites within different operons, leading to diverse regulatory patterns . Plant Mol Biol, 2001 Aug, 46(6), 705 - 15 Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex; Drea SC et al.; We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species . Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal . Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity . PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma . In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A . thaliana . Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum . GUS expression was observed in seeds, flowers, root tips and young leaves . GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves . Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence . Database searches on ptlpd2 sequences identified a second A . thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P . sativum. Plant Mol Biol, 2001 Aug, 46(6), 651 - 60 cDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site; Lee Y et al.; The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector . ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively . The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast targeting proteins . The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea and Arabidopsis thaliana . However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported . Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a . Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity . In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity . The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis . When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly . It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site. J Clin Microbiol, 2001 Oct, 39(10), 3712 - 7 PCR for specific detection of H7 flagellar variant of fliC among extraintestinal pathogenic Escherichia coli; Johnson JR et al.; A newly developed PCR-based assay for the H7 variant of the Escherichia coli flagellin gene, fliC, was 100% sensitive and specific in comparison with serology and probe hybridization . It revealed broad conservation of the H7 fliC variant among phylogenetically diverse lineages of extraintestinal pathogenic E . coli (ExPEC) and superseded serotyping for certain isolates with ambiguous or non-H7 serotyping results . The H7 primers functioned well when incorporated into a multiplex PCR assay for diverse virulence-associated genes of ExPEC. J Biol Chem, 2001 Dec 7, 276(49), 45694 - 703 Epub 2001 Sep 26. A novel carbamoyl-phosphate synthetase from Aquifex aeolicus; Ahuja A et al.; Aquifex aeolicus, an extreme hyperthermophile, has neither a full-length carbamoyl-phosphate synthetase (CPSase) resembling the enzyme found in all mesophilic organisms nor a carbamate kinase-like CPSase such as those present in several hyperthermophilic archaea . However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains . The glutaminase, CPS.A, and CPS.B homologs from A . aeolicus were cloned, overexpressed in Escherichia coli, and purified to homogeneity . The isolated proteins could catalyze several partial reactions but not the overall synthesis of carbamoyl phosphate . However, a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia . The inclusion of the glutaminase subunit resulted in the formation of a 171-kDa complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised . Molecular modeling, using E . coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that all of the residues known to be essential for function are conserved and properly positioned . A steady state kinetic study at 78 degrees C indicated that although the substrate affinity was similar for bicarbonate, ammonia, and glutamine, the K(m) for ATP was appreciably higher than that of any known CPSase . The A . aeolicus complex, with a split gene encoding the major synthetase domains and relatively inefficient coupling of amidotransferase and synthetase functions, may be more closely related to the ancestral precursor of contemporary mesophilic CPSases. EMBO J, 2001 Oct 1, 20(19), 5521 - 31 Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase; Guarne A et al.; Human MutLalpha, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair . Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer . We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPgammaS and ADP at 1.95, 2.7 and 2.7 A resolution, respectively . The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein-protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP . Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding . NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLalpha, and vice versa . The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLalpha. EMBO J, 2001 Oct 1, 20(19), 5503 - 12 Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA; Diges CM et al.; Escherichia coli DbpA is a member of the DEAD/H family of proteins which has been shown to have robust ATPase activity only in the presence of a specific region of 23S rRNA . A series of bimolecular RNA substrates were designed based on this activating region of rRNA and used to demonstrate that DbpA is also a non-processive, sequence-specific RNA helicase . The high affinity of DbpA for the RNA substrates allowed both single and multiple turnover helicase assays to be performed . Helicase activity of DbpA is dependent on the presence of ATP or dATP, the sequence of the loop of hairpin 92 of 23S rRNA and the position of the substrate helix with respect to hairpin 92 . This work indicates that certain RNA helicases require particular RNA structures in order for optimal unwinding activity to be observed. EMBO J, 2001 Oct 1, 20(19), 5392 - 9 Repression of deoP2 in Escherichia coli by CytR: conversion of a transcription activator into a repressor; Shin M et al.; In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes . The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components . We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site . Interestingly, we also found that deoP2 carries an "UP-element" immediately upstream of the downstream cAMP-CRP site . The UP-element overlaps with the DNA site for CytR . However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the alpha-subunit as well as with intact RNA polymerase . The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of alpha, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2. Vet Microbiol, 2001 Nov 26, 83(3), 275 - 86 Organisation and in vitro expression of esp genes of the LEE (locus of enterocyte effacement) of bovine enteropathogenic and enterohemorrhagic Escherichia coli; Goffaux F et al.; Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of attaching and effacing (AE) lesions on intestinal epithelial cells . Secretion of extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the AE lesions by human EPEC . In this study, we show that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system . Bovine EPEC and EHEC strains present two different secretion profiles of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR . We also demonstrate that genes encoding secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69. J Biochem (Tokyo), 2001 Oct, 130(4), 527 - 33 Functions and ATP-binding responses of the twelve histidine residues in the TF1-ATPase beta subunit; Tozawa K et al.; The C2 proton signals of all (twelve) histidine residues of the TF1 beta subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experiments and site-directed substitution of histidines by glutamines . pH and ligand titration experiments were carried out for these signals . Furthermore, the ATPase activity of the reconstituted alpha3beta3gamma complex was examined for the twelve mutant beta subunits . Two of three conserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity . The former fixes the N-terminal domain to the central domain . His-324 is involved in the formation of the interface essential for the alpha3beta3gamma complex assembly . The other conserved residue, His-363, showed a very low pK(a), suggesting that it is involved in the tertiary structure formation . On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted . These histidines are located in the hinge region, and its proximity, of the beta subunit . This observation provided further support for the conformational change of the beta monomer from the open to the closed form on the binding of a nucleotide proposed by us {Yagi et al . (1999) Biophys . J . 77, 2175-2183} . This conformational change should be one of the essential driving forces in the rotation of the alpha3beta3gamma complex. J Biochem (Tokyo), 2001 Oct, 130(4), 471 - 4 Accelerated refolding of subtilisin BPN' by tertiary-structure-forming mutants of its propeptide; Kojima S et al.; The propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease . We previously showed that the successive amino acid replacements Ala47-->Phe, Gly13-->Ile, and Val65-->Ile in the propeptide to increase its hydrophobicity resulted in formation of a tertiary structure, accompanied by increased ability to bind to the protease and increased resistance to proteolysis . In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide . The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221-->Ala mutation in the catalytic residue . Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluorescence . The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 x 10(-3) s(-1) in the equation describing an intramolecular process . The Ala47-->Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding . When the additional replacement Gly13-->Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential process composed of a fast phase with a rate constant of 2.1 x 10(-2) s(-1) and a slow phase with a rate constant of 4.5 x 10(-3) s(-1) . The rate constant of the fast phase was increased slightly by a further replacement, Val65-->Ile . Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'. Genome Biol . 2001;2(9):RESEARCH0035 . Epub 2001 Aug 20. A functional update of the Escherichia coli K-12 genome; Serres MH et al.; BACKGROUND: Since the genome of Escherichia coli K-12 was initially annotated in 1997, additional functional information based on biological characterization and functions of sequence-similar proteins has become available . On the basis of this new information, an updated version of the annotated chromosome has been generated . RESULTS: The E . coli K-12 chromosome is currently represented by 4,401 genes encoding 116 RNAs and 4,285 proteins . The boundaries of the genes identified in the GenBank Accession U00096 were used . Some protein-coding sequences are compound and encode multimodular proteins . The coding sequences (CDSs) are represented by modules (protein elements of at least 100 amino acids with biological activity and independent evolutionary history) . There are 4,616 identified modules in the 4,285 proteins . Of these, 48.9% have been characterized, 29.5% have an imputed function, 2.1% have a phenotype and 19.5% have no function assignment . Only 7% of the modules appear unique to E . coli, and this number is expected to be reduced as more genome data becomes available . The imputed functions were assigned on the basis of manual evaluation of functions predicted by BLAST and DARWIN analyses and by the MAGPIE genome annotation system . CONCLUSIONS: Much knowledge has been gained about functions encoded by the E . coli K-12 genome since the 1997 annotation was published . The data presented here should be useful for analysis of E . coli gene products as well as gene products encoded by other genomes. Exp Neurol, 2001 Oct, 171(2), 329 - 41 Hypothalamic pituitary adrenal axis and hypothalamic-neurohypophyseal responsiveness in water-deprived rats; Grinevich V et al.; The differential effects of osmotic stimulation on magnocellular and parvocellular hypothalamic neurons were studied by analysis of corticotropin-releasing hormone (CRH) and vasopressin (VP) expression in controls and 48-h water-deprived rats subjected to either restraint for 1 h or a single lipopolysaccharide injection (250 microg/100 g) . Water deprivation reduced basal CRH mRNA levels but the increments following 4 h of restraint or 6 h lipopolysaccharide (LPS) injection were similar to those in controls . In contrast, water deprivation had no effect on basal VP heteronuclear RNA (hnRNA) and mRNA levels in parvocellular neurons, but responses to restraint or LPS injection were reduced . VP expression in magnocellular paraventricular and supraoptic nuclei, and plasma sodium and vasopressin were higher in water-deprived rats, changes which were unaffected by restraint . LPS injection reduced VP mRNA but not hnRNA levels in magnocellular neurons and increased plasma vasopressin levels only in water-deprived rats independently of changes in plasma sodium . This was accompanied by an increase in vasopressin mRNA content in the posterior pituitary . The data show that the blunted ACTH responses to acute stress during chronic osmotic stimulation are correlated with the inability of parvocellular neurons to increase VP rather than CRH expression . In addition, LPS-induced endotoxemia causes disturbances of the magnocellular vasopressinergic system with an unexpected potentiation of osmotic simulated VP secretion . The lack of increase in VP transcription after LPS and changes in VP mRNA distribution suggest that endotoxemia affect the secretory process at the levels of the neurohypophyseal axon terminal . J Clin Laser Med Surg, 2000 Aug, 18(4), 209 - 13 Newly induced beta-galactosidase molecules have a higher activity than the basally expressed enzyme; Craig DB et al.; OBJECTIVE: The objective of this study was to compare the activities of individual molecules of induced and basally expressed Escherichia coli beta-galactosidase . BACKGROUND DATA: Single-molecule assays of enzymes have determined that individual molecules are not identical . They differ with respect to catalytic rate . The structural cause and cellular role of this microheterogeneity is as yet unknown . METHODS: E . coli were grown and induced to produce beta-galactosidase by treatment with isopropyl-beta-D-thiogalactopyranoside . Cells were lysed and the beta-galactosidase assayed with capillary electrophoresis instrumentation utilizing post-column, laser-induced fluorescence detection . The enzyme obtained from treated cells were compared to that from untreated cells . RESULTS: The activity of newly induced beta-galactosidase was found to be approximately 20% greater than that of the basally expressed enzyme . This measured difference is statistically significant . CONCLUSIONS: Production of beta-galactosidase in E . coli under differing conditions results in differences in the activities of the individual enzyme molecules. Biochem Soc Symp, 2001, (68), 69 - 82 Defining the structure of the substrate-free state of the DnaK molecular chaperone; Swain JF et al.; Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell . Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism . Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also . At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs . At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time . We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH . The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well . In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain. Nat Struct Biol, 2001 Oct, 8(10), 879 - 82 Quantitative protein stability measurement in vivo; Ghaemmaghami S et al.; The equilibrium between the native and denatured states of a protein can be key to its function and regulation . Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers . However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability . Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX) . The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro . However, when the E . coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced . The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights. Nat Struct Biol, 2001 Oct, 8(10), 858 - 63 UDP-galactopyranose mutase has a novel structure and mechanism; Sanders DA et al.; Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi . UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase) . The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target . The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear . We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution . The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues . Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD . Assay results establish that the enzyme is active only when flavin is reduced . We conclude that mutase most likely functions by transient reduction of substrate. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11388 - 93 Contribution of individual random mutations to genotype-by-environment interactions in Escherichia coli; Remold SK et al.; Numerous studies have shown genotype-by-environment (GxE) interactions for traits related to organismal fitness . However, the genetic architecture of the interaction is usually unknown because these studies used genotypes that differ from one another by many unknown mutations . These mutations were also present as standing variation in populations and hence had been subject to prior selection . Based on such studies, it is therefore impossible to say what fraction of new, random mutations contributes to GxE interactions . In this study, we measured the fitness in four environments of 26 genotypes of Escherichia coli, each containing a single random insertion mutation . Fitness was measured relative to their common progenitor, which had evolved on glucose at 37 degrees C for the preceding 10,000 generations . The four assay environments differed in limiting resource and temperature (glucose, 28 degrees C; maltose, 28 degrees C; glucose, 37 degrees C; and maltose, 37 degrees C) . A highly significant interaction between mutation and resource was found . In contrast, there was no interaction involving temperature . The resource interaction reflected much higher among mutation variation for fitness in maltose than in glucose . At least 11 mutations (42%) contributed to this GxE interaction through their differential fitness effects across resources . Beneficial mutations are generally thought to be rare but, surprisingly, at least three mutations (12%) significantly improved fitness in maltose, a resource novel to the progenitor . More generally, our findings demonstrate that GxE interactions can be quite common, even for genotypes that differ by only one mutation and in environments differing by only a single factor. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11259 - 64 Disordered to ordered folding in the regulation of diphtheria toxin repressor activity; Twigg PD et al.; Understanding how metal binding regulates the activity of the diphtheria toxin repressor protein (DtxR) requires information about the structure in solution . We have prepared a DtxR mutant construct with three additional N-terminal residues, Gly-Ser-His-DtxR(Cys-102 --> Asp), that retains metal-binding capabilities, but remains monomeric in solution and does not bind DNA under conditions that effect dimerization and DNA binding in the functional DtxR(Cys-102 --> Asp) construct . Although the interaction properties of this inactive mutant in solution are very different from that of active repressors, crystallization imposes the same dimeric structure as observed in all crystal forms of the active repressor with and without bound metal . Our solution NMR analyses of active and inactive metal-free diphtheria toxin repressors demonstrate that whereas the C-terminal one-third of the protein is well ordered, the N-terminal two-thirds exhibits conformational flexibility and exists as an ensemble of structural substates with undefined tertiary structure . Fluorescence binding assays with 1-anilino naphthalene-8-sulfonic acid (ANS) confirm that the highly alpha-helical N-terminal two-thirds of the apoprotein is molten globule-like in solution . Binding of divalent metal cations induces a substantial conformational reorganization to a more ordered state, as evidenced by changes in the NMR spectra and ANS binding . The evident disorder to order transition upon binding of metal in solution is in contrast to the minor conformational changes seen comparing apo- and holo-DtxR crystal structures . Disordered to ordered folding appears to be a general mechanism for regulating specific recognition in protein action and this mechanism provides a plausible explanation for how metal binding controls the DtxR repressor activity. J Biol Chem, 2001 Dec 21, 276(51), 48243 - 9 Epub 2001 Sep 25. In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit; Rotem D et al.; EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds . Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer . Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer . To identify the functional unit of EmrE, a novel approach was developed . In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity . Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells . Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits . The hetero-oligomers thus formed display a decreased affinity to substrates . In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer . It is concluded that, in EmrE, the oligomer is the functional unit. J Biol Chem, 2001 Dec 14, 276(50), 47185 - 94 Epub 2001 Sep 25. Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme; Leu FP et al.; The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA . The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA . These proteins are functionally and structurally conserved in all cells . The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp . The delta subunit of the E . coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces . This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta . The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta . The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma . The implications of these actions for the workings of the E . coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed. Cell, 2001 Sep 21, 106(6), 655 - 60 Opening of the clamp: an intimate view of an ATP-driven biological machine; Ellison V et al.; DNA polymerases require tethering to an accessory factor, typically a ring-shaped clamp, to remain bound to DNA during replication . Three recent structural studies provide unique insight into how these clamps are loaded onto DNA by the clamp loader machinery. Fiziol Zh, 2001, 47(4), 19 - 24 {Modulating effects of exogenous histamine, serotonin, and heparin on leukocytic reaction during inflammation}; Klymenko MO et al.; On the model of E . coli-induced acute infectious peritonitis in rats previously depleted on mast cells of peritoneal cavity the replacing effects of various doses of exogenous histamine, serotonin and heparin and their complex on leukocytic reaction of inflammatory focus and blood were studied . In was shown that all of the used substances had a meaning in the mechanisms of modulating effect of mast cells on leukocytes . The analogous on direction to mast cells ones effects on leukocytes were produced mostly by histamine and especially by the complex of substances. Gene Ther, 2001 Sep, 8(17), 1281 - 90 Development of formulations that enhance physical stability of viral vectors for gene therapy; Croyle MA et al.; This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites . We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions . Adenovirus and adeno-associated virus expressing E . coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants . X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations . Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at -20 and 4 degrees C . Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degrees C . Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degrees C . Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degrees C for 1 year after an initial loss of 0.5 log due to processing . Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product . Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization . In summary, lyophilized preparations that can be shipped and stored at 25 degrees C offer a solution to the current problem of distribution of viral vectors for clinical trials. J Biol Chem, 2001 Nov 30, 276(48), 44598 - 603 Epub 2001 Sep 24. Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact; Heyduk E et al.; The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence . To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E . coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization . The alpha dimer and DNA formed a 1:1 complex in solution . Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed . The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero . Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation . No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation . The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed. Appl Environ Microbiol, 2001 Oct, 67(10), 4504 - 11 Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi; Zappa S et al.; This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon . An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli . Analysis of the sequence showed conservation of the active site and structural elements of the E . coli AP . The recombinant AP was purified and characterized . Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa . Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively . Thus far, P . abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively . Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity . The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate . Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P . abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds . In addition, P . abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively. Appl Environ Microbiol, 2001 Oct, 67(10), 4426 - 31 EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R . flavefaciens protein via a dockerin domain; Rincon MT et al.; The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp . bind to cellulose are not fully understood . The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose . The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences . EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain . Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca . 130 kDa present among supernatant proteins from Avicel-grown R . flavefaciens that attach to cellulose . The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y . Ding et al., J . Bacteriol . 183:1945-1953, 2001) . It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex. Biochemistry, 2001 Oct 2, 40(39), 11955 - 64 A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity; Lobo S et al.; Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria . Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII . Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps . The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme . The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases . The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities . Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E . coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM) . In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM) . A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity . No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate . An ACT activity with ACP has not previously been observed for thiolases and in the case of the S . collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)) . The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII. Biochemistry, 2001 Oct 2, 40(39), 11777 - 84 Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides; Lavrik IN et al.; Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system . The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites . Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues . The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome . Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation. Biochemistry, 2001 Oct 2, 40(39), 11734 - 41 Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides . Appendix: thermodynamic interpretation of the activation by concentrated salts; Huang X et al.; Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease . PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions . In addition, PSA does not react readily with prototypical serine protease inactivators . We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid . Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4) . This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-) . The nature of the cation has little effect on salt activation . The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions . Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations . These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA . CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations . Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions . Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo. Protein Expr Purif, 2001 Oct, 23(1), 207 - 17 Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp . PCC 7120; Desplancq D et al.; Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes . While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp . PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources . We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp . PCC 7120 . The 24-kDa N-terminal domain of the E . coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter . The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide . The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E . coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium . Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E . coli sample, demonstrating that it was of sufficient quality for 3D-structure determination . Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp . PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies . Protein Expr Purif, 2001 Oct, 23(1), 191 - 7 Purification of a model substrate for transcription factor phosphorylation by ERK2; Waas WF et al.; ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects on proliferation and differentiation by growth factors and hormones . While its cellular function has been under intense scrutiny since its initial discovery nearly 15 years ago, little progress has been made in understanding its kinetic mechanism . Such an understanding is important for the development of potent and specific inhibitors . A contributory factor has been the lack of a protein substrate suitable for rigorous mechanistic studies . Here we report the expression, purification, and characterization of the N-terminus (residues 1 through 138) of the transcription factor Ets-1, an excellent model substrate for ERK2 mechanistic studies . (His(6)-tagged)Ets-1(1-138) was expressed in Escherichia coli and rapidly purified in two steps by nickel-agarose-affinity chromatography, followed by high-resolution Mono-Q anion-exchange chromatography . A yield of 60 mg of the purified protein per liter of culture was obtained and could be stored conveniently at -80 degrees C in water . Rigorous characterization demonstrated that under the assay conditions, (His(6)-tagged)Ets-1(1-138) is exclusively phosphorylated on residue Thr-38 by ERK2 with the following Michaelis parameters: k(cat) = 17 s(-1), K(ATP)(m) = 140 microM, K(ATP)(i) = 68 microM, K(Ets-1)(m) = 19 microM, and K(Ets-1)(i) = 9.3 microM . Protein Expr Purif, 2001 Oct, 23(1), 134 - 41 Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli; Jonsson CB et al.; Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome . The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA . The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography . We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants . Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L) . The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications . Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography . Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant . The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay . The N protein-vRNA complex had an apparent dissociation constant of 140 nM . Protein Expr Purif, 2001 Oct, 23(1), 106 - 12 Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein; Bonifacio MJ et al.; Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag . The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E . coli, was purified by calmodulin affinity chromatography and gel filtration . Up to 16 mg of pure recombinant enzyme was recovered per liter of culture . Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 {246, 609} microM and 531 {330, 732} microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 {9, 45} microM and 38 {21, 55} microM, respectively) . In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 {44, 397} nM and 74 {38, 143} nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1) . The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 {75, 597} microM and 439 {168, 711} microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 {19, 101} nM and 61 {33, 111} nM, respectively) . This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme . This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies . Protein Expr Purif, 2001 Oct, 23(1), 97 - 105 Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520; Christodoulou E et al.; A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described . Chi40 was overproduced in the cytosolic and secreted forms . The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts . The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis . The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time . The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied . Thermostability measurements showed that Chi40 has a T(m) of 60.7 degrees C at neutral pH . (13)C-(15)N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21trxB(DE3) cells grown in minimal medium in the presence of the paramagnetic elements {(13)C}glucose and (15)NH(4)Cl . The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy . Protein Expr Purif, 2001 Oct, 23(1), 75 - 83 Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag; Rumlova M et al.; The efficiencies of different procedures for purification of the capsid protein (CA) of Mason-Pfizer monkey virus are compared . Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for affinity chromatography purification were prepared . CA was expressed in Escherichia coli (i) as a wild-type protein, (ii) C-terminally extended with a six-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (CA 6aa6His) . Electron microscopy was used for comparison of the resulting proteins, as CA is a structural protein with no enzymatic activity . We have found that these C-terminal fusions dramatically influenced the properties and morphology of structures formed by CA protein in E . coli . The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His proteins formed organized structures . CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form . Both six-histidine-tagged proteins were purified using affinity chromatography under either native (CA 6His) or denaturing (CA 6aa6His) conditions . CA protein was purified under denaturing conditions using gel-filtration chromatography followed by refolding . All proteins were obtained at a purity >98% . Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form organized structures within E . coli . We show here that the widely used histidine anchor may significantly alter the properties of the protein of interest . Protein Expr Purif, 2001 Oct, 23(1), 33 - 7 High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli; Chen XG et al.; Surface antigen 1 (SAG1) of Toxoplasma gondii is a good candidate for diagnosis and vaccine development, but recombinant SAG1 produced in Escheichia coli often loses its specific immunogenicity due to the incorrect folding . In the present study, a truncated SAG1 was highly expressed in E . coli as a fusion protein, about 30% of the total protein of the cell lysate . After a simple purification and refolding procedure, purified rSAG1 can be recognized by human Toxoplasma-infective serum, and ELISA kits constructed by rSAG1 can sensitively and specifically detect toxoplasma infection . Protein Expr Purif, 2001 Oct, 23(1), 14 - 21 Expression and complement d activity of porcine adipsin; Miner JL et al.; To learn how signals from adipocytes might be involved in regulation of energy intake and storage, we have begun to characterize the porcine complement protein, adipsin . Adipsin was originally identified as a protein that is produced rather specifically by adipocytes, is secreted, and is nearly absent in several obese rodent models . We now report that porcine adipsin mRNA sequence is 74% identical to rat and predicts a protein that has 82 and 68% identity to human and rat forms, respectively . Porcine adipsin has none of the asparagine glycosylation consensus sites which make recombinant expression of mouse adipsin in Escherichia coli impractical . We present a method for engineering the porcine cDNA to facilitate expression by E . coli and provide a protocol for refolding and purifying porcine adipsin protein and for immunoassay . We have found that in addition to adipose tissue, adipsin mRNA is present in gut tissues . Coupled with the fact that adipsin is required for processing of complement C3a-desArg, and that C3a-desArg is a potent stimulant of fatty acid acylation in adipocytes, the production of adipsin in the gut may be related to a mechanism for adipocyte removal of lipid from chylomicrons . Protein Expr Purif, 2001 Oct, 23(1), 8 - 13 A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone; Thomson CA et al.; Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions . We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain . Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C . The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol . Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies . Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used . The ability to form a trimer may be important for its role as a chaperone . The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments . Illustrative data on collagen binding by (35)S-Hsp47 are shown . Protein Expr Purif, 2001 Oct, 23(1), 1 - 7 Functionally active VEGF fusion proteins; Backer MV et al.; Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) acting via endothelial cell-specific receptors, such as VEGFR-2, that are overexpressed at the sites of angiogenesis . If VEGF retains activity as a fusion protein with a large N-terminal extension, it would facilitate development of VEGF-based vehicles for receptor-mediated delivery of therapeutic and diagnostic agents to the sites of angiogenesis . We have constructed, expressed in Escherichia coli, and purified VEGF fusion proteins containing a 158-amino acid N-terminal extension fused to human VEGF(121), VEGF(165), and VEGF(189) . We report here that VEGF fusion proteins induce tyrosine autophosphorylation of VEGFR-2 and its downstream targets, as well as cell contraction in cells overexpressing VEGFR-2 . Although N-terminal extensions decrease the affinity of VEGF fusion proteins to VEGFR-2, at saturating concentrations these proteins are as efficient as correct size VEGF(165) . We hypothesize that VEGF fusion proteins may be employed for targeting endothelial cells at the sites of angiogenesis . Am J Med Sci, 2001 Sep, 322(3), 141 - 4 Nonspecific human IgG reduces survival in neonatal rats infected with Escherichia coli; Cole CW et al.; BACKGROUND: Human intravenous IgG (IVIG) containing specific antibodies protects neonatal rats from septic death . However, IVIG has immunosuppressive properties and clinical trials of IVIG in neonates at risk for sepsis have yielded conflicting results . HYPOTHESIS: This study was designed to test the hypothesis that nonspecific antibodies in IVIG reduce survival in neonatal rats infected with Escherichia coli . METHODS: Specific antibodies were adsorbed from IVIG with E . coli to produce IVIG/anti-E . coli- . After transthoracic administration of E . coli, survival was determined in neonatal rats injected intraperitoneally with phosphate-buffered saline, IVIG/anti-E . coli- (500 mg/kg) or IVIG containing anti-E . coli antibodies (IVIG/anti-E . coli+) . Complement-mediated hemolytic activity of neonatal rat serum was quantified using sensitized sheep erythrocytes . RESULTS: Compared with placebo, intraperitoneal IVIG/anti-E . coli- reduced neonatal survival after E . coli infection . In contrast, IVIG/anti-E . coli+ protected infected animals . Both IVIG/anti-E . coli- and IVIG/anti-E . coli+ impaired the complement-mediated hemolytic activity of neonatal rat serum . CONCLUSIONS: IVIG contained (1) nonspecific antibodies that reduced survival in neonatal rats infected with E . coli and (2) protective anti-E . coli antibodies that enhanced survival in neonatal rats infected with E . coli . We speculate that in clinical trials of IVIG to treat or prevent neonatal sepsis, inconsistent results may be caused, in part, by lot-to-lot variations in the ratio of immunosuppressive, nonspecific antibodies to protective, specific antibodies. Curr Genet, 2001 Aug, 40(1), 82 - 90 Group I intron lateral transfer between red and brown algal ribosomal RNA; Bhattacarya D et al.; How group I introns originate in nuclear ribosomal (r)RNA genes is an important question in evolutionary biology . Central to this issue is the multitude of group I introns present in evolutionarily distantly related plant, fungal, and protist lineages, together with an understanding of their origin and lateral transfer from one exon to another, between cell organelles, and between cells . These introns vary considerably in primary and secondary structure; and their provenance from a few or perhaps many mobile elements that have spread in rRNAs is unknown . Here we show that a novel lineage of group IC1 introns inserted at position 516 (Escherichia coli gene numbering) in the small subunit rRNA in bangiophyte red algae and a brown alga (Aureoumbra lagunensis) are specifically related, although their host cells are not . These bangiophyte and Aureoumbra introns are the only known cases that have a helical insertion in the P5b helix . The highly conserved primary and secondary structure of the extra P5b helix suggests that it is important, although its specific function is unknown . Our study attempts to understand the origin and movement of these IC1 introns. Curr Genet, 2001 Aug, 40(1), 2 - 12 Mechanisms involved in metalloid transport and tolerance acquisition; Tamas MJ et al.; Toxic metalloids such as arsenic and antimony have always been an integral part of the natural environment . To survive in such a hostile habitat, it is crucial to develop strategies to exclude toxic substances from the cell and to acquire tolerance . Cells remove metalloids from the cytosol either by active efflux or by sequestration in an internal organelle . Controlling the influx appears to be another way of maintaining a low intracellular metalloid content . Inside the cell, the metalloid can be reduced to a form that is recognised by the expulsion system(s) . In addition, metalloid complexation and compartmentalisation contributes to enhanced cellular tolerance . Finally, the presence of metalloids activates transcription of various cellular defence genes . Metalloid-containing drugs are currently used to treat protozoan infections and promyelocytic leukaemia . Since metalloid resistance hampers efficient treatment, interest in identifying the mechanisms involved in tolerance acquisition has arisen . The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model system to investigate the basis of metalloid tolerance at a molecular level . This review describes the recent progress made in elucidating the mechanisms involved in metalloid transport and tolerance in yeast and other organisms. Biotechniques, 2001 Sep, 31(3), 560, 562, 564 - 8, passim Universal SNP genotyping assay with fluorescence polarization detection; Hsu TM et al.; The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr) . FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr . We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays . In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs) . This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions . Even without optimization, approximately 70% of all SNP markers tested yielded robust assays . The addition of an E . coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90% . Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100% . In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers) . With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays . When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes) . The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design. Biotechniques, 2001 Sep, 31(3), 534, 536, 538 - 40 Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper; Timiryasova TM et al.; Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy . The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses . Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses . In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection . In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome . In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV . The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E . coli beta-galactosidase . An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation. Anal Chem, 2001 Sep 1, 73(17), 4154 - 61 Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry; Hernandez H et al.; Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry . From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed . The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer . The masses of the monomeric species were consistent with the presence of a {2Fe-2S} cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond . Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra . Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer . Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact . Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment . A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+. Avian Dis, 2001 Jul-Sep, 45(3), 670 - 9 Genotypic and phenotypic characterization of potential virulence of intestinal avian Escherichia coli strains isolated in Algeria; Mellata M et al.; In order to characterize potential pathogenic Escherichia coli strains isolated from diarrheic hens and chickens originating from intensive battery rearing in North Algeria, the presence of a large range of virulence factors and markers was studied in 50 strains by DNA-DNA hybridization on colonies and phenotypic tests . The sequences we focused on were those coding for adhesins F5, F41, F17, Pap, Afa, and Sfa; intimin Eae; and toxins STa, STb, LT1, Stx1, Stx2, CNF1, and CNF2 . The phenotypes explored were the colicins, aerobactin, hemolysins, and hemagglutinin production and serum resistance . The genotypic and phenotypic tests enabled us to categorize the isolates into two distinct groups: those with a potential to invade the host (27 strains were serum resistant and/or produced aerobactin), among which three strains were also potentially diarrheagenic, one strain was LT1 + F17+ Afa+ Pap+ (enterotoxigenic E . coli) and the two others were Stx1 (verotoxigenic E . coli) . Twenty-three strains were colicinogenic, including 19 strains producing colicin V . This latter factor was also detected in isolates negative for the other virulence factors . On the basis of the type of erythrocytes agglutinated, we established 14 mannose-resistant hemagglutination patterns among the 37 strains tested, including 22 serum-resistant and/or aerobactin producing strains and 15 strains negative for these two characters . None of the strains produced alpha hemolysin, whereas two strains produced beta hemolysin and enterohemolysin, respectively . Congo red fixation was observed in 25 strains . No relationship could be detected between Congo red fixation and the presence of other virulence markers, such as serum resistance and aerobactin production . This study shows that among isolates originating from the feces of diarrheic chickens, the proportion of potentially diarrheagenic E . coli strains is low. Genes Genet Syst, 2001 Jun, 76(3), 189 - 98 Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression; Yoshioka Y et al.; We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene . Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine . In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities . The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity . We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA . thaliana . AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots . Significant expression of this gene was also observed in trichomes after bolting . Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas . These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner. Am J Obstet Gynecol, 2001 Sep, 185(3), 716 - 24 A high-throughput study of gene expression in preterm labor with a subtractive microarray approach; Muhle RA et al.; OBJECTIVE: We propose that elucidation of the pathophysiology of preterm labor can be achieved with genome-scale analyses of differential gene expression . STUDY DESIGN: CD-1 mice on day 14.5 of a 19- to 20-day gestation were assigned to one of 4 treatment groups modeling different clinical conditions (n = 5 per group): group A, infection with labor (intrauterine injection of 10(10) heat-killed Escherichia coli, which causes delivery within an average of 20 hours); group B, infection without labor (intrauterine injection of 10(7) heat-killed E coli, which leads to normal delivery at term); group C, labor without infection (ovariectomy, which causes delivery within an average of 27 hours); and group D, no infection and no labor (intrauterine injection of vehicle) . Total pooled myometrial RNA was prepared 3.5 hours after surgery for groups A, B, and D and 5 hours after surgery for group C . The relative expression of 4963 genes was assayed in these pools by using DNA microarrays . Transcripts specifically involved in infection-induced labor were identified by subtracting from the list of differentially regulated genes in group A those with common expression in groups B and C . RESULTS: In group A 68 differentially expressed transcripts (>or=2-fold upregulation or downregulation) were identified . Among these are 39 characterized genes . Fourteen (45%) are involved in inflammatory responses, 7 (18%) are involved in growth-differentiation-oncogenesis, and 3 (8%) are involved in apoptosis . Subtraction identified 13 gene products most likely to be important for bacterially induced labor, as opposed to labor without infection or bacterial exposure without labor . CONCLUSION: This study demonstrates the potential of the subtractive DNA microarray technique to identify transcripts important specifically for bacterially induced preterm labor. J Biosci, 2001 Sep, 26(3), 315 - 24 Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis); Anathy V et al.; A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated . Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence . The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids . The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively . The predicted amino acid sequence of H . fossils GH shared 98% homology with other catfishes . Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors . The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone. Biofactors, 2001, 14(1-4), 179 - 90 Cloning of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as an anti-apoptotic and growth promoting gene of Burkitt lymphoma cells; Brielmeier M et al.; Burkitt Lymphoma (BL) cells are highly sensitive to suboptimal growth conditions and undergo apoptosis when seeded at low cell density or reduced serum concentration . Irradiated fibroblasts or a mix of pruvate, alpha-thioglycerol, and bathocuproine disulfonate can protect BL cells from apoptosis induced by lowering cell density or serum concentration by promoting cystine uptake in the cells . The availability of cystine is the limiting factor for glutathione biosynthesis in BL cells and thus for the ability of the cells to cope with oxidative stress . We have set up an expression cloning strategy to clone genes that protect BL cells from apoptosis induced by low cell density and/or serum . Using this approach we have cloned among others the cDNA for Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx). Biofactors, 2001, 14(1-4), 69 - 74 Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis; Lacourciere GM et al.; Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP . Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic . Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo . The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide . Recently, three E . coli NifS-like proteins, CsdB, CSD, and IscS, were characterized . All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively . Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis. Biofactors, 2001, 14(1-4), 61 - 8 An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure; Engelberg-Kulka H et al.; The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading . Interesting alternative relates to the function of the UGA codon . Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide . UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure . Here we discuss our investigation on the E . coli SECIS . This includes the follows: 1) The nature of the minimal E . coli SECIS . We found that in E . coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA {34}; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation {34} because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency {35} . This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it . These two regions act independently and additively and probably through different mechanisms . The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes . These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites . Finally, we present a model for the E . coli fdhF SECIS as a multifunctional RNA structure containing three functional elements . Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain. J Biol Chem, 2001 Dec 14, 276(50), 47285 - 90 Epub 2001 Sep 21. Structure of crystalline D-Tyr-tRNA(Tyr) deacylase . A representative of a new class of tRNA-dependent hydrolases; Ferri-Fioni ML et al.; Cell growth inhibition by several d-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules . Escherichia coli and yeast cells express an enzyme, d-Tyr-tRNA(Tyr) deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA and d-amino acid . Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases . Orthologs of the deacylase are found in many cells . In this study, the crystallographic structure of dimeric E . coli d-Tyr-tRNA(Tyr) deacylase at 1.55 A resolution is reported . The structure corresponds to a beta-barrel closed on one side by a beta-sheet lid . This barrel results from the assembly of the two subunits . Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the beta-strands . The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases. J Appl Physiol, 2001 Oct, 91(4), 1701 - 7 Normovolemic hemodilution improves oxygen extraction capabilities in endotoxic shock; Creteur J et al.; We studied the effects of normovolemic hemodilution on tissue oxygen extraction capabilities in a canine model of endotoxic shock . Eighteen anesthetized and mechanically ventilated dogs underwent normovolemic hemodilution with 6% hydroxyethyl starch solution to reach hematocrit (Hct) levels around 40, 30, or 20% before the administration of 2 mg/kg of Escherichia coli endotoxin . Cardiac tamponade was then induced by repeated injections of normal saline into the pericardial sac to reduce cardiac output and study whole body oxygen extraction capabilities . Whole body critical oxygen delivery was lower in the Hct 20% and 30% groups (8.4 +/- 0.4 and 10.4 +/- 0.7 ml . kg(-1) . min(-1), respectively) than in the Hct 40% group (12.8 +/- 0.8 ml . kg(-1) . min(-1)) (both P < 0.005) . The whole body critical oxygen extraction ratio was higher in the Hct 30% and 20% groups (49.1 +/- 8.2 and 55.2 +/- 4.6%, respectively) than in the Hct 40% group (37.1 +/- 4.4 %) (both P < 0.05) . Liver critical oxygen extraction ratio was also higher in the Hct 30% and 20% groups than in the Hct 40% group . The arterial lactate concentrations and the gradient between ileum mucosal PCO(2) and arterial PCO(2) were lower in the Hct 20% and 30% groups than in the Hct 40% group . We conclude that, during an acute reduction in blood flow during endotoxic shock in dogs, normovolemic hemodilution is associated with improved tissue perfusion and increased oxygen extraction capabilities. Comp Biochem Physiol B Biochem Mol Biol, 2001 Oct, 130(3), 299 - 312 Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2; Weebadda WK et al.; Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus) . We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E . coli O2 . Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E . coli O2 isolate . A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose . An analogous protein in air sac fluid proteins bound to intact E . coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc) . The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients . However, endogenous amino acid sequences were unrelated to other known proteins . LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins . These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E . coli responsible for respiratory disease. Vaccine, 2001 Oct 12, 20(1-2), 115 - 20 Improved vectors for expression library immunization--application to Mycoplasma hyopneumoniae infection in pigs; Moore RJ et al.; Expression library immunization (ELI) has previously been used in a number of disease models in mice . Here, we describe the first example of the application of ELI to a large animal model with the immunization of pigs against enzootic pneumonia, a disease caused by Mycoplasma hyopneumoniae . The development of new plasmid vectors and library screening methods facilitated the application of ELI to this disease by allowing random libraries to be screened for clones expressing recombinant proteins . In this way the vast majority of clones in random libraries that are unproductive can be eliminated, meaning that libraries are more likely to give protection and are subsequently easier to further screen and analyze . By using this approach we have used one library screen and two animal trials to progress from an original library of 20,000 clones to a group of just 96 clones. Vaccine, 2001 Oct 12, 20(1-2), 12 - 5 Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response; Morris CB et al.; Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified . In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes . Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses . This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine. Acta Crystallogr D Biol Crystallogr, 2001 Oct, 57(Pt 10), 1397 - 404 Epub 2001 Sep 21. NMR trial models: experiences with the colicin immunity protein Im7 and the p85alpha C-terminal SH2-peptide complex; Pauptit RA et al.; Two cases of successful molecular replacement using NMR trial models are presented . One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a specific PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85alpha subunit of human phosphatidylinositol 3-OH kinase . In both cases, molecular replacement was non-trivial . Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more flexible portions had been excised . Use of maximum-likelihood refinement proved critical to be able to refine the poor starting models . The challenges typical of the use of NMR trial models in molecular replacement are discussed. Protein Sci, 2001 Oct, 10(10), 2131 - 7 A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data; Morelli XJ et al.; We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy . This method, termed "restrained soft-docking," is validated for several known protein complexes . These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources . The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed. Protein Sci, 2001 Oct, 10(10), 2114 - 22 Origin of fibronectin type II (FN2) modules: structural analyses of distantly-related members of the kringle family idey the kringle domain of neurotrypsin as a potential link between FN2 domains and kringles; Ozhogina OA et al.; Analysis of complete genome sequences has made it clear that fibronectin type II (FN2) modules are present only in the vertebrate lineage, raising intriguing questions about the origin of this module type . Kringle domains display many similarities to FN2 domains; therefore it was suggested previously that they are highly divergent descendants of the same ancestral protein-fold . Since kringles are present in arthropodes, nematodes, and invertebrate chordates as well as in vertebrates, it is suggested that the FN2 domain arose in the vertebrate lineage through major structural modification of the more ancestral kringle fold . To explore this structural transition, in the present work we compare key structural features of two highly divergent kringle domains (the kringle of Caenorhabditis elegans Ror receptor tyrosine kinase and the kringle of rat neurotrypsin) with those of plasminogen kringles and FN2 domains . Our NMR conformation fingerprinting analysis indicates that characteristic (1)H-NMR markers of kringle or FN2 native folding, such as the dispersion of Trp aromatic connectivities and shifts of the Leu(46)/Thr(16) methyl signals, both decrease in the order kringles > neurotrypsin kringle > FN2 domains . These results suggest that the neurotrypsin kringle may represent an intermediate form between typical kringles and FN2 domains. Protein Sci, 2001 Oct, 10(10), 1980 - 8 Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription; Schubot FD et al.; Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined . In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution . The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices . Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC' . This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter . Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site. Protein Sci, 2001 Oct, 10(10), 1962 - 9 Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: function of the Asn-His interaction in the catalytic triad; Snijder HJ et al.; Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad . In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate . Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant . Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA . The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain . Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant. J Biol Chem, 2001 Dec 7, 276(49), 46319 - 25 Epub 2001 Sep 20. Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli; Frueauf JB et al.; Structural prediction of several bacterial and plant ADP-glucose pyrophosphorylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized pyrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and Bourne, Y . (1999) EMBO J . 18, 4096-4107; Blankenfeldt, W., Asuncion, M., Lam, J . S., and Naismith, J . H . (2000) EMBO J . 19, 6652-6663) . This comparison led to the discovery of highly conserved residues throughout the superfamily of pyrophosphorylases despite the low overall homology . One of those residues, Asp(142) in the ADP-glucose pyrophosphorylase from Escherichia coli, was predicted to be near the substrate site . To elucidate the function that Asp(142) might play in the E . coli ADP-glucose pyrophosphorylase, aspartate was replaced by alanine, asparagine, or glutamate using site-directed mutagenesis . Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specific activity of up to 4 orders of magnitude . Comparison of other kinetic parameters, i.e . the apparent affinities for substrates and allosteric effectors, showed no significant changes, excluding this residue from the specific role of ligand binding . Only the D142E mutant exhibited altered K(m) values but none as pronounced as the decrease in specific activity . These results show that residue Asp(142) is important in the catalysis of the ADP-glucose pyrophosphorylase from E . coli. J Biol Chem, 2001 Nov 30, 276(48), 44590 - 7 Epub 2001 Sep 20. B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase; Sarin J et al.; The B-subunit of phosphate-specific transporter (PstB) is an ABC protein . pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli . The overexpressed protein was found to be in inclusion bodies . The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography . The molecular mass of the protein was approximately 31 kDa . The eluted protein showed ATP-binding ability and exhibited ATPase activity . Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M . tuberculosis PstB-ATPase . The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein . Divalent cation like manganese was inhibitory to the ATPase activity . Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme . The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm . Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase . Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria. J Bacteriol, 2001 Oct, 183(20), 6144 - 7 Escherichia coli minicell membranes are enriched in cardiolipin; Koppelman CM et al.; The phospholipid composition of Escherichia coli minicells has been studied as a model for the cell division site . Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol . Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells. J Bacteriol, 2001 Oct, 183(20), 6140 - 3 Novel motility mutants of synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis; Bhaya D et al.; We screened for transposon-generated mutants of Synechocystis sp . strain PCC 6803 that exhibited aberrant phototactic movement . Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes . These novel genes and their possible roles in motility are discussed. J Bacteriol, 2001 Oct, 183(20), 6126 - 34 Growth phase and growth rate regulation of the rapA gene, encoding the RNA polymerase-associated protein RapA in Escherichia coli; Cabrera JE et al.; The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family . We have studied the rapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro . We have found that the expression of rapA is growth phase dependent, peaking at the early log phase . The growth phase control of rapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E . coli promoters . We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro . Furthermore, we have shown that a GC-rich or discriminator sequence between the -10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP. J Bacteriol, 2001 Oct, 183(20), 6095 - 106 Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant; Bylund GO et al.; The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes . A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits . The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA . Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA . Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene . Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription . The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA . Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators . All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains . The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C . Here, we show that nusA is also essential at 37 degrees C. J Bacteriol, 2001 Oct, 183(20), 6065 - 73 RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus; Maisnier-Patin S et al.; The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations . Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival . Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability . The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains . In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus . This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites . Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways. J Bacteriol, 2001 Oct, 183(20), 6017 - 27 Regulatory interactions of Csr components: the RNA binding protein CsrA activates csrB transcription in Escherichia coli; Gudapaty S et al.; The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions . Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively . CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity . We have further examined the regulatory interactions of CsrA and CsrB RNA . The 5' end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed . CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains . CsrA levels exhibited modest or negligible effects of these mutations . The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA . In contrast, CsrB levels were drastically decreased (~10-fold) in the csrA mutants . CsrB transcript stability was unaffected by csrA . The expression of a csrB-lacZ transcriptional fusion containing the region from -242 to +4 bp of the csrB gene was decreased ~20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro . These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription . Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB. J Bacteriol, 2001 Oct, 183(20), 5974 - 81 RpoS-dependent transcriptional control of sprE: regulatory feedback loop; Ruiz N et al.; The stationary-phase response exhibited by Escherichia coli upon nutrient starvation is mainly induced by a decrease of the ClpXP-dependent degradation of the alternate primary sigma factor RpoS . Although it is known that the specific regulation of this proteolysis is exercised by the orphan response regulator SprE, it remains unclear how SprE's activity is regulated in vivo . Previous studies have demonstrated that the cellular content of SprE itself is paradoxically increased in stationary-phase cells in an RpoS-dependent fashion . We show here that this RpoS-dependent upregulation of SprE levels is due to increased transcription . Furthermore, we demonstrate that sprE is part of the two-gene rssA-sprE operon, but it can also be transcribed from an additional RpoS-dependent promoter located in the rssA-sprE intergenic region . In addition, by using an in-frame deletion in rssA we found that RssA does not regulate either SprE or RpoS under the conditions tested. J Bacteriol, 2001 Oct, 183(20), 5911 - 7 The C terminus of sigma(32) is not essential for degradation by FtsH; Tomoyasu T et al.; A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH . Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases . We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins . Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro . Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded . The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo . These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones. J Bacteriol, 2001 Oct, 183(20), 5885 - 95 In vivo synthesis of the periplasmic domain of TonB inhibits transport through the FecA and FhuA iron siderophore transporters of Escherichia coli; Howard SP et al.; The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane . The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins . In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA . Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage phi80, and killing of cells by colicin M via FhuA . Transport of ferrichrome by FhuADelta5-160 was also inhibited by TonB(33-239), although FhuADelta5-160 lacks the TonB box which is involved in TonB binding . The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional . In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited . Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions. J Bacteriol, 2001 Oct, 183(20), 5870 - 6 Regulation of osmC gene expression by the two-component system rcsB-rcsC in Escherichia coli; Davalos-Garcia M et al.; The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase . The gene is transcribed from two overlapping promoters, osmCp(1) and osmCp(2) . Several factors regulating these promoters have been reported . The leucine-responsive protein Lrp represses osmCp(1) and activates osmCp(2), the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor sigma(s) specifically recognizes osmCp(2) . This work reports the identification of an additional regulatory element, the two-component system rcsB-rcsC, affecting positively the distal promoter osmCp(1) . The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp(2) . Deletion analysis located the site necessary for RcsB activation just upstream of osmCp(1) . In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp(1). EMBO J, 2001 Sep 17, 20(18), 5290 - 301 The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control mechanism; Moulinier L et al.; The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction . The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit . However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine . The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions . Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism. EMBO J, 2001 Sep 17, 20(18), 5060 - 9 Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria; Palmieri L et al.; The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle . Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains . Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+) . Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle . These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans . The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized . These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle. Biol Reprod, 2001 Oct, 65(4), 1092 - 101 An aspartic proteinase expressed in the yolk sac and neonatal stomach of the mouse; Chen X et al.; A murine aspartic proteinase, described herein, is intermediate in amino acid sequence identity between the placentally produced pregnancy-associated glycoproteins (PAGs) and gastric pepsins . While PAGs are secreted products of placental trophoblast tissue of ungulates and most are not believed to function proteolytically, pepsins are digestive enzymes . The cDNA for this aspartic proteinase was amplified by reverse transcription-polymerase chain reaction from RNA extracted from murine placentas and neonatal stomachs . The open reading frame encoded a 387-amino acid polypeptide with a 15-residue signal sequence . The enzyme most resembled pepsinogen F (a protein identified in the stomachs of neonatal rabbits and rats) and PAG-like proteins cloned from equine and feline placentae . In the stomach, both its mRNA and protein were expressed in gastric chief cells of preweaned neonates . Within the placenta, its mRNA was present in both the parietal and visceral yolk sacs . However, the protein was most prevalent in the visceral yolk sac, with little detectable in the parietal yolk sac . The recombinant protein was expressed in Escherichia coli . This protein was capable of self-activation and exhibited proteolytic activity toward casein . The presence of this enzyme in two organs involved in the selective transcellular transport of proteins suggests that it has specialized digestive functions. Biomol Eng, 2001 Oct 15, 18(3), 117 - 24 Impact of epitope permutations in the antibody response of mice to a multi-epitope polypeptide of the V3 loop of human immunodeficiency virus type 1; Aguilar A et al.; Our group have produced in Escherichia coli and evaluated the immunogenicity of different multi-epitope polypeptides (MEPs) bearing one copy of V3 loop sequential B cell epitopes from several isolates of human immunodeficiency virus type 1 (HIV-1) gp120 . One of these MEPs called TAB9 comprises the 15 central amino acids of the V3 loop from isolates LR150, JY1, RF, MN, BRVA and IIIB in this order . Antibodies against all V3 regions were elicited after immunization of rabbits, macaques and humans with TAB9 . In contrast, mice immunized with this protein only developed antibodies against epitopes JY1, LR150 and MN in that order (JY1>LR150>MN>>>RF, BRVA, IIIB) resembling an immunodominant gradient from the N-terminus to the C-terminal portion of this construction . To assess what role the location of the V3 epitopes in TAB9 could play, we constructed the protein TAB16, by altering the position of V3 epitopes in TAB9 primary structure and compared the pattern of antibodies elicited by both MEPs in H-2(d) Balb/c mice . The MEP TAB16 elicited antibody titers comparable to that of the sera from mice immunized with TAB9 . There were no statistical differences in antibody titers between both groups (P>0.05) . JY1, LR150 and MN V3 epitopes were again immunodominant in mice immunized with TAB16 fusion protein . The highest antibody titers detected in both groups among V3 epitopes corresponded to JY1, now located at the C-terminus of the permuted chimera . Antibodies against V3 epitopes RF, BRVA and IIIB were again not detected . Additionally, the MN V3 epitope showed to be significantly more immunogenic in its new orientation in TAB16, possibly as a result of a higher degree of accessibility in the surface of the protein . The results of the present investigation strongly suggest that the sequential order or the intramolecular position of V3 epitopes inside the primary structure of TAB9 and TAB16 MEPs does not interfere with the global immunogenicity or with the hierarchy of immunodominance of these regions. Biochim Biophys Acta, 2001 Sep 28, 1533(2), 119 - 27 Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase; Psylinakis E et al.; Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense . HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A) . HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques . The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands . The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy . The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation . Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation . The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features. Biochim Biophys Acta, 2001 Sep 10, 1549(1), 73 - 87 Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani(1); Jensen AT et al.; To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L . donovani amastigotes . Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identified and characterised . The GRP78 gene was localised to chromosome 15 in L . donovani, L . major, and L . mexicana by pulse-field gel electrophoresis . The Leishmania GRP78 protein contain a carboxy-terminal endoplasmic reticulum retention signal sequence (MDDL) as does the Trypanosoma cruzi GRP78 . Immunofluorescence using antibodies to the recombinant DNA-derived GRP78 protein showed staining localised to reticular material throughout the cytoplasm and in the perinuclear region of promastigotes, suggesting that the protein is localised in the endoplasmic reticulum . The protective efficacy of GRP78 was assessed in mice vaccine experiments . A GRP78 DNA vaccine primed for an immune response that protected C57Bl/6 and C3H/He mice against infection with L . major . Similarly vaccination with a recombinant form of GRP78 purified from Escherichia coli and administered with Freund's as adjuvant induced protective immunity in C57Bl/6 mice. Biochim Biophys Acta, 2001 Sep 21, 1520(3), 242 - 6 Molecular cloning and characterization of a cDNA encoding soybean nodule IMP dehydrogenase; Cao Y et al.; Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo purine biosynthesis and is a postulated key enzyme in nitrogen assimilation in ureide-exporting nodules . A 2016 bp cDNA for IMPDH, designated as IMPDH, was cloned from a soybean nodule cDNA library . IMPDH encodes a polypeptide of 502 amino acids with a predicted molecular weight of 53000 and a pI of 5.54 . The deduced IMPDH is 70.5% identical to that in Arabidopsis, with a 100% homology in the putative active-site region . Expressing the cloned cDNA in Escherichia coli mutant strain KLC381 (DeltaguaB) restored IMPDH activity, permitting bacterial growth on minimal medium . Southern blot analysis suggested a single copy of IMPDH gene in the soybean genome . Northern blot analysis showed that the expression of IMPDH gene is apparently nodule-specific. J Microbiol Methods, 2001 Oct, 47(1), 73 - 82 Effects of humic substances on fluorometric DNA quantification and DNA hybridization; Bachoon DS et al.; DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities . Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification . The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent . Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent . A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances . In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence . The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample . Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane . Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes . DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes . Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification. FEBS Lett, 2001 Sep 14, 505(2), 291 - 5 Role of the sequence of the rne-dependent site in 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P; Sim S et al.; The 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps . The precursor of M1 RNA has an rne-dependent site downstream of the processing site, whose sequence variation affects the processing efficiency . In this study, we showed that the sequence itself of the rne-dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites. FEBS Lett, 2001 Sep 14, 505(2), 245 - 8 Is Ffh required for export of secretory proteins? Kim J, Rusch S, Luirink J, Kendall DA. In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)-dependent pathway or the Sec-dependent pathway . Membrane proteins utilize the SRP-dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery . To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes . We show that depletion of SRP, but not trigger factor, affects all the mutants examined . These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export. FEBS Lett, 2001 Sep 14, 505(2), 240 - 4 Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli; Borecky J et al.; The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes . Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent . Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids . Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3 . This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP. Structure (Camb), 2001 Sep, 9(9), 817 - 26 The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP; Sakai N et al.; BACKGROUND: In Escherichia coli, the cell division site is determined by the cooperative activity of min operon products MinC, MinD, and MinE . MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the supressor of MinC . MinD plays a multifunctional role . It is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE . MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed by fluorescent microscopy . RESULTS: The three-dimensional structure of the MinD-2 from Pyrococcus horikoshii OT3 (PH0612) has been determined at 2.3 A resolution by X-ray crystallography using the Se-Met MAD method . The molecule consists of a beta sheet with 7 parallel and 1 antiparallel strands and 11 peripheral alpha helices . It contains the classical mononucleotide binding loop with bound ADP and magnesium ion, which is consistent with the suggested ATPase activity . CONCLUSIONS: Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the P loop-containing nucleotide triphosphate hydrolase superfamily of proteins . Unlike nitrogenase or other member proteins that normally work as a dimer, MinD was present as a monomer in the crystal . Both the 31P NMR and Malachite Green method exhibited relatively low levels of ATPase activity . These facts suggest that MinD may work as a molecular switch in the multiprotein complex in bacterial cell division. Biochemistry (Mosc), 2001 Aug, 66(8), 832 - 9 P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes; Ferapontova EE et al.; Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied . Recombinant forms of HRP were produced by a genetic engineering approach using an E . coli expression system . According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface . However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme . Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET . Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase. Arch Biochem Biophys, 2001 Oct 1, 394(1), 54 - 60 Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells; Lario PI et al.; The human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis . The purified protein exists as a tetramer at a concentration approximately 1 mg/ml from light scattering measurement and has a Km of 2 microM in hydrolyzing cAMP . In comparison, a partially purified PDE4A330-723 expressed in Escherichia coli has an apparent Km of 10 microM . The EC50 values for the Mg2+- or Co2+-mediated cAMP hydrolysis between the two enzymes differed by less than twofold . In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors . Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site . An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described . Arch Biochem Biophys, 2001 Oct 1, 394(1), 21 - 8 The role of cytochrome 2B1 substrate recognition site residues 115, 294, 297, 298, and 362 in the oxidation of steroids and 7-alkoxycoumarins; Domanski TL et al.; At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s . The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation . Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile . In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V . Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates . Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites . The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families . Cytokines Cell Mol Ther, 2000 Dec, 6(4), 177 - 87 Glutathione depletion is associated with augmenting a proinflammatory signal: evidence for an antioxidant/pro-oxidant mechanism regulating cytokines in the alveolar epithelium; Haddad JJ; Chemioxyexcitation {deltapO2/reactive oxygen species (ROS)} constitutes a potential signaling mechanism for regulating an inflammatory signal associated with oxidative stress . Exposure of fetal alveolar type II epithelial cells to an ascending deltaPO2 regimen with or without the hydroxyl radical (OH) or the superoxide radical anion (O2*-) induces a dose-dependent release of pro-inflammatory cytokines . Similarly, the Escherichia coli-derived lipopolysaccharide (LPS) upregulates cytokine biosynthesis in a dose- and time-dependent manner . Irreversible inhibition by L-buthionine-(S,R)-sulfoximine (BSO) of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), induces intracellular accumulation of ROS and augments chemioxyexcitation and LPS-mediated release of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) . Analysis of the molecular mechanism implicated reveals an inhibitory kappaB (IkappaB-alpha)/nuclear factor kappaB (NF-kappaB)-independent pathway mediating the redox-dependent regulation of inflammatory cytokines . Although BSO stabilizes cytosolic IkappaB-alpha and downregulates its phosphorylation, thereby blockading NF-kappaB activation, it augments cytokine biosynthesis in a dose-dependent manner . These results indicate that glutathione depletion is associated with augmentation of an oxidative stress-mediated pro-inflammatory state in an ROS-dependent mechanism and that the IkappaB-alpha/NF-kappaB pathway is otherwise not necessarily indispensable for redox-mediated regulation of cytokines. J Biomol Struct Dyn, 2001 Aug, 19(1), 141 - 58 RADACK, a stochastic simulation of hydroxyl radical attack to DNA; Begusova M et al.; RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes . It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling . The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively . The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed . The calculated relative probabilities of OH* radical attack to all reaction sites are compared to experimental FSB and ARB values . By a fitting procedure, the relative efficiencies of conversion of the C4' and C5'-centred radicals into FSB, epsilon (C4'): epsilon (C5'), and the relative efficiencies of base radicals- to- ARB conversion, epsilon(T) : epsilon(A) : epsilon(C) : epsilon(G), are then deduced for each DNA form . The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems : the lac repressor-lac operator complex and the nuclcosome core. Mol Cell Biol, 2001 Oct, 21(20), 6999 - 7009 A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines; Matsuguchi T et al.; We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages) . Three other presumed splice variant isoforms have also been identified for MKP-M . The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein . The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase . It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence . Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined . MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation . Immunocytochemical analysis showed MKP-M to be present within cytosol . When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) >> p38 = extracellular signal-regulated kinase . Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages. J Mol Endocrinol, 2001 Oct, 27(2), 239 - 47 Purification and molecular characterization of recombinant rat betacellulin; Dunbar AJ et al.; A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy . Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC . Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa . Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography . Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources. Chem Biol, 2001 Sep, 8(9), 899 - 912 Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit; Chen H et al.; BACKGROUND: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism . The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance . One newly discovered PK/NRP product, epothilone D from Sorangium cellulosum, has shown great potential as an anti-tumor agent . RESULTS: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase . The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp . The four-domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carrier protein domains: Cy-A-Ox-PCP) was also expressed and purified in soluble form from E . coli . Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate . When acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB, generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB . Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy-S-EpoB . When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB acyl enzymes were produced . CONCLUSIONS: These experiments establish chain transfer across a PKS and NRPS interface . Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth. Chem Biol, 2001 Sep, 8(9), 891 - 8 Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes; Raillard S et al.; BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems . We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space . RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines . The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent . It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme . CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed . The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution. Immunol Cell Biol, 2001 Oct, 79(5), 454 - 61 Molecular and immunological characterization of Mycobacterium avium 65 kDa heat shock protein (Hsp65); Nagabhushanam V et al.; The heat shock protein Hsp65 has been characterized previously in several mycobacterial species . This is the first report of the complete sequence of the coding region of the Mycobacterium avium homologue . The sequence was highly homologous to the Hsp65 of other mycobacterial species, as well as being related closely to the murine and human homologues . Recombinant Hsp65 (rHsp65) was expressed in Escherichia coli to high levels and the recombinant protein tested for its immunogenicity in a murine model of M . avium infection . Although mice infected with M . avium produced antibodies that reacted with rHsp65, they showed low proliferative T-cell responses and no cytokine production in response to the same antigen . However, immunization with rHsp65 in the adjuvant dimethydioctadecylammonium bromide (DDA), induced T cells that responded to native Hsp65 with proliferation and IFN-gamma production, indicating that the recombinant and native forms of the protein were antigenically similar . Therefore, the findings indicate that Hsp65 is not a dominant T-cell antigen during M . avium infection. Br J Haematol, 2001 Sep, 114(4), 794 - 9 Pharmacokinetics of native Escherichia coli asparaginase (Asparaginase medac) and hypersensitivity reactions in ALL-BFM 95 reinduction treatment; Muller HJ et al.; Repeated asparaginase treatment has been associated with hypersensitivity reactions against the bacterial macromolecule in a considerable number of patients . Immunological reactions may range from anaphylaxis without impairment of serum asparaginase activity to a very fast decline in enzyme activity without any clinical symptoms . Previous investigations on a limited number of patients have shown high interindividual variability of asparaginase activity time courses and hypersensitivity reactions in about 30% of patients during reinduction treatment . Therefore, monitoring of reinduction treatment was performed prospectively in 76 children with newly diagnosed acute lymphoblastic leukaemia (ALL) . According to the ALL-Berlin-Frankfurt-Munster (BFM) 95 protocol, 10 000 U/m2 body surface area of native Escherichia coli asparaginase (Asparaginase medac) was given on d 8, 11, 15 and 18 . In 45/76 children, trough and peak activities were determined with every dose, and also on d 4 and d 11 after the last administration . Data on asparaginase activity were not available from the remaining 31 patients, but information with regard to hypersensitivity reactions only was given . Eighteen out of 76 patients (24%) suffered a clinical hypersensitivity reaction; however, no silent inactivation was observed . Activity in the therapeutic range of greater than 100 U/l for at least 14 d was determined in 43 of the 45 patients who were analysed for enzyme activity. Biochem J, 2001 Oct 1, 359(Pt 1), 23 - 34 Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean; Hamada S et al.; The nature and enzymic properties of starch-branching enzyme (SBE) are two of the dominant factors influencing the fine structure of starch . To understand the role of this enzyme's activity in the formation of starch in kidney bean (Phaseolus vulgaris L.), a study was undertaken to identify the major SBE sequences expressed during seed development and to characterize the enzymic properties of the coded recombinant enzymes . Two SBE cDNA species (designated pvsbe2 and pvsbe1) that displayed significant similarity (more than 70%) to other family A and B SBEs respectively were isolated . Northern blot analysis revealed that pvsbe1 and pvsbe2 were differentially expressed during seed development . pvsbe2 showed maximum steady-state transcript levels at the mid-stage of seed maturation, whereas pvsbe1 reached peak levels at a later stage . Western blot analysis with antisera raised against both recombinant proteins (rPvSBE1 and rPvSBE2) showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean . PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with the starch granule fraction . The differences in location suggest that these two SBE isoenzymes have different roles in amylopectin synthesis in kidney bean seeds . rPvSBE1 and rPvSBE2 were purified from Escherichia coli and their kinetic properties were determined . The affinity of rPvSBE2 for amylose (K(m) 1.27 mg/ml) was lower than that of rPvSBE1 (0.46 mg/ml) . The activity of rPvSBE2 was stimulated more than 3-fold in the presence of 0.3 M citrate, whereas rPvSBE1 activity was not affected . The implications of the enzymic properties and the distribution of SBEs and amylopectin structure are discussed. Biochemistry (Mosc), 2001 Jul, 66(7), 803 - 7 Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase; Kononova SV et al.; The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone--protein SecB . Secretion increases in the presence of this chaperone at 30 degrees C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme . This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase--protein SecA. J Med Chem, 2001 Sep 27, 44(20), 3216 - 22 Synthesis of a tetronic acid library focused on inhibitors of tyrosine and dual-specificity protein phosphatases and its evaluation regarding VHR and cdc25B inhibition; Sodeoka M et al.; Selective inhibitors of protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs) are expected to be useful tools for clarifying the biological functions of the PTPs themselves and also to be candidates for novel therapeutics . We planned a library approach for the identification of PTP/DSP inhibitors in which 3-acyltetronic acid is used as a "core" phosphate mimic . A series of novel tetronic acid derivatives were synthesized and evaluated as inhibitors of the dual-specificity protein phosphatases VHR and cdc25B . Several compounds are found to be potent inhibitors of cdc25B, which is a key enzyme for cell-cycle progression . The promising results described herein strongly indicated that this tetronic acid library is potent as a library focused on the PTP/DSP-selective inhibitor. Biochem Biophys Res Commun, 2001 Sep 28, 287(3), 682 - 7 Catalase-peroxidase from synechocystis is capable of chlorination and bromination reactions; Jakopitsch C et al.; Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity . Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner . Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity . Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested . Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A . Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH . The halogenation activity of R119A was about 7-9% that of the wild-type enzyme . Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased . The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions . That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride . Biochem Biophys Res Commun, 2001 Sep 28, 287(3), 636 - 41 Role of the connectivity of secondary structure segments in the folding of alpha(1)-antitrypsin; Lee C et al.; The native form of serpins (serine protease inhibitors) is metastable, which is critical to their biological functions . Spontaneous conversion from the native form of serpins into a more stable conformation, called the "latent" form, is restricted . To examine whether the connectivity of strand 1 of beta-sheet C to the hydrophobic core is critical to the serpin's preferential folding to the metastable native conformation, we designed a circularly-permuted mutant of alpha(1)-antitrypsin, the prototype serpin, in which strand 1C is disconnected from the hydrophobic core . Conformation of the circular permutant was similar to that of the latent form, as revealed by equilibrium unfolding, limited proteolysis, and spectroscopic properties . Our results support the notion that rapid folding of the hydrophobic core with concomitant incorporation of strand 1C into beta-sheet C traps the serpin molecule into its native metastable conformation . Appl Biochem Biotechnol, 2001 Jun, 94(3), 243 - 55 Cloning and expression of thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102 and characterization of recombinant enzyme; Xiangyuan H et al.; The gene coding for beta-glycosidase (EC 3.2.1.21) from Thermus nonproteolyticus HG102 was cloned and expressed in Escherichia coli . The gene open reading frame was 1311 bp, and it codes for 437 amino acids . The deduced amino acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I . The enzyme had high content of Arg and Pro . The recombinant enzyme was purified to homogeneity with heat precipitation, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared slab polyacrylamide gel electrophoresis . The enzyme was monomeric and had a molecular mass of 48,900 Daltons and a pI of 5.2 . The enzyme showed optimum activity at pH 5.6 and 90 degrees C . It catalyzed the hydrolysis of beta-D-glucoside, beta-D-galactoside, beta-D-fucoside, and beta-D-mannoside . Lineweaver-Burk plots showed that the kcat/Km ratio for beta-D-glucoside and beta-D-fucoside was higher than for beta-D-mannoside and beta-D-galactoside . The enzyme was extremely thermostable, with a half-life of 2.5 h at 90 degrees C, and was stable over a wide range of pH . It also had transglycosidic activity at high temperature. Nucleosides Nucleotides Nucleic Acids, 2001 Apr-Jul, 20(4-7), 785 - 8 Cyclohexene nucleic acids (CeNA) form stable duplexes with RNA and induce RNase H activity; Wang J et al.; Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry . Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex . CeNA is stable against degradation in serum . A CeNA/RNA hybrid is able to activate E . Coli RNase H . resulting in cleavage of the RNA strand. Nucleosides Nucleotides Nucleic Acids, 2001 Apr-Jul, 20(4-7), 441 - 9 RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1; Darfeuille F et al.; We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome . We selected aptameric RNA hairpins giving rise to kissing complexes with TAR . The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range . However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E . coli which is specific for kissing complexes. Int J Mol Med, 2001 Oct, 8(4), 397 - 404 Dimerization of small GTPase Rab5; Daitoku H et al.; Rab proteins are small GTPases, localized to distinct cellular compartments, regulating specific steps of intracellular membrane trafficking . One member of the Rab family, Rab5, consists of three isoforms, Rab5a, Rab5b, and Rab5c, which have been shown to play an important role in early events of endocytosis . Using the yeast two-hybrid system, we have identified several cytosolic proteins that interact with the Rab5b Q79L (decreasing intrinsic and GTPase-activating protein-stimulated GTPase activities) . Surprisingly, most positive clones were Rab5b or Rab5c, indicating that Rab5 could dimerize among extra-isoforms in the yeast two-hybrid system . In vitro and in vivo chemical cross-linking assays demonstrated that lipid-unmodified wild-type Rab5b purified from Escherichia coli, wild-type Rab5b, or a dominant active form Rab5b Q79L expressed in human 293T cells dimerized . Furthermore, the same assays using a Rab5b R81A substitution mutant showed that the Arg81 in the Switch II region {the second GTP/GDP binding motif (residues 74-93)} was essential for Rab5b dimerization . These results suggest that Rab5 isoforms can be dimerized depending upon the GTP-bound conformation, but independent upon a lipid modification. J Gen Virol, 2001 Oct, 82(Pt 10), 2579 - 88 Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively; Nurkiyanova KM et al.; Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed . The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast . The protein encoded by ORF4 was assessed also for RNA-binding properties . This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography . Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios . Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA . UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl . Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes . The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed. J Gen Virol, 2001 Oct, 82(Pt 10), 2559 - 67 Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus; Pant V et al.; The complete nucleotide sequence of the blackgram isolate of mungbean yellow mosaic virus, IMYMV-Bg, which infects legumes in India, was determined and compared at the amino acid level with those of other whitefly-transmitted geminiviruses . The genome organization of IMYMV-Bg was similar to that of the begomoviruses . A unique feature of the genome organization was the sequence divergence of the common region (CR) between DNA-A and DNA-B . In order to understand the mechanism of viral DNA replication, the replication initiator protein, Rep, of IMYMV-Bg was overexpressed in E . coli . The recombinant and refolded Rep bound to CR-sequences of IMYMV-Bg in a specific manner . In this study, evidence is presented for ATP-upregulated cleavage function and ATP-mediated conformational change of Rep . It is hypothesized that, although ATP is not required for cleavage, ATP-mediated conformational changes may result in better access of Rep to the DNA-cleavage site . Evidence is also presented for a site-specific topoisomerase function of Rep, which has not been demonstrated before . The Rep protein can be classified as a type-I topoisomerase because of its nicking activity and sensitivity towards camptothecin, a topoisomerase type-I inhibitor. J Gen Virol, 2001 Oct, 82(Pt 10), 2425 - 35 Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (G(L)): induction of virus-neutralizing antibody and assessment of protection in ponies; Castillo-Olivares J et al.; An Escherichia coli-expressed recombinant protein (6hisG(L)ecto) comprising the entire ectodomain (aa 18-122) of equine arteritis virus (EAV) glycoprotein G(L), the immunodominant viral antigen, induced higher neutralizing antibody titres than other G(L)-derived polypeptides when compared in an immunization study in ponies . The potential of the recombinant G(L) ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 microg of protein . All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1-2 weeks after the administration of a booster on week 5 (VNAb titres of 1.8-3.1), 13 (VNAb titres of 1.4-2.9) or 53 (VNAb titres of 1.2-2.3) . Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb . Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx . The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion . These results provide the first evidence that a sub-unit vaccine protects horses against EAV . The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11615 - 20 Epub 2001 Sep 18. An unsuspected autoregulatory pathway involving apocytochrome TorC and sensor TorS in Escherichia coli; Gon S et al.; Trimethylamine N-oxide (TMAO) respiration is carried out mainly by the Tor system in Escherichia coli . This system is encoded by the torCAD operon and comprises a periplasmic TMAO reductase (TorA) and a c-type cytochrome (TorC), which shuttles electrons to TorA . Expression of the tor operon is positively controlled by the TorS/TorR phosphorelay system in response to TMAO availability and negatively regulated by apocytochrome TorC . Interaction studies showed that, when immature, TorC can no longer bind TorA efficiently but can bind the periplasmic detector region of sensor TorS . ApoTorC negative autoregulation and TMAO induction are thus mediated by the same sensor protein . As apocytochromes related to TorC could not down-regulate the tor operon, we concluded that this negative control is highly specific . Moreover, the N-terminal half of apoTorC played no role in this control but the immature C-terminal domain of TorC strongly down-regulated the tor operon and interacted with the TorS detector region . This sophisticated autoregulatory pathway thus involves the C-terminal domain of apoTorC and allows optimal TorC biogenesis by preventing from saturation the c-type cytochrome maturation machinery. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11120 - 5 Epub 2001 Sep 18. Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation; Feinberg JS et al.; Translocation of the tRNA-mRNA complex is a fundamental step in the elongation cycle of protein synthesis . Our studies show that the ribosome can translocate a P-site-bound tRNA(Met) with a break in the phosphodiester backbone between positions 56 and 57 in the TPsiC-loop . We have used this fragmented P-site-bound tRNA(Met) to identify two 2'-hydroxyl groups at positions 71 and 76 in the 3'-acceptor arm that are essential for translocation . Crystallographic data show that the 2'-hydroxyl group at positions 71 and 76 contacts the backbone of 23S rRNA residues 1892 and 2433-2434, respectively, in the ribosomal E site . These results establish a set of functional interactions between P-site tRNA and 23S rRNA that are essential for translocation. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11248 - 53 Epub 2001 Sep 18. Creating multiple-crossover DNA libraries independent of sequence identity; Lutz S et al.; We have developed, experimentally implemented, and modeled in silico a methodology named SCRATCHY that enables the combinatorial engineering of target proteins, independent of sequence identity . The approach combines two methods for recombining genes: incremental truncation for the creation of hybrid enzymes and DNA shuffling . First, incremental truncation for the creation of hybrid enzymes is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion . This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers . SCRATCHY libraries were created from the glycinamide-ribonucleotide formyltransferase (GART) genes from Escherichia coli (purN) and human (hGART) . The developed modeling framework eSCRATCHY provides insight into the effect of sequence identity and fragmentation length on crossover statistics and draws contrast with DNA shuffling . Sequence analysis of the naive shuffled library identified members with up to three crossovers, and modeling predictions are in good agreement with the experimental findings . Subsequent in vivo selection in an auxotrophic E . coli host yielded functional hybrid enzymes containing multiple crossovers. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11163 - 8 Epub 2001 Sep 18. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation; Park H et al.; Peptide:N-glycanase (PNGase) cleaves oligosaccharide chains from glycopeptides and glycoproteins . Recently the deduced amino acid sequence of a cytoplasmic PNGase has been identified in various eukaryotes ranging from yeast to mammals, suggesting that deglycosylation may play a central role in some catabolic process . Several lines of evidence indicate that the cytoplasmic enzyme is involved in the quality control system for newly synthesized glycoproteins . Two-hybrid library screening by using mouse PNGase as the target yielded several PNGase-interacting proteins that previously had been implicated in proteasome-dependent protein degradation: mHR23B, ubiquitin, a regulatory subunit of the 19S proteasome, as well as a protein containing an ubiquitin regulatory motif (UBX) and an ubiquitin-associated motif (UBA) . These findings by using the two-hybrid system were further confirmed either by in vitro binding assays or size fractionation assays . These results suggest that PNGase may be required for efficient proteasome-mediated degradation of misfolded glycoproteins in mammalian cells. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11218 - 23 Epub 2001 Sep 18. Structure of rat BCKD kinase: nucleotide-induced domain communication in a mitochondrial protein kinase; Machius M et al.; Mitochondrial protein kinases (mPKs) are molecular switches that down-regulate the oxidation of branched-chain alpha-ketoacids and pyruvate . Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branched-chain ketoaciduria, and primary lactic acidosis . We report a three-dimensional structure of a member of the mPK family, rat branched-chain alpha-ketoacid dehydrogenase kinase (BCK) . BCK features a characteristic nucleotide-binding domain and a four-helix bundle domain . These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems . Unlike PHKs, BCK dimerizes through direct interaction of two opposing nucleotide-binding domains . Nucleotide binding to BCK is uniquely mediated by both potassium and magnesium . Binding of ATP induces disorder-order transitions in a loop region at the nucleotide-binding site . These structural changes lead to the formation of a quadruple aromatic stack in the interface between the nucleotide-binding domain and the four-helix bundle domain, where they induce a movement of the top portion of two helices . Phosphotransfer induces further ordering of the loop region, effectively trapping the reaction product ADP, which explains product inhibition in mPKs . The BCK structure is a prototype for all mPKs and will provide a framework for structure-assisted inhibitor design for this family of kinases. J Biol Chem, 2001 Nov 30, 276(48), 44563 - 9 Epub 2001 Sep 18. Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporters; Senn ME et al.; Plant K(+) transporters of the HAK family belong to four rather divergent phylogenetic clusters, although most of the transporters belong to clusters I or II . A simple phylogenetic analysis of fungal and plant HAK transporters suggests that an original HAK gene duplicated even before fungi and plants diverged, generating transporters that at present fulfill different functions in the plant . The HvHAK1 transporter belongs to cluster I and mediates high-affinity K(+) uptake in barley roots, but no function is known for the cluster II transporter, HvHAK2, which is not functional in yeast . The function of HvHAK2 was investigated by constructing HvHAK1-HAK2 chimeric transporters, which were not functional even when they included only short fragments of HvHAK2 . Then, amino acids characteristic of cluster II in the N terminus and in the first transmembrane domain were introduced into HvHAK1 . All of these changes increased the Rb(+) K(m), introducing minimal changes in the Na(+) K(m), which suggested that HvHAK2 is a low-affinity, Na(+)-sensitive K(+) transporter . Using a K(+)-defective Escherichia coli mutant, we functionally expressed HvHAK2 and found that the predicted characteristics were correct, as well as discovering that the bacterial expression of HvHAK2 is functional at pH 5.5 but not at 7.5 . We discuss whether HvHAK2 may be a tonoplast transporter effective for vacuolar K(+) depletion in K(+) starved plants. J Biol Chem, 2001 Nov 23, 276(47), 43580 - 8 Epub 2001 Sep 18. Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii . Biochemical and genetic characterization of key enzymes; Empadinhas N et al.; The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase . The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P . horikoshii genome . This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli . The recombinant product of gene PH0927 catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute alpha-mannosylglycerate . The MPG synthase and the MPG phosphatase were specific for these substrates . Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphate-guanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923) . Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to alpha-mannosylglycerate synthesis . Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100 degrees C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100 degrees C and between pH 5.2 and 6.4 . This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon. Comb Chem High Throughput Screen, 2001 Nov, 4(7), 535 - 43 Identification of enzyme inhibitors from phage-displayed combinatorial peptide libraries; Kay BK et al.; In recent years, there have been a growing number of examples of the successful isolation of peptide ligands for enzymes from phage-displayed combinatorial peptide libraries . These peptides typically bind at or near the active site of the enzymes and can inhibit their activity . We review the literature on peptide ligands that have been isolated for enzymes other than proteases as well as present data on peptide ligands we have identified for E . coli dihydrofolate reductase (DHFR) which bind at, or near, the same site as the known inhibitors methotrexate or trimethoprim . Thus, while the peptide ligand isolated from phage-displayed libraries may not resemble the chemical structure of the normal substrate of the enzyme, the peptide can be used as an inhibitor to evaluate the function of the enzyme or for drug discovery efforts (i.e., as a lead compound for peptidomimetic design or as displaceable probe in high-throughput screens of libraries of small molecules). Comb Chem High Throughput Screen, 2001 Sep, 4(6), 525 - 33 Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening; Deng SJ et al.; The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening . Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y . A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation . The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds . Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified . Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2 . Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1) . Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors . Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay. Bioconjug Chem, 2001 Sep-Oct, 12(5), 770 - 5 A ribonuclease H-oligo DNA conjugate that specifically cleaves hepatitis B viral messenger RNA; Walton CM et al.; Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity . However, the formation of DNA-RNA hybrids does require specific sequence recognition . On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H . The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA . A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E . coli RNase H using a water soluble cross-linker . A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase . Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner . Eighty-five percent of substrate was cleaved under optimal conditions . Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity . RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts. Int Immunopharmacol, 2001 Sep, 1(9-10), 1867 - 75 Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-receptors; Azuma Y et al.; Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes . In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis . Moreover, histamine at l0(-5) M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate (PMA) stimulated macrophages and histamine at a concentration range of 10(-7) to 10(-5) M significantly inhibited phagocytosis of Escherichia coli by macrophages . In addition, H2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E . coli by macrophages . On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFalpha and IL-12 by macrophages . These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-histamine receptors . reserved. Epidemiol Infect, 2001 Aug, 127(1), 27 - 36 Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals; Asakura H et al.; Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank . The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC . On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC . Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat . In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans. Environ Technol, 2001 Aug, 22(8), 897 - 904 Remediation and toxicity removal from Kraft E1 paper mill effluent by ozonization; Freire RS et al.; The degradation of Kraft E1 pulp mill effluent was studied by four different ozonization oxidation systems (O3/pH3, O3/pH11, O3/pH11/H2O2, O3/pH11/UV) . The investigation was focused on the reduction of total organic carbon (TOC), total phenols, color and acute toxicity (monitoring by inhibition of Escherichia coli respiration) . For a reaction time of 90 minutes, the O3/pH11/UV was the most effective process for decoloration (45%) . The O3/pH11/H2O2, O3/pH11/UV and O3/pH11 processes showed the best results for total phenols reduction (approximately/= 90%) . None of the studied processes showed a significant TOC reduction . The O3/pH11/UV and O3/pH11 processes were effective for the acute toxicity reduction . Different kinetic parameters were also determined in order to quantify the reactivity of the effluent towards the applied oxidation systems. Eur J Cell Biol, 2001 Aug, 80(8), 527 - 38 A novel tetratricopeptide repeat-containing J-protein localized in a plasma membrane-bound protein complex of the phytopathogenic oomycete Phytophthora megasperma; Porschewski P et al.; Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones . To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins . Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain . Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth . Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex . In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches . Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain . The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions . By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp. Mol Cells, 2001 Aug 31, 12(1), 91 - 6 Molecular cloning and functional expression of bovine brain GABA transaminase; Jeon SG et al.; We isolated a cDNA that encodes the bovine brain gamma-aminobutyrate transaminase (GABA-T; EC 2.6.1.19) from the lambda gt 11 cDNA library, which showed a high degree of sequence similarity to the corresponding enzymes from various sources . Northern blot analysis revealed two differentially expressed GABA-T transcripts of approximately 2.0 and 6.0 kb in the bovine tissues . Southern blot analysis indicates that the two GABA-T transcripts are encoded in a greater-than 10-kb, single-copy gene . Bovine GABA-T cDNA was expressed in E . coli using the pGEX bacterial- expression vector system . The overexpressed GABA-T was enzymatically active after purification, and it had very similar kinetic parameters when compared with those of other mammalian GABA-Ts. Mol Cells, 2001 Aug 31, 12(1), 50 - 6 Immune induction and modulation in mice following immunization with DNA encoding F protein of respiratory syncytial virus; Park EK et al.; Respiratory syncytial virus (RSV) is one of the principal agents of bronchiolitis and pneumonia in young children . Thus, there is a strong need to make a safe and effective vaccine against the RSV infection . DNA immunization is very effective at inducing both cellular and humoral immune responses . In this study, we inserted the RSV-F gene into expression vectors, pcDNA3.1 and pQE . These constructs were transformed into C2C12 and E . coli M15 cells, respectively . The expression of the RSV-F protein was confirmed by SDS-PAGE, followed by Western blot analyses . The immunization of pcDNA3.1-RSV-F elicited both anti-RSV-F titer in mouse sera and CTL activities with mouse splenocytes . Especially, the co-administration of IL-4, or the GM-CSF gene with the RSV-F gene construct, enhanced the production of anti-RSV-F Ab . However, this enhancement disappeared by the simultaneous injection of the Th1 and Th2 type cytokine genes . The CTL activities were affected by the co-delivery of the IFN-gamma gene, but not by Th2-type cytokines. J Biol Chem, 2001 Nov 16, 276(46), 43040 - 8 Epub 2001 Sep 17. Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex; Belden WJ et al.; Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting . Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast . Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable . Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p . To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed . The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences . COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently . These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER . The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER. J Biol Chem, 2001 Nov 9, 276(45), 42146 - 52 Epub 2001 Sep 17. Recombinant mouse muscle adenylosuccinate synthetase: overexpression, kinetics, and crystal structure; Iancu CV et al.; Vertebrates possess two isozymes of adenylosuccinate synthetase . The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle . Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate synthetase . The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K(m) parameters for GTP, IMP, and l-aspartate (12, 45, and 140 microm, respectively) similar to those of the enzyme from Escherichia coli . The mouse muscle and E . coli enzymes have similar polypeptide folds, differing primarily in the conformation of loops, involved in substrate recognition and stabilization of the transition state . Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure . In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the active site . Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes. Biochemistry, 2001 Sep 25, 40(38), 11596 - 603 Chemical communication across the zinc tetrathiolate cluster in Escherichia coli Ada, a metalloactivated DNA repair protein; Sun LJ et al.; The Escherichia coli Ada protein repairs methylphosphotriesters in DNA through direct, irreversible transfer to a cysteine residue on the protein, Cys 69 . Methylation of Cys 69 increases the sequence-specific DNA-binding activity of Ada by 10(3)-fold, enabling the methylated protein to activate transcription of a methylation-resistance regulon . The thiolate sulfur atom of Cys 69 is coordinated to a tightly bound zinc ion in the Ada N-terminal domain, and this metal-ligand interaction plays a direct role in promoting the DNA repair chemistry . Ada is thus the founding member of a mechanistic class of proteins that employ metalloactivated thiolates as nucleophiles, other examples of which include protein prenyltransferases and cobalamin-independent methionine synthase . Here we have probed the role of the three other Cys residues in Ada that together with Cys 69 coordinate the zinc through mutation to the alternative ligand residues Asp and His . All of the mutant proteins folded properly and bound zinc, but none of them exhibited measurable levels of DNA repair activity . Significantly, the Cys-to-His mutant proteins retained nearly wild-type sequence-specific DNA-binding activity in the unmethylated state . These findings demonstrate that the three "spectator" Cys ligands communicate chemically with Cys 69 through the bound metal ion. Biochemistry, 2001 Sep 25, 40(38), 11586 - 95 DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA; Stern JC et al.; The TraI protein has two essential roles in transfer of conjugative plasmid F Factor . As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell . TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer . As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence . The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity . TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold . The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies . In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site . The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer. Biochemistry, 2001 Sep 25, 40(38), 11543 - 51 Multiple autophosphorylation is essential for the formation of the active and stable homodimer of heme-regulated eIF2alpha kinase; Bauer BN et al.; In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI) . Activation of HRI is accompanied by its phosphorylation . We have investigated the role of autophosphorylation in the formation of active and stable HRI . Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE . Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues . Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI . Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation . Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II . We further separated and characterized these two species biochemically . We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer . Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism . In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation . Although species I is an active autokinase, it is still without eIF2alpha kinase activity . Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme. Biochemistry, 2001 Sep 25, 40(38), 11433 - 41 Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3; Oz G et al.; The brain specific member of the metallothionein (MT) family of proteins, metallothionein-3, inhibits the growth and survival of neurons, in contrast to the ubiquitous mammalian MT isoforms, MT-1 and MT-2, that are found in most tissues and are thought to function in metal ion homeostasis and detoxification . Solution NMR was utilized to determine the structural and dynamic differences of MT-3 from MT-1 and 2 . The high-resolution solution structure of the C-terminal alpha-domain of recombinant mouse MT-3 revealed a tertiary fold very similar to MT-1 and 2, except for a loop that accommodates an acidic insertion relative to these isoforms . This loop was distinguished from the rest of the domain by dynamics of the backbone on the nano- to picosecond time-scale shown by (15)N relaxation studies and was identified as a possible interaction site with other proteins . The N-terminal beta-domain contains the region responsible for the growth inhibitory activity, a CPCP tetrapeptide close to the N-terminus . Because of exchange broadening of a large number of the NMR signals from this domain, homology modeling was utilized to calculate models for the beta-domain and suggested that while the backbone fold of the MT-3 beta-domain is identical to MT-1 and 2, the second proline responsible for the activity, Pro9, may show structural heterogeneity . (15)N relaxation analyses implied fast internal motions for the beta-domain . On the basis of these observations, we conclude that the growth inhibitory activity exhibited by MT-3 is a result of a combination of local structural differences and global dynamics in the beta-domain. Biochemistry, 2001 Sep 25, 40(38), 11382 - 9 ATP-mediated changes in cross-subunit interactions in the RecA protein; Logan KM et al.; RecA protein undergoes ATP- and DNA-induced conformational changes that result in different helical parameters for free protein filaments versus RecA/ATP/DNA nucleoprotein filaments . Previous mutational studies of a particular region of the RecA oligomeric interface suggested that cross-subunit contacts made by residues K6 and R28 were more important for stabilization of free protein oligomers than nucleoprotein filaments {Eldin, S., et al . (2000) J . Mol . Biol . 299, 91-101} . Using mutant proteins with specifically engineered Cys substitutions, we show here that the efficiency of cross-subunit disulfide bond formation at certain positions in this region changes in the presence of ATP or ATP/DNA . Our results support the idea that specific cross-subunit interactions that occur within this region of the subunit interface are different in free RecA protein versus RecA/ATP/DNA nucleoprotein filaments. Biochemistry, 2001 Sep 25, 40(38), 11372 - 81 A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability; Shah AM et al.; DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis . In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen . Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta . This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays . M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors . Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta . Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding . However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved . X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity . Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain . Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability . The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282 . Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair . The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity. Biochemistry, 2001 Sep 25, 40(38), 11364 - 71 Site-bound water and the shortcomings of a less than perfect transition state analogue; Snider MJ et al.; Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) {Snider, M . J., et al . (2000) Biochemistry 39, 9746-9753} . In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants . The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy . The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis . The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia. Biochemistry, 2001 Sep 25, 40(38), 11353 - 63 Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes; Argyrou A et al.; The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity . The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer . The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism . Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small {(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07} when D,L-lipoamide is the oxidant but large and equivalent {(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03} when 5-hydroxy-1,4-naphthoquinone is the oxidant . Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01 . Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15 . All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step . Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant. Biochemistry, 2001 Sep 25, 40(38), 11327 - 37 Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4; Roberts SA et al.; The nitrophorins are a family of proteins that use ferric heme to transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation . We have refined atomic resolution structures of nitrophorin 4 (NP4) from Rhodnius prolixus complexed with NO (1.08 A) and NH(3) (1.15 A), yielding a highly detailed picture of the iron coordination sphere . In NP4-NO, the NO nitrogen is coordinated to iron (Fe-N distance = 1.66 A) and is somewhat bent (Fe-N-O angle = 156 degrees ), with bending occurring in the same plane as the proximal histidine ring . The Fe(NO)(heme)(His) coordination geometry is unusual but consistent with an Fe(III) oxidation state that is stabilized by a highly ruffled heme . Heme ruffling occurs in both structures, apparently due to close contacts between the heme and leucines 123 and 133, but increases on binding NO even though the steric contacts have not changed . We also report the structure of NP4 in complexes with histamine (1.50 A) and imidazole (1.27 A) . Unexpectedly, two mobile loops that rearrange to pack against the bound NO in NP4-NO, also rearrange in the NP4-imidazole complex . This conformational change is apparently driven by the nonpolar nature of the NO and imidazole (as bound) ligands . Taken together, the desolvation of the NO binding pocket through a change in protein conformation, and the bending of the NO moiety, possibly through protein-assisted heme ruffling, may lead to a nitrosyl-heme complex that is unusually resistant to autoreduction. J Virol, 2001 Oct, 75(20), 9939 - 46 Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein; Pallaoro M et al.; The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction . Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction . We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor . Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction . This truncated protein was efficiently expressed and processed in Escherichia coli . The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies . This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor . Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein . However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants . Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing . Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad . We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors. J Virol, 2001 Oct, 75(20), 9925 - 38 Cellular membrane-binding ability of the C-terminal cytoplasmic domain of human immunodeficiency virus type 1 envelope transmembrane protein gp41; Chen SS et al.; The amphipathic alpha-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity . Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity . However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood . In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli beta-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region . We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells . Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability . Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes . High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the beta-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences . Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated beta-galactosidase and enhanced green fluorescence protein to the ER . Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction. J Virol, 2001 Oct, 75(20), 9633 - 43 Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication; Matusan AE et al.; The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein . The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae . DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli . The effects of 10 mutations on ATPase and RNA helicase activity were examined . Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis . The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA . Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity . Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R(376)KNGK(380), abolished helicase activity only . No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication . The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication . For the two nonreplicating viruses encoding clustered changes at R(184)KR(186) and D(436)GEE(439), we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex . Two of the replicating viruses displayed a temperature-sensitive phenotype . One contained a clustered mutation at D(334)EE(336) and grew too poorly for further characterization . However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37 degrees C) and showed reduced RNA synthesis at the higher temperature. J Agric Food Chem, 2001 Sep, 49(9), 4224 - 30 Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification; Tzeng SS et al.; A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host . After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E . coli . The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography . The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa) . Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11 . No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C . They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B. Chem Res Toxicol, 2001 Sep, 14(9), 1247 - 53 Formation of the 1,N2-glyoxal adduct of deoxyguanosine by phosphoglycolaldehyde, a product of 3'-deoxyribose oxidation in DNA; Awada M et al.; Oxidation of deoxyribose in DNA results in the formation of a variety of electrophilic products that have the potential to react with nucleobases to form adducts . We now report that 2-phosphoglycolaldehyde, a model for the 3'-phosphoglycolaldehyde residue generated by 3'-oxidation of deoxyribose in DNA, reacts with dG and DNA to form the diastereomeric 1,N2-glyoxal adducts of dG, 3-(2-deoxy-beta-D-erythro-pentofuransyl)-6,7-dihydro-6,7-dihydroxyimidazo{1,2-a}purine-9(3H)-one . The glyoxal adducts were the predominant species formed under biological conditions (pH 7.4 and 37 degrees C), with several minor fluorescent adducts, including 1,N6-ethenoadenine . The adducts were fully characterized by HPLC, mass spectrometry, and UV and NMR spectroscopy . The reaction of 2-phosphoglycolaldehyde with dG occurred with a rate constant of 10(-6) M(-1) s(-1) compared to the rate constants of 0.08 and approximately 10(-9) M(-1) s(-1) for the reactions of glyoxal and glycolaldehyde with dG, respectively . The kinetic results rule out contamination of 2-phosphoglycolaldehyde preparations with glyoxal as the basis for our observations . The rate constant for the formation of glyoxal from 2-phosphoglycolaldehyde (10(-8) s(-1)) is consistent with glyoxal generation being the rate-limiting step in the formation of dG adducts in reactions with 2-phosphoglycolaldehyde . Mechanistic studies were also undertaken to define the basis for the different oxidation states of glyoxal and 2-phosphoglycolaldehyde . Although 2-phosphoglycolaldehyde produced a weak ESR signal consistent with generation of hydroxyl radicals and it caused DNA strand breaks at high concentrations, the formation of the glyoxal adducts of dG was insensitive to radical quenchers (e.g., sorbitol) and independent of molecular oxygen . In contrast, the formation of glyoxal-dG adducts with glycolaldehyde was dependent on molecular oxygen and quenched by sorbitol, and the glycolaldehyde-glyoxal rearrangement produced a strong ESR signal characteristic of alkyl radicals . These observations are consistent with a model in which glyoxal is generated from 2-phosphoglycolaldehyde by a nonradical, oxygen-independent mechanism that is currently under investigation . Our results provide a mechanistic basis for the observation by Murata-Kamiya et al . {(1995) Carcinogenesis 16, 2251-2253} that oxidation of DNA with the Fe(II)-EDTA complex results in the formation of the glyoxal adducts of dG. Methods, 2001 Sep, 25(1), 62 - 77 Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy; Bailey MF et al.; Prior to undergoing postsynthetic 3'-5' editing (proofreading), a defective DNA primer terminus must be transferred from the 5'-3' polymerase active site to a remote 3'-5' exonuclease site . To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase I . The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees) . Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees ) . If both polymerase and exonuclease binding modes are populated (lower limit approximately 5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape . Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e)) and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively . The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K(pe) (equal to x(b)/x(e)) . The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K(pe) . Methods, 2001 Sep, 25(1), 44 - 53 Luminescence resonance energy transfer analysis of RNA polymerase complexes; Heyduk T; Resonance energy transfer allows measurement of atomic-scale distances under a variety of solution conditions . Luminescence resonance energy transfer (LRET) is a variant of energy transfer measurement in which lanthanide chelates are used as the probes . The unusual properties of lanthanide emission, in particular their long microsecond-scale lifetimes, offer several advantages for energy transfer measurements with biological samples . One of the unique features of LRET is the ability to measure energy transfer under conditions where severe heterogeneity of labeled macromolecules exists . This feature of LRET is the special emphasis of this article . We describe here LRET methodology with a particular attention to using sensitized acceptor emission to determine efficiency of energy transfer . Although we employed this technique in the characterization of Escherichia coli RNA polymerase complexes it is readily compatible with the study of essentially any protein . Poult Sci, 2001 Sep, 80(9), 1314 - 22 Effect of early handling of turkey poults on later responses to multiple dexamethasone-Escherichia coli challenge . 2 . Resistance to air sacculitis and turkey osteomyelitis complex; Huff GR et al.; Dexamethasone (DEX)-induced immunosuppression facilitates Escherichia coli pathogenesis leading to lesions of air sacculitis and turkey osteomyelitis complex (TOC) . The purpose of this study was to determine if early handling could affect resistance to disease in this model . Seven hundred twenty male turkey poults were handled 0, 1 (1x), or 2 (2x) times daily for the first 10 d after hatch . Handling consisted of gently catching each individual poult, holding it for 10 s, and placing it into a basket . Starting on Day 11 after hatch, half of the birds from each handling treatment were treated with three injections of 2 mg DEX/kg BW on alternating days . On the day of the third DEX treatment, duplicate pens of birds were also inoculated in the air sac with 0 or 50 cfu of E . coli . All DEX-treated birds were given a second series of DEX injections at 5 wk of age, and 10 birds per pen were necropsied 3 wk later . Surviving birds were treated with a third series of DEX injections at 10 wk of age . Two weeks later, all surviving turkeys were necropsied . All mortalities and necropsied birds were scored for air sacculitis and examined for TOC lesions . All livers, air sacs, and TOC lesions were cultured for bacteria . There was increased mortality after the first series of DEX treatments of birds handled 2x . After the second series of DEX treatments, birds handled 1x had increased mortality, incidence of air sacculitis, and recovery of E . coli from tissues, whereas 2x handled birds were identical to unhandled controls . After the third series of DEX treatments, handling 1x resulted in decreased air sacculitis scores and decreased incidence of mortality, green liver, TOC lesions, and recovery of E . coli from tissues . The effects of early handling of turkey poults were variable, depending on the number of DEX treatments and the age of the birds . These results suggest that early handling can affect the susceptibility of stressed turkeys to E . coli air sacculitis and TOC and that differences in susceptibility may be influenced by age and individual variability in the stress response. Poult Sci, 2001 Sep, 80(9), 1305 - 13 Effect of early handling of turkey poults on later responses to a dexamethasone-Escherichia coli challenge . 1 . Production values and physiological response; Huff GR et al.; The stress responses of mice and rats has been shown to be permanently altered by brief, gentle handling during the first 10 d of life, resulting in increased BW and resistance to stress-induced immunosuppression . The purpose of this study was to determine whether early handling of turkey poults could permanently affect production values and physiology of adult turkeys . Turkey poults were handled 0, 1 (1x), or 2 (2x) times daily for the first 10 d after hatch . Handling consisted of gently catching each poult and holding it for 10 s . On Day 11 after hatch, half of the birds from each handling treatment were treated with three injections of 2 mg dexamethasone (DEX)/kg BW on alternating days . On the day of the third DEX injection, duplicate pens of birds were also inoculated in the airsac with 0 or 50 cfu of Escherichia coli . The same birds were treated with a second series of DEX injections at 5 wk of age . Two weeks later, all birds were weighed, and 3 wk later four birds per pen were bled and 10 birds per pen were necropsied; relative organ weights were then determined . Surviving birds were treated with a third series of DEX injections at 10 wk of age; 2 wk later, all surviving turkeys were bled, weighed, and necropsied . Feed consumption was determined weekly . There were no differences due to handling treatment on the body weights or on the relative organ weights of birds that died after the first DEX treatment . Birds treated with a second DEX injection at 5 wk of age and handled 1x daily had decreased BW . Those handled 1x or 2x daily had higher feed conversion ratios . Surviving birds that were given a third DEX treatment had higher BW and no difference in feed conversion when handled 1x or 2x daily . Relative liver, heart, and spleen weights were affected by handling of DEX-E . coli-treated birds, as were serum chemistry values for calcium, iron, glucose, total protein, blood urea nitogen, uric acid, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and gamma-glutamyltransferase . Handling also affected the numbers of white blood cells of DEX-treated birds . These results indicate that brief and gentle handling of turkey poults during the first 10 d after hatch has lasting effects on production values and physiology of adult turkeys and that these effects can be positive or negative . These results suggest a genetic divergence in the response to stress and its effect on production values and physiology of commercial turkey populations. Chem Pharm Bull (Tokyo), 2001 Sep, 49(9), 1128 - 31 Application of combined reagent solution to the oxidative refolding of recombinant human interleukin 6; Harada T et al.; Human interleukin 6 (hIL-6), which is a cytokine involved in diverse biological activities, consists of a four-helix bundle with two disulfide bonds . For the clinical use of hIL-6 in cancer therapy, designing of commercial-scale production systems of recombinant hIL-6 (rhIL-6) expressed by E . coli has been attempted . Since rhIL-6 has been produced as inclusion bodies in the expression systems reported to date, establishment of a strategy to achieve a high yield of refolding of this recombinant protein is quite desirable . It has been reported that oxidation of rhIL-6 under a completely denaturing condition suppresses aggregation during the refolding process {Ejima et al., Biotechnol . Bioeng., 62, 301-310 (1999)} . In this protocol, however, small but significant amounts of unidentified by-products unavoidably arose, which might be problematic in the therapeutic use of rhIL-6 . In the present study, detailed characterization of the individual by-products has been performed on inspection of peptide maps, and the by-products found to originate from improperly formed disulfide bonds, most of which are disulfide-linked dimers . In order to minimize these by-products, combined solutions of urea and LiCl were used for oxidative refolding of rhIL-6 . It was demonstrated that combined use of 1-2 M urea and 1-3 M LiCl effectively suppresses the formation of the by-products as well as aggregates . We propose that the use of the combined reagents can be an alternative method for refolding of rhIL-6 for clinical purposes. Biol Pharm Bull, 2001 Sep, 24(9), 1064 - 7 Metabolism and interaction of bisphenol A in human hepatic cytochrome P450 and steroidogenic CYP17; Niwa T et al.; The metabolism of bisphenol A (BPA) was determined for 11 forms of human hepatic cytochromes P450 (CYPs) expressed in the yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli . Additionally, the effect of BPA on the progesterone 17alpha-chydroxylase activity of CYP17 was investigated . CYP2C18 catalyzed BPA metabolism most efficiently, followed by CYP2C19 and CYP2C9 . CYP2C9 and CYP2C18 exhibited the highest affinity (Km=3.9 microM) for BPA metabolism . The Vmax of CYP2C18 (8.10 nmol x min(-1) x nmol CYP(-1)) was 5 times higher than that of CYP2C9 . Although the Vmax of CYP2C19 was 1.5 times higher than that of CYP2C18, the affinity of CYP2C19 was 12 times lower than that of CYP2C9 and CYP2C18 . Therefore the intrinsic clearance (Vmax/Km) of CYP2C18 was more than 5 times higher than that of CYP2C9 and CYP2C19 . On the other hand, BPA exhibited a competitive-type inhibition of the progesterone 17alpha-hydroxylase activity of CYP17 with a Ki value of 71 microM, whereas no metabolism of BPA by CYP17 was detected . These results suggest that BPA is mainly metabolized by the CYP2C subfamily in human liver, and that BPA inhibits human steroidogenic CYP17 activities. Mikrobiologiia, 2001 Jul-Aug, 70(4), 487 - 94 {Putrescine as a oxidative stress protecting factor in Escherichia coli}; Tkachenko AG et al.; The level of expression of oxyR, the gene that protects Escherichia coli against oxidative stress, was enhanced by physiological concentrations of the biogenic amine putrescine . This level was directly proportional to the degree of negative DNA supercoiling . 1,4-Diamino-2-butanone (DAB), a specific inhibitor of ornithine decarboxylase, the key enzyme of polyamine synthesis, produced an inhibitory effect on the level of oxyR expression under oxidative stress, which was relieved by the addition of putrescine . The direct relationship between putrescine concentration and the degree of negative DNA supercoiling suggests that the mechanism of action of putrescine as the modulator of oxyR transcription activity is based on both its direct influence on the gene expression level and its indirect effect mediated by topological DNA changes . Putrescine was shown to produce a protective effect if the DNA is damaged by reactive oxygen species. Bioorg Khim, 2001 Jul-Aug, 27(4), 282 - 90 {Extensive complementarity of the Shine-Dalgarno region and 3'-terminal sequence of 16S ribosomal RNA is inefficient for translation in vivo}; Komarova AV et al.; Translation initiation in Escherichia coli involves as a rule complementary interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD) . The translation efficiency is believed to be directly affected by the affinity of the ribosome to the mRNA initiation region . Earlier, high-affinity RNA ligands to E . coli ribosomes were selected by the SELEX approach, with the ligands containing an extended SD-sequence well represented . In this work, we examined the ability of artificial ribosome binding sites (RBSs) containing such an extended (10-nt) SD-sequence (superSD) to drive translation in vivo, as well as its ability to form the translation initiation complex in vitro . Toe print experiments showed the formation of a ternary initiation complex on mRNA comprising superSD . Moreover, they proved the formation of an extended SD-duplex in the binary 30S-mRNA complex . Nevertheless, the superSD appeared to be inefficient in translation in vivo . We believe that the initiation complex involving a superSD-element is too stable to be functional; it may impede the transition from initiation to elongation, thus disrupting the transcription-translation coupling and inhibiting the formation of polysomes. Bioorg Khim, 2001 Jul-Aug, 27(4), 257 - 64 {Structural-functional study of recombinant forms of onconase}; Vorob'ev II et al.; A method for expression of an onconase gene leading to a soluble form of the protein was developed . The enzymatic and cytotoxic properties of the protein's recombinant forms were studied . Recombinant onconase with an additional N-terminal Met residue isolated in nondenaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity . The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase . The method proposed is useful for the onconase structure-function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy. Yakugaku Zasshi, 2001 Sep, 121(9), 701 - 5 {Colony and phage-plaque direct sequencings by dye-terminator methods}; Sasho C et al.; Direct sequencing using lambda phage DNA and E . coli colonies with plasmid DNA is a very powerful technique . Almost all of the reported direct sequencing methods involve either radioactive sequencing or fluorescent dye-primer sequencing . We present a direct colony sequencing strategy that uses a dye terminator (BigDye terminator kit) together with dye primer sequencing . We found that single-colony sequencing with the terminator yielded about 500 base pairs of sequence information . Signal strength was not improved when the number of cycles increased to 40 . The colony used for the sequencing was estimated to contain about 5.6 x 10(7) cells . In addition, although a single plaque consisted of 2 x 10(6) cells, the pfu was not high enough to read with single-cycle sequencing, and only about 300 base pairs of sequence information were obtained from a single plaque using two cycle-sequencing reactions (re-cycle sequencing) . The optimal amounts of the template were 500 ng of purified lambda DNA and 1 x 10(7) pfu of the lambda phage suspension, but with BigDye terminator it was possible to detect as little as 50 ng of purified lambda DNA and 2 x 10(6) pfu for lambda phage suspensions . Thus, colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries. Science, 2001 Sep 14, 293(5537), 2040 - 4 Pathway databases: a case study in computational symbolic theories; Karp PD; A pathway database (DB) is a DB that describes biochemical pathways, reactions, and enzymes . The EcoCyc pathway DB (see describes the metabolic, transport, and genetic-regulatory networks of Escherichia coli . EcoCyc is an example of a computational symbolic theory, which is a DB that structures a scientific theory within a formal ontology so that it is available for computational analysis . It is argued that by encoding scientific theories in symbolic form, we open new realms of analysis and understanding for theories that would otherwise be too large and complex for scientists to reason with effectively. Nucleic Acids Res, 2001 Sep 15, 29(18), 3873 - 81 Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator; Lutz R et al.; The mode of action of regulated promoters is largely determined by kinetic parameters which govern the interaction between promoters and proteins involved in induction and repression of transcription . To gain insight into the interplay between positively and negatively acting transcriptional regulators, in this case AraC and LacR, we have generated a panel of promoter sequences derived from P(lac), the promoter of the Escherichia coli lac operon . The function of these promoters is limited at different steps and to various extents within the pathway of RNA polymerase (RNAP)/promoter interaction . Moreover, in all promoters the cAMP receptor protein binding site was replaced by the binding motif of AraC to prevent pleiotropic effects in vivo upon activation . Analyzing the activation of these promoters by AraC in vivo under conditions of repression by LacR and derepression yielded a three step model of transcription initiation which reveals mechanisms of AraC and LacR action . Our data show three distinct rate limiting steps at which AraC can exert its function . In general, the activator accelerates the formation of the first stable complex between RNAP and promoter . At most promoter sequences, however, its main impact is on the conversion of the closed to the open complex . However, AraC is also capable of eliminating limitations at steps following open complex formation. Nucleic Acids Res, 2001 Sep 15, 29(18), 3822 - 34 Nuclear DNA polymerase beta from Leishmania infantum . Cloning, molecular analysis and developmental regulation; Taladriz S et al.; We have identified a novel polymerase beta (Pol beta)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans . This protein, named Li Pol beta, shows a nuclear localization that contrasts with the mitochondrial localization of Pol beta from Crithidia fasciculata, a closely related parasite, the only polymerase beta described so far in Trypanosomatidae . Li Pol beta, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol beta-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved . In agreement with this, Li Pol beta, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity . Cell synchronization experiments showed a correlation between both Li Pol beta mRNA and protein levels along the parasite cell cycle . Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol beta at the infective phase of the parasite . The data suggest a role of Li Pol beta in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. J Biol Chem, 2001 Nov 16, 276(46), 42965 - 70 Epub 2001 Sep 13. Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins; Panagotopulos SE et al.; Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis . Unfortunately, the structural basis of this phenomenon is not fully understood . Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes . The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains . To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain . We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model . In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I . For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart . In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains . For the picket fence model, maximal quenching should occur at two different levels in the bilayer . The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 A from the center of the bilayer . This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs. J Biol Chem, 2001 Nov 16, 276(46), 42915 - 22 Epub 2001 Sep 13. Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance; Nakajima H et al.; Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome . Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown . In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis . A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface . Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased . In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer . It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer . This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect . The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected . The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate . The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins. Free Radic Biol Med, 2001 Sep 15, 31(6), 816 - 23 Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals; Sidorkina O et al.; The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes . We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals . These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent . Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities . We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity . FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg . The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity . FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all . Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained . The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines . Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues . The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes. Mech Ageing Dev, 2001 Oct, 122(15), 1771 - 86 Effects of age and food restriction on oxidative DNA damage and antioxidant enzyme activities in the mouse aorta; Guo ZM et al.; In this study, DNA damage in mouse aortic cells was measured using the comet assay . The tail moment of the comet assay in aortic cells obtained from 26-month-old mice fed ad libitum (O-AL) was significantly increased as compared to 6-month-old mice fed ad libitum (Y-AL) after the cells were incubated with formamidopyrimidine-DNA glycosylase (Fpg), which specifically recognizes oxidized purines, endonuclease III (Endo III), which specifically recognizes oxidized pyrimidines, or the combination of Endo III and Fpg . The tail moment in aortic cells obtained from 26-month-old mice fed a food-restricted diet (O-FR) was significantly reduced as compared to O-AL mice after the cells were incubated with the combination of Endo III and Fpg . These results indicate that oxidative DNA lesions, i.e . the Endo III- and Fpg-sensitive sites, increase with age in mouse aortic cells and that FR attenuates the age-related increase in oxidative DNA damage . To determine if the changes in oxidative DNA damage in mouse aortic cells are related to the antioxidant status in these cells, we measured the activities of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, extracellular-SOD, catalase and glutathione peroxidase-1 in the mouse aorta . We observed that the activities of all antioxidant enzymes studied were significantly increased with age and that FR attenuated the age-related increase . These data indicate that the age-related increase and FR-induced decrease in oxidative DNA damage, i.e . the Endo III- and Fpg-sensitive sites, in mouse aortic cells is not due to alteration of the antioxidant defense system. Vet Microbiol, 2001 Nov 8, 83(2), 147 - 59 Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene; Chiers K et al.; A PCR assay for the detection of Actinobacillus pleuropneumoniae was developed based on the amplification of a dsbE-like gene . All of 157 field isolates of A . pleuropneumoniae reacted in the PCR by the amplification of a 342bp product . No reaction was observed with related bacterial species or other bacterial species isolated from pigs, except for A . lignieresii . The lower detection limit of the PCR was 10(2) CFU per PCR test tube and was not affected by the addition of 10(6) CFU Escherichia coli . The PCR was evaluated on mixed bacterial cultures from nasal and tonsillar swabs as well as suspensions of nasal conchae and tonsils obtained from specific pathogen-free (SPF) pigs, experimentally infected pigs, and pigs from farrow-to-finish herds . The results of the new PCR were compared with a PCR based on the detection of the omlA gene coding for an outer membrane protein, with a commercially available PCR (Adiavet APP, Adiagene, Saint-Brieuc, France), and with conventional culturing . No positive reactions were observed with any of the PCR methods in samples of SPF animals . In samples of the other animals, no or low significant differences between nasal swabs and suspensions as well as tonsillar swabs and suspensions were observed in any method . In general, more positive results were obtained from tonsillar samples in comparison to nasal samples . Interassay sensitivity and specificity values were assessed for each test by pair wise comparisons between assays . The agreement between tests was evaluated by calculating Cohen's kappa coefficient . From these analyses the three PCR assays showed a good agreement . The dsbE-based PCR proved to be highly sensitive (95 and 93%) and specific (82 and 74%) in comparison to the omlA-based PCR and the commercially available PCR, respectively . It was concluded that the dsbE-like gene-based PCR is a reliable diagnostic assay for demonstration of A . pleuropneumoniae . Furthermore, it was demonstrated that tonsillar swabs can be used for the detection of the pathogen in healthy carrier animals. FEMS Microbiol Lett, 2001 Sep 11, 203(1), 95 - 102 Characterization of the Mycobacterium tuberculosis region containing the mpt83 and mpt70 genes; Juarez MD et al.; Like the products of the genes mpt83 and mpt70, the putative protein encoded by the gene located between these genes was undetectable in Mycobacterium tuberculosis with an antiserum raised against the recombinant protein . The protein showed 100% homology with M . tuberculosis Rv2874 and similarities with CcdA and DipZ proteins involved in cytochrome-c biogenesis in bacteria . Expression analysis by RT-PCR and transcriptional fusions of Rv2874 and their neighbor genes Rv2871, Rv2872, mpt83 and mpt70 with lacZ suggest that these genes are part of an operon and their transcription is driven by promoter regions located 5' upstream of mpt83 and of Rv2874 genes. FEMS Microbiol Lett, 2001 Sep 11, 203(1), 41 - 7 The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila; Steinert M et al.; The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila . In this study, we correlated the pigment production of two lly-positive L . pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant . The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD) . This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment . One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases . By screening a genomic library of L . pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E . coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence . DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene . The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA . Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence. FEMS Microbiol Lett, 2001 Sep 11, 203(1), 29 - 33 Binding of lactoferrin and free secretory component to enterotoxigenic Escherichia coli; de Oliveira IR et al.; The ability of two glycoproteins of human milk, lactoferrin and free secretory component, to bind to Escherichia coli colonization factors (CFAs) was investigated using immunocytochemistry assays of enriched fimbrial extracts . The results revealed that lactoferrin binds to fimbrial CFA I adhesin but not to CFA II adhesin (CS1 and CS3), while free secretory component interacts with both CFA I and CFA II adhesins . Our data indicate that lactoferrin and free secretory component, which are very abundant proteins of human milk, could play an important role against infant enteric disease by blocking bacterial adhesion. Biochim Biophys Acta, 2001 Oct 1, 1514(2), 239 - 52 Lipopolysaccharide can activate BK channels of arterial smooth muscle in the absence of iNOS expression; Yakubovich N et al.; The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca(2+)-dependent K(+) channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells . Confocal laser scanning microscopy and patch clamp recordings were utilised . Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries . This staining was absent in cells fixed immediately after donor rat sacrifice . LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2-4 h of donor rat sacrifice . It was found that LPS (10-100 microg/ml) rapidly and reversibly increased the open probability of BK channels in these patches . This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N(omega)-nitro-L-arginine . LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice . LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice . These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells. Biochim Biophys Acta, 2001 Oct 1, 1514(2), 230 - 8 The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking; Ding PZ et al.; The melibiose carrier of Escherichia coli is a transmembrane protein that comprises 12 transmembrane helices connected by periplasmic and cytoplasmic loops, with both the N- and C-termini located on the cytoplasmic side . Our previous studies of second-site revertants suggested proximity between several helices, including helices XI and I . In this study, we constructed six double cysteine mutants, each having one cysteine in helix I and the other in helix XI: three mutants, K18C/S380C, D19C/S380C, and F20C/S380C, have their cysteine pairs near the cytoplasmic side of the carrier, and the other three, T34C/G395C, D35C/G395C, and V36C/G395C, have their cysteine pairs near the periplasmic side . In the absence of substrate, disulfide formations catalyzed by iodine and copper-(1,10-phenanthroline)(3) indicate that helix I and helix XI are in immediate proximity to each other on the periplasmic side but not on the cytoplasmic side, as shown by protease cleavage analyses . We infer that the two helices are tilted with respect to each other, with the periplasmic sides in close proximity. Biochim Biophys Acta, 2001 Oct 1, 1514(2), 165 - 9 A high-throughput screen for MscL channel activity and mutational phenotyping; Maurer JA et al.; A novel fluorescence-based screen for bacterial mechanosensitive ion-channel activity has been developed . This assay is capable of clearly distinguishing the previously observed gain of function and loss of function phenotypes for the Escherichia coli mechanosensitive channel of large conductance (Ec-MscL) . The method modifies Molecular Probes' Live/Dead BacLight bacterial viability assay to monitor MscL channel activity as a function of bacterial survival from osmotic downshock. Arch Biochem Biophys, 2001 Sep 15, 393(2), 329 - 38 Expressed CYP4A4 metabolism of prostaglandin E(1) and arachidonic acid; Aitken AE et al.; Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their omega-hydroxylated products . Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the omega-hydroxylated product of arachidonate, is a potent vasoconstrictor . Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization . A CYP4A4 construct was prepared and expressed in Escherichia coli . The enzyme was purified, and its activity with substrates prostaglandin E(1) (PGE(1)) and AA was examined in the presence and absence of cytochrome b(5) (cyt b(5)) and with a heme-depleted form of cyt b(5) (apo b(5)) . The stimulatory role played by cyt b(5) in this system is not dependent on electron transfer from cyt b(5) to the CYP4A4 as similar stimulation was observed with apo b(5) . Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result . Both flavin and heme reduction rates were constant in the absence and presence of cyt b(5) or apo b(5) . CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5) . Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4 . The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE(1) or AA metabolism . All but 10-UDYA were effective inhibitors of CYP4A4 . Arch Biochem Biophys, 2001 Sep 15, 393(2), 290 - 6 Osmotic stress induced by carbohydrates enhances expression of foreign proteins in Escherichia coli; Kagawa N et al.; Arabinose has been serendipitously observed to enhance the expression of P450s in Escherichia coli . To understand the mechanism of this arabinose-dependent enhancement, the effects of various carbohydrates were investigated . Surprisingly, a series of sugars, including pentoses and hexoses, enhanced the foreign gene expression in a manner similar to arabinose . Furthermore, glycerol, a poor carbon source, also enhanced P450 expression . These results indicate that the enhancement is independent of the specific efficiency of the carbon source and also suggest the involvement of osmotic stress . Therefore, the effect of the sigma(s) (also termed sigma(38)) factor, a sigma subunit of RNA polymerase that plays a central role in regulating the expression of osmotic stress response genes, has been examined . We found that the glycerol-dependent increase in P450 expression was not observed in sigma(s)-deficient E . coli, indicating that carbohydrates enhance the foreign gene expression in E . coli via the induction of the osmotic stress response . The results suggest the important role of the osmotic stress response in posttranscriptional processes required for producing functional proteins . Arch Biochem Biophys, 2001 Sep 15, 393(2), 255 - 61 Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity; Hanna IH et al.; Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen . In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6 . An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems . Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase . The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301 . Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol . Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme . These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase . Arch Biochem Biophys, 2001 Sep 15, 393(2), 187 - 91 1,4-Dihydro-l-phenylalanine-its synthesis and behavior in the phenylalanine ammonia-lyase reaction; Skolaut A et al.; 1,4-Dihydro-l-phenylalanine, a nonaromatic derivative of l-phenylalanine, has been isolated for the first time . It was synthesized as a yet unobserved minor product in the Birch reduction of l-phenylalanine . This is unexpected because it has an electron donor substituent at a reduced sp(3)-carbon atom of the ring system . Kinetic measurements with phenylalanine ammonia-lyase showed that 1,4-dihydro-l-phenylalanine is no substrate but a moderately good competitive inhibitor of the enzymatic reaction . This is in agreement with its predicted behavior and provides further evidence for the plausibility of the recently proposed mechanism of action of phenylalanine ammonia-lyase . Appl Immunohistochem Mol Morphol, 2001 Sep, 9(3), 276 - 80 Immunocytochemical localization of mitochondrial single-stranded DNA-binding protein in mitochondria-rich cells of normal and neoplastic human tissue; Muller-Hocker J et al.; Mitochondrial single-stranded DNA-binding protein (mtSSB) is necessary for mtDNA replication . So far the protein has been studied mainly in Escherichia coli and in cell cultures of lower mammals . In this investigation, the authors studied the expression of mtSSB in normal and neoplastic human tissue by light and electron immunocytochemistry . mtSSB has been detected in various tissues and particularly in mitochondria-rich tissues such as external eye muscles and parietal cells of the stomach and in mitochondria-rich tumors (oncocytomas) of various origins . Ultraimmunocytochemistry disclosed the specific distribution of immunoreactive mtSSB over the mitochondria . The staining intensity was heterogeneous . Forty-five percent had a labeling index (silver grains/microm2) greater than 1 and less than 3, approximately 20% had an index of 3 or greater, and 15% of mitochondria remained unstained . The mean labeling index was 1.83 . Immunolabeling showed a linear correlation with the mitochondrial profile area (r = 0.82) . In conclusion, mtSSB is regularly expressed in normal and neoplastic human tissue of different origin, function, and differentiation . The heterogeneous staining pattern most probably reflects the functional heterogeneity of mitochondria. Arch Virol, 2001 Jul, 146(7), 1391 - 7 Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus; Qiu T et al.; The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined . It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa . Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus . The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY) . The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1. Arch Virol, 2001 Jul, 146(7), 1355 - 67 Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene; Ribeiro BM et al.; We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters . The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A . gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated . The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene . Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase . Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter . The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay. Arch Virol, 2001 Jul, 146(7), 1297 - 306 Inhibition of potato virus Y NIa activity: preparation of monoclonal antibody directed against PVY NI protein that inhibits cleavage of PVY polyprotein; Rouis S et al.; A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY) . Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen . Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP) . These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins . When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm . In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products . It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI. Rapid Commun Mass Spectrom, 2001, 15(18), 1752 - 9 Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry; Walters JJ et al.; Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53 . SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes . When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information . Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction . The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work . Two known polymorphisms of the p53 gene were genotyped . A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis . Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis . Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS . This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A) . Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products . Rapid Commun Mass Spectrom, 2001, 15(18), 1693 - 700 Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry; Ma Y et al.; A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented . In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra . The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein . In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer . This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides . After {(32)P} labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein . Hum Mol Genet, 2001 Sep 1, 10(18), 1971 - 82 Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease; Mukherji M et al.; Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia . Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid . Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate . Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis . The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands . The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation . The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes . Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding . The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation . The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation. Reprod Domest Anim, 2001 Aug, 36(3-4), 157 - 61 Postovulatory effect of intravenous administration of lipopolysaccharide (Escherichia coli, O55:B5) on the endocrine status of recently ovulated sows; Mwanza AM et al.; The effects of lipopolysaccharide (Escherichia coli, O55:B5), administered 18 h after ovulation in the second oestrus after weaning on the hormonal profiles in 14 Swedish cross-bred (Landrace x Yorkshire) multiparous sows were studied . The endotoxin group (E-group) sows were administered with 300 ng/kg of lipopolysaccharide (LPS) whereas the control group (C-group) sows were administered 5 ml of saline intravenously via an indwelling jugular cannula . Blood samples for hormonal analyses were collected from all sows until slaughter . In the E-group, progesterone, cortisol and prostaglandin F(2 alpha) metabolite levels increased significantly (p < 0.05) following LPS compared with the C-group . It can be concluded from this study that apart from elevating cortisol and prostaglandin F(2 alpha) metabolite, LPS also elevates progesterone levels. Oral Microbiol Immunol, 2001 Oct, 16(5), 290 - 5 Characterization of serum antibody to Actinobacillus actinomycetemcomitans GroEL-like protein in periodontitis patients and healthy subjects; Tabeta K et al.; Recent evidence suggests that molecular mimicry between bacterial and human heat shock protein 60 (hsp60) is involved in various conditions of autoimmune and infectious diseases . Many periodontopathic bacteria have been reported to express GroEL-like protein that is homologous to human hsp60 . In this study, the presence of antibodies to the hsp60 of Actinobacillus actinomycetemcomitans in the sera of periodontitis patients and periodontally healthy control subjects was evaluated by enzyme-linked immunosorbent assay using a recombinant A . actinomycetemcomitans GroEL as an antigen . Furthermore, their cross-reactivity with Escherichia coli GroEL and Mycobacterium bovis BCG hsp65 was examined . The mean values of antibody were 0.624 (range 0.088-1.113) and 0.728 (range 0.217-1.296) in control subjects and periodontitis patients, respectively . The antibody levels to A . actinomycetemcomitans after absorption with E . coli GroEL and M . bovis BCG clearly decreased in both control subjects and periodontitis patients . The remaining antibody levels to A . actinomycetemcomitans GroEL after absorption with M . bovis BCG hsp65 were higher than those with E . coli GroEL, indicating higher cross-reactivity with E . coli GroEL . These results suggest that not only periodontitis patients but also periodontally healthy subjects may be infected with A . actinomycetemcomitans but that the part of the antibody could be derived from the cross-reactivity with E . coli GroEL . Any relationship of the antibody to the disease, however, remains to be determined. Mol Microbiol, 2001 Sep, 41(5), 1187 - 98 Novel mode of transcription regulation by SdiA, an Escherichia coli homologue of the quorum-sensing regulator; Yamamoto K et al.; SdiA, an Escherichia coli homologue of the quorum-sensing regulator, controls the expression of the ftsQAZ operon for cell division . Transcription of ftsQ is under the control of two promoters, upstream ftsQP2 and downstream ftsQP1, which are separated by 125 bp . SdiA activates transcription from ftsQP2 in vivo . Here, we demonstrate that SdiA facilitates the RNA polymerase binding to ftsQP2 and thereby stimulates transcription from P2 . Gel shift and DNase I footprinting assays indicated that SdiA binds to the ftsQP2 promoter region between -51 and -25 with respect to the P2 promoter . Activation of ftsQP2 transcription by SdiA was observed with a mutant RNA polymerase containing a C-terminal domain (CTD)-deleted alpha-subunit (alpha 235) but not with RNA polymerase containing sigma(S) or a CTD-deleted sigma(D) (sigma(D)529) . In good agreement with the transcription assay, no protection of P2 was observed with the RNA polymerase holoenzymes, E sigma(S) and E sigma(D)529 . These observations together indicate that: (i) SdiA supports the RNA polymerase binding to ftsQP2; and (ii) this recruitment of RNA polymerase by SdiA depends on the presence of intact sigmaCTD . This is in contrast to the well-known mechanism of RNA polymerase recruitment by protein-protein contact between class I factors and alpha CTD . In addition to the P2 activation, SdiA inhibited RNA polymerase binding to the ftsQP1 promoter and thereby repressed transcription from P1 . Gel shift assays indicate weak binding of SdiA to the P1 promoter region downstream from -13 (or +112 with respect to P2) . Neither alpha CTD nor sigma CTD are required for this inhibition . Thus, the transcription repression of P1 by SdiA may result from its competition with the RNA polymerase in binding to this promoter. Mol Microbiol, 2001 Sep, 41(5), 1101 - 11 Escherichia coli DNA glycosylase Mug: a growth-regulated enzyme required for mutation avoidance in stationary-phase cells; Mokkapati SK et al.; The Escherichia coli DNA glycosylase Mug excises 3,N(4)-ethenocytosines (epsilon C) and uracils from DNA, but its biological function is obscure . This is because epsilon C is not found in E . coli DNA, and uracil-DNA glycosylase (Ung), a distinct enzyme, is much more efficient at removing uracils from DNA than Mug . We find that Mug is overexpressed as cells enter stationary phase, and it is maintained at a fairly high level in resting cells . This is true of cells grown in rich or minimal media, and the principal regulation of mug is at the level of mRNA . Although the expression of mug is strongly dependent on the stationary-phase sigma factor, sigma(S), when cells are grown in minimal media, it shows only a modest dependence on sigma(S) when cells are grown in rich media . When mug cells are maintained in stationary phase for several days, they acquire many more mutations than their mug(+) counterparts . This is true in ung as well as ung(+) cells, and a majority of new mutations may not be C to T . Our results show that the biological role of Mug parallels its expression in cells . It is expressed poorly in exponentially growing cells and has no apparent role in mutation avoidance in these cells . In contrast, Mug is fairly abundant in stationary-phase cells and has an important anti-mutator role at this stage of cell growth . Thus, Mug joins a very small coterie of DNA repair enzymes whose principal function is to avoid mutations in stationary-phase cells. Genes Cells, 2001 Sep, 6(9), 803 - 14 HscA is involved in the dynamics of FtsZ-ring formation in Escherichia coli K12; Uehara T et al.; BACKGROUND: FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non-dividing Escherichia coli . However, it assembles in cytokinetic rings at the early stages of septation . Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined . RESULTS: At 42 degrees C, JE10715 mutant bacteria formed multinucleated filaments with a highly reduced number of FtsZ-rings at potential division sites . The JE10715 phenotype resulted from a mis-sense mutation in the hscA gene which encodes a heat shock Hsp70 family protein, with a single alanine-to-valine substitution at position 192 within the ATPase domain . Both JE10715 and the hscA knockout strain of JE10715 were completely complemented by a plasmid-born, wild-type hscA gene, but not by a mutant-type hscA715 gene . An hscA conditional knockout of the wild-type strain under non-permissive conditions exhibited longer rod cells with an abnormal localization of FtsZ . The over-expression of dnaK partially complemented the JE10715 mutation . In vitro, the ATPase activity of the mutant protein HscA715 was reduced to 63% of wild-type HscA activity . HscA co-sedimented with FtsZ-polymers in the presence of GTP . CONCLUSION: HscA is involved in FtsZ-ring formation, through a chaperon-like interaction with FtsZ . Defects in hscA, however, can partially be compensated for by redundant genes, including the wild-type dnaK. J Mol Biol, 2001 Sep 14, 312(2), 405 - 18 Crystal structures of human gephyrin and plant Cnx1 G domains: comparative analysis and functional implications; Schwarz G et al.; The molybdenum cofactor (Moco) consists of a unique and conserved pterin derivative, usually referred to as molybdopterin (MPT), which coordinates the essential transition metal molybdenum (Mo) . Moco is required for the enzymatic activities of all Mo-enzymes, with the exception of nitrogenase and is synthesized by an evolutionary old multi-step pathway that is dependent on the activities of at least six gene products . In eukaryotes, the final step of Moco biosynthesis, i.e . transfer and insertion of Mo into MPT, is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals . Gephyrin is ubiquitously expressed, and was initially found in the central nervous system, where it is essential for clustering of inhibitory neuroreceptors in the postsynaptic membrane . Gephyrin and Cnx1 contain at least two functional domains (E and G) that are homologous to the Escherichia coli proteins MoeA and MogA, the atomic structures of which have been solved recently . Here, we present the crystal structures of the N-terminal human gephyrin G domain (Geph-G) and the C-terminal Arabidopsis thaliana Cnx1 G domain (Cnx1-G) at 1.7 and 2.6 A resolution, respectively . These structures are highly similar and compared to MogA reveal four major differences in their three-dimensional structures: (1) In Geph-G and Cnx1-G an additional alpha-helix is present between the first beta-strand and alpha-helix of MogA . (2) The loop between alpha 2 and beta 2 undergoes conformational changes in all three structures . (3) A beta-hairpin loop found in MogA is absent from Geph-G and Cnx1-G . (4) The C terminus of Geph-G follows a different path from that in MogA . Based on the structures of the eukaryotic proteins and their comparisons with E . coli MogA, the predicted binding site for MPT has been further refined . In addition, the characterized alternative splice variants of gephyrin are analyzed in the context of the three-dimensional structure of Geph-G . Biochem Biophys Res Commun, 2001 Sep 21, 287(2), 519 - 21 Residues 137 and 153 of XylS influence contacts with the C-terminal domain of the RNA polymerase alpha subunit; Ruiz R et al.; XylS and XylS1 are transcriptional regulators that stimulate transcription from the Pm promoter for the meta-cleavage pathway operon for alkylbenzoate degradation . These regulators that differ in five amino acids interact with alpha-CTD domain of RNA polymerase . These interactions take place preferentially through residues 291 in XylS and 289 in XylS1 . Substitution at position 137 and 153 in XylS influence the interactions with alpha-CTD because single and double mutants in these positions turned preferential interactions to residue 289 . Biochem Biophys Res Commun, 2001 Sep 21, 287(2), 348 - 54 Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35; Lim WJ et al.; The gene encoding an intracellular isoamylase from the Pectobacterium chrysanthemi PY35 was cloned in Escherichia coli DH5alpha and sequenced . The isoamylase gene (amyX) had an open reading frame of 1974 bp encoding 657 amino acid residues with a calculated molecular weight of 74,151 Da . The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel . Isoamylase from P . chrysanthemi PY35 had 59% pairwise amino acid identity with glycogen debranching enzyme from E . coli and contained the four regions conserved among all amylolytic enzymes . The isoamylase was optimally active at pH 7 and 40 degrees C . AmyX hydrolyzed alpha-1,6-glycosidic linkages of amylopectin, while did not hydrolyze alpha-1,4-glycosidic linkages of amylose . Anal Biochem, 2001 Sep 15, 296(2), 270 - 8 Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse; Wilson CJ et al.; An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments . YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used . Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid . The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts . YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background . Int J Med Microbiol, 2001 Aug, 291(3), 227 - 30 Distribution of the outer membrane haem receptor protein ChuA in environmental and human isolates of Escherichia coli; Hoffmann H et al.; ChuA, the haem receptor of Escherichia coli, is thought to contribute to the pathogenicity of E . coli strains causing extraintestinal infections . We investigated the prevalence and distribution of chuA in E . coli analysing 304 strains from different origins . 30% of E . coli strains isolated from the environment and about 70% of E . coli strains isolated from human sources carried chuA . No difference in chuA prevalence was found between commensals isolated from the intestine of healthy volunteers and isolates from extraintestinal infections . Our results indicate that ChuA might be involved in the colonization of human hosts. Nucleosides Nucleotides Nucleic Acids, 2001 Aug, 20(8), 1449 - 61 Synthesis of a new 5'-O-triphosphate analog of 5-methyl 2'-O-deoxycytidine . Preliminary in vitro labelling for non-radioactive detection of DNA; Rodriguez-Tanty C et al.; We report the synthesis of the triphosphate of 5-methyl 4-N-{6-(p-bromobenzamido)hex-1-yl}-2'-O-deoxycytidine 3A . We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-{n-{6-(p-bromobenzamido) caproyl amino}alk-1-yl}-2'-deoxycytidine compounds, and confirmed their presence by 1H-NMR studies . In vitro DNA labeling with modified nucleotides is preliminarily evaluated. Antioxid Redox Signal, 2001 Aug, 3(4), 597 - 609 The OGG1 gene encodes a repair enzyme for oxidatively damaged DNA and is involved in human carcinogenesis; Shinmura K et al.; 8-Hydroxyguanine (oh8G) is a major base lesion produced by reactive oxygen species . oh8G in DNA causes G:C to T:A transversions and, thus, could be responsible for mutations that lead to carcinogenesis . A human DNA glycosylase/AP lyase encoded by the OGG1 gene has an activity to remove directly oh8G from DNA, and suppresses the mutagenic effect of oh8G . OGG1 protein has a helix-hairpin-helix-GPD motif as a domain for both DNA binding and catalysis, a nuclear localization signal, and a mitochondria targeting signal . Among multiple OGG1 isoforms, OGG1-type la is expressed predominantly in human cells and repairs chromosomal DNA in the nucleus . Inactivation of the OGG1 gene in yeast and mice leads to elevated spontaneous mutation frequency in the cells . The human OGG1 gene maps to chromosome 3p26.2, and allelic deletions of this region occur frequently in a variety of human cancers . Moreover, the OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among human populations . Repair activities of some mutated and polymorphic OGG1 proteins are lower than those of wild-type OGG1-type la-Ser326 protein and, thus, could be involved in human carcinogenesis. Antioxid Redox Signal, 2001 Aug, 3(4), 535 - 48 Oxidation of zinc finger transcription factors: physiological consequences; Webster KA et al.; Redox-sensitive cysteine residues are present in the interaction domains of many protein complexes . There are examples in all of the major categories of transcription factors, including basic region, leucine zipper, helix-loop-helix, and zinc finger . Zinc finger structures require at least two zinc-coordinated cysteine sulfhydryl groups, and oxidation or alkylation of these can eliminate DNA-binding and transcriptional functions . We review here the evidence for oxidation of zinc finger cysteines, the pathways and reactive oxygen intermediates involved, and the functional and physiological consequences of these reactions . Despite skepticism that the strongly reducing intracellular environment would permit significant oxidation of cysteine residues within zinc finger transcription factors, there is compelling evidence that oxidation occurs both in vitro and in vivo . Early reports demonstrating reversible oxidation of zinc-coordinated cysteines with loss of binding function in vitro were shown to reflect accurately the changes in intact cells, and these in turn have been shown to correlate with physiological changes . In particular, the accumulation of oxidized Spl zinc fingers during aging, and estrogen receptors in tamoxifen-resistant breast cancers are dramatic examples of what may be a general sensitivity of zinc finger factors to changes in the redox state of the cell. Prog Nucleic Acid Res Mol Biol, 2001, 68, 365 - 86 Properties and functions of human uracil-DNA glycosylase from the UNG gene; Krokan HE et al.; The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences . PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2 . Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease . The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out . Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine . These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E . coli chromosome and were used to examine biological consequences of AP sites . It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination . However, one major role of UNG2 is to remove misincorporated dUMP residues . Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell . We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution. Prog Nucleic Acid Res Mol Biol, 2001, 68, 305 - 14 Crystallizing thoughts about DNA base excision repair; Hollis T et al.; Chemically damaged bases are removed from DNA by glycosylases that locate the damage and cleave the bond between the modified base and the deoxyribose sugar of the DNA backbone . The detection of damaged bases in DNA poses two problems: (1) The aberrant bases are mostly buried within the double helix, and (2) a wide variety of chemically different modifications must be efficiently recognized and removed . The human alkyladenine glycosylase (AAG) and Escherichia coli Alka DNA glycosylases excise many different types of alkylated bases from DNA . Crystal structures of these enzymes show how substrate bases are exposed to the enzyme active site and they suggest mechanisms of catalytic specificity . Both enzymes bend DNA and flip substrate bases out of the double helix and into the enzyme active site for cleavage . Although AAG and AlkA have very different overall folds, some common features of their substrate-binding sites suggest related strategies for the selective recognition of a chemically diverse group of alkylated substrates. Prog Nucleic Acid Res Mol Biol, 2001, 68, 257 - 71 Enzymology of mitochondrial base excision repair; Bogenhagen DF et al.; A number of laboratories have shown that those types of DNA damage that are generally reparable by base excision repair are efficiently repaired in mtDNA . In contrast, most types of damage that require other sorts of repair machinery are not effectively repaired in mtDNA . We have shown that a set of highly purified mitochondrial proteins, including AP endonuclease (APE), DNA polymerase gamma, and mtDNA ligase, is capable of efficiently repairing abasic (AP) sites in mtDNA . These three enzymes appear to conduct all four steps in a conventional BER mechanism: incision, removal of the 5'-deoxyribosephosphate by dRP lyase, polymerization, and ligation . Both DNA polymerase gamma and mtDNA ligase possess some dRP lyase activity . DNA polymerase gamma is a member of the family A of DNA polymerases, with clear homology to DNA pol I of E . coli, while mtDNA ligase is an alternatively expressed form of DNA ligase III . The dRP lyase activities discovered in these mitochondrial enzymes are not unique, but are found in all representatives tested of the family-A DNA polymerases and of the ATP-dependent DNA ligases . These dRP lyase activities have low turnover rates that may have important implications for the overall process of BER . All proteins involved in maintenance of mtDNA are encoded in the nuclear genome and must be directed to mitochondria in order to act on mtDNA . Thus, it is evident that the scope of DNA repair activities undertaken within mitochondria is determined by the set of nucleus-encoded DNA repair enzymes that are capable of being imported into the organelle . A review of DNA repair proteins that may be imported into mitochondria in various organisms will be presented. Prog Nucleic Acid Res Mol Biol, 2001, 68, 223 - 34 Mechanism of action of Escherichia coli formamidopyrimidine N-glycosylase: role of K155 in substrate binding and product release; Rabow L et al.; Escherichia coli formamidopyrimidine N-glycosylase (fpg) is a DNA glycosylase with an associated beta,delta-lyase activity . We have recently shown that the highly conserved lysine residue K155 is important for base recognition . Incubation of a double-stranded DNA containing an abasic site with the wild-type fpg protein generated only beta,delta-product . However, incubation of a double-stranded DNA containing an abasic site opposite a small gap with fpg protein generated predominantly beta-product . These data suggested that the induction of a double-strand break by fpg led to the destabilization of the protein-DNA covalent intermediate, causing the fpg protein to prematurely dissociate from the DNA substrate . Furthermore, when a double-stranded DNA containing an abasic site opposite an A was used as a substrate, K155A mutant fpg protein yielded a mixture of beta- and beta,delta-products . These data suggested that K155 is essential for maintaining the stability of the intermediary protein-DNA covalent complex . Pre-steady-state burst kinetics showed that mutation in K155 led to the apparent disappearance of the initial burst, suggesting that the rate of product release from K155A is much greater than the rate of chemical reaction catalyzed by the mutant enzyme . This is consistent with the idea that K155A dissociates prematurely from the covalent complex, leading to a higher turnover number observed for K155A for DNA substrate containing an AP site. Prog Nucleic Acid Res Mol Biol, 2001, 68, 193 - 205 Multiple DNA glycosylases for repair of 8-oxoguanine and their potential in vivo functions; Hazra TK et al.; 8-Oxoguanine (8-oxoG) is a critical mutagenic lesion because of its propensity to mispair with A during DNA replication . All organisms, from bacteria to mammals, express at least two types of 8-oxoguanine-DNA glycosylase (OGG) for repair of 8-oxoG . The major enzyme class (OGG1), first identified in Escherichia coli as MutM (Fpg), and later in yeast and humans, excises 8-oxoG when paired with C, T, and G but rarely with A . In contrast, a distinct and less abundant OGG, OGG2, prefers 8-oxoG when paired with G and A as a substrate, and has been characterized in yeast and human cells . Recently, OGG2 activity was detected in E . coli which was subsequently identified to be Nei (Endo VIII) . In view of the ubiquity of OGG2, we have proposed a model named "bipartite antimutagenic processing of 8-oxoguanine" and is an extension of the original "GO model." The GO model explains the presence of OGG1 (MutM) that excises 8-oxoG from nonreplicated DNA . If 8-oxoG mispairs with A during replication, MutY excises A and provides an opportunity for insertion of C opposite 8-oxoG during subsequent repair replication . Our model postulates that whereas OGG1 (MutM) is responsible for global repair of 8-oxoG in the nonreplicating genome, OGG2 (Nei) repairs 8-oxoG in nascent or transcriptionally active DNA . Interestingly, we observed that MutY and MutM reciprocally inhibited each other's catalytic activity but observed no mutual interference between Nei and MutY . This suggests that the recognition sites on the same substrate for Nei and MutY are nonoverlapping . Human OGG1 is distinct from other oxidized base-specific DNA glycosylases because of its extremely low turnover, weak AP lyase activity, and nonproductive affinity for the abasic (AP) site, its first reaction product . OGG1 is activated nearly 5-fold in the presence of AP-endonuclease (APE) as a result of its displacement by the latter . These results support the "handoff" mechanism of BER in which the enzymatic steps are coordinated as a result of displacement of the DNA glycosylase by APE, the next enzyme in the pathway . The physiological significance of multiple OGGs and their in vivo reaction mechanisms remain to be elucidated by further studies. Prog Nucleic Acid Res Mol Biol, 2001, 68, 165 - 88 Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts; Sanderson RJ et al.; The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay . Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene . Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E . coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred . Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides . The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts . The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C . Uracil-initiated BER DNA synthesis in extracts of E . coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined . Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4) . A reduced, but detectable level of BER was observed in extracts of E . coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold. Prog Nucleic Acid Res Mol Biol, 2001, 68, 107 - 23 Mammalian Ogg1/Mmh gene plays a major role in repair of the 8-hydroxyguanine lesion in DNA; Nishimura S; 8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage . MutM of E . coli and OGG1 of Saccharomyces cerevisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions . Recently, cDNA clones of four isoforms (types 1a, 1b, 1c, and type 2) of human OGG1 homologs (hMMH) were isolated . However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells . We have chosen two approaches to clarify this issue . First, using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we found that hMMH type 1a protein is in fact expressed in many types of human cells, showing that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity . Furthermore, we have shown that upon antibody-mediated depletion of hMMH type 1a protein in a whole-cell extract, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesion in human cells . In our second approach we have generated a mouse line carrying a mutant Mmh allele by targeted gene disruption . Mmh homozygous mutant mice were found to be physically normal in appearance, but to have lost the nicking activity for substrate DNA containing 8-OH-G in liver extracts . In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mutant mice at 8 weeks of age was 3-fold higher compared with wild-type or heterozygous mice . A further increase of 8-OH-G up to 7-fold was observed in 14-week-old animals . These results indicate that exposure of DNA to internal oxidative species constantly produces the mutagenic DNA adduct 8-OH-G in mice, and that Mmh plays an essential role in the repair of this type of oxidative DNA damage. Proc Natl Acad Sci U S A, 2001 Sep 11, 98(19), 10560 - 5 Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host; Tooley AJ et al.; The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp . PCC6803) was reconstituted in Escherichia coli . Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter . Genes for the apoprotein (C-phycocyanin alpha subunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were expressed from the trc promoter on a second plasmid . Upon induction, recombinant E . coli used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria . About a third of the apo-CpcA was converted to holo-CpcA . No significant bilin addition took place in a similarly engineered E . coli strain that lacks cpcE and cpcF . This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis . These studies also demonstrate the feasibility of generating constructs of these proteins in situ for use as fluorescent protein probes in living cells. Mol Biol Cell, 2001 Sep, 12(9), 2800 - 12 Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast; Thrower DA et al.; We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction . Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein . Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase . In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase . On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation . Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome . These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage . The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA. Mol Biol Cell, 2001 Sep, 12(9), 2756 - 66 The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission; Fukushima NH et al.; Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes . In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide . Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself . In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro . Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo . With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin . The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules . Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission . Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission . Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission. J Biol Chem, 2001 Nov 16, 276(46), 42881 - 6 Epub 2001 Sep 11. Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein; Kopriva S et al.; Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants . Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins . UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent . This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a {4Fe-4S} cluster as the cofactor . EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01 . Therefore, Mossbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic {4Fe-4S}(2+) cluster . This cluster was unusual because only three of the iron sites exhibited the same Mossbauer parameters . The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites . Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a {4Fe-4S} center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria. Infect Immun, 2001 Oct, 69(10), 6537 - 40 Cloning, nucleotide sequence, and expression of the Brucella melitensis sucB gene coding for an immunogenic dihydrolipoamide succinyltransferase homologous protein; Zygmunt MS et al.; The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced . The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus and Escherichia coli, respectively . Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein. Infect Immun, 2001 Oct, 69(10), 6515 - 9 Cytotoxic necrotizing factor type 1-positive Escherichia coli causes increased inflammation and tissue damage to the prostate in a rat prostatitis model; Rippere-Lampe KE et al.; Infection of rat prostates with cytotoxic necrotizing factor type 1 (CNF1)-positive uropathogenic Escherichia coli caused more inflammation-mediated morphological and histological tissue damage than did infection with isogenic CNF1-negative mutants . These striking differences occurred despite the finding that bacterial counts for the strain pairs were indistinguishable . We conclude that CNF1 contributes to E . coli virulence in a model of acute prostatitis . To our knowledge, the results of this study provide the first demonstration of a role for any uropathogenic E . coli virulence factor in acute prostatitis. Infect Immun, 2001 Oct, 69(10), 6483 - 94 Neospora caninum microneme protein NcMIC3: secretion, subcellular localization, and functional involvement in host cell interaction; Naguleswaran A et al.; In apicomplexan parasites, host cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules . Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the host cell and thus mediate and establish the physical interaction between the parasite and the host cell surface . This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date . NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains . We determined the subcellular localization of NcMIC3 prior to and following host cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following host cell lysis in a temperature-dependent manner . Surface-exposed NcMIC3 could be detected up to 2 to 3 h following host cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes . In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system . Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants . Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N . caninum tachyzoites to adhere to Vero cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains . NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero cells showed that the protein binds to the Vero cell surface . In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient . Our data are indicative of a functional role of NcMIC3 in host cell infection. Infect Immun, 2001 Oct, 69(10), 6140 - 7 Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression; Thorpe CM et al.; Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS) . Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS . We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response . We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs . We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78 . Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1 . Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis . Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA . We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved . We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins . Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection. Infect Immun, 2001 Oct, 69(10), 5991 - 6 Impaired pulmonary NF-kappaB activation in response to lipopolysaccharide in NADPH oxidase-deficient mice; Koay MA et al.; Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-kappaB . We investigated the role of NADPH oxidase in the NF-kappaB activation pathway by utilizing knockout mice (p47phox-/-) lacking the p47phox component of NADPH oxidase . Wild-type (WT) controls and p47phox-/- mice were treated with intraperitoneal (i.p.) Escherichia coli lipopolysaccharide (LPS) (5 or 20 microg/g of body weight) . LPS-induced NF-kappaB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox-/- mice 90 min after treatment with 20 but not 5 microg of i.p . LPS per g . In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox-/- mice compared to WT mice following treatment with aerosolized LPS . In contrast to NF-kappaB activation in p47phox-/- mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice . We conclude that LPS-induced NF-kappaB activation is deficient in the lungs of p47phox-/- mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response. Bioorg Med Chem, 2001 Sep, 9(9), 2479 - 84 Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain; Camarero JA et al.; Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell . A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells . The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure . This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research. Bioorg Med Chem, 2001 Sep, 9(9), 2387 - 93 Designed potent multivalent chemoattractants for Escherichia coli; Gestwicki JE et al.; Bacterial chemotactic responses are initiated when certain small molecules (i.e., carbohydrates, amino acids) interact with bacterial chemoreceptors . Although bacterial chemotaxis has been the subject of intense investigations, few have explored the influence of attractant structure on signal generation and chemotaxis . Previously, we found that polymers bearing multiple copies of galactose interact with the chemoreceptor Trg via the periplasmic binding protein glucose/galactose binding protein (GGBP) . These synthetic multivalent ligands were potent agonists of Escherichia coli chemotaxis . Here, we report on the development of a second generation of multivalent attractants that possess increased chemotactic activities . Strikingly, the new ligands can alter bacterial behavior at concentrations 10-fold lower than those required with the original displays; thus, they are some of the most potent synthetic chemoattractants known . The potency depends on the number of galactose moieties attached to the oligomer backbone and the length of the linker tethering these carbohydrates . Our investigations reveal the plasticity of GGBP; it can bind and mediate responses to several carbohydrates and carbohydrate derivatives . These attributes of GGBP may underlie the ability of bacteria to sense a variety of ligands with relatively few receptors . Our results provide insight into the design and development of compounds that can modulate bacterial chemotaxis and pathogenicity. Bioorg Med Chem, 2001 Sep, 9(9), 2373 - 9 Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo; Pastrnak M et al.; The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities . Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities . To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized . Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope . The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection. Bioorg Med Chem, 2001 Sep, 9(9), 2237 - 42 Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol; Huang Q et al.; Taxadiene, the key intermediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl diphosphate in cell-free extracts of Escherichia coli by overexpressing genes encoding isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase and taxadiene synthase . In addition, by the expression of three genes encoding four enzymes on the terpene biosynthetic pathway in a single strain of E . coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield of 1.3mg per liter of cell culture . The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production of taxoids by non-paclitaxel producing organisms through pathway engineering. Eur J Anaesthesiol, 2001 Sep, 18(9), 572 - 5 Hypotension during endotoxemia in aged humans; Krabbe KS et al.; BACKGROUND: and objective The aim of this study was to determine possible age-associated differences in human blood pressure regulation during an immunological challenge in healthy subjects . METHODS: Eight healthy young volunteers (median age 24 yr) and nine healthy elderly volunteers (median age 66 yr) received an intravenous bolus injection of Escherichia coli endotoxin (2 ng kg(-1)) . Blood pressure, heart rate and core temperature were monitored during the first 7 h . Plasma catecholamine concentrations were measured at hourly intervals . RESULTS: The elderly showed a significantly more pronounced decrease in mean arterial pressure 4-7 h after endotoxin administration compared with the young controls (ANOVA; age x time; P < 0.0005) . This mainly reflected a decrease in the systolic blood pressure in the elderly . The heart rate of both groups increased without difference between groups . Increased plasma epinephrine concentrations were found 2-3 h after endotoxin administration in both groups . Five hours after the endotoxin challenge, the epinephrine concentration had returned to control values in the elderly group only, in spite of decreased blood pressure . CONCLUSION: In conclusion, healthy elderly subjects fail to maintain a constant mean arterial pressure in response to the immunological challenge of endotoxemia. Cell Microbiol, 2001 Sep, 3(9), 579 - 85 Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development; Tobe T et al.; Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas . Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies . In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony . Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract . In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells . These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell. J Am Chem Soc, 2001 Sep 19, 123(37), 8895 - 901 Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy; Serber Z et al.; Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells . Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm . Less relevant is the selective incorporation of (15)N . Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed . We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications. J Mol Graph Model, 2001, 19(6), 495 - 513 Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modelling; Dolan MA et al.; Structure models for each of the secondary structure regions from the Escherichia coli 16S rRNA (58 separate elements) were constructed using a constraint satisfaction modelling program to determine which helices deviated from classic A-form geometry . Constraints for each rRNA element included the comparative secondary structure, H-bonding conformations predicted from patterns of base-pair covariation, tertiary interactions predicted from covariation analysis, chemical probing data, rRNA-rRNA crosslinking information, and coordinates from solved structures . Models for each element were built using the MC-SYM modelling algorithm and subsequently were subjected to energy minimization to correct unfavorable geometry . Approximately two-thirds of the structures that result from the input data are very similar to A-form geometry . In the remaining instances, the presence of internal loops and bulges, some sequences (and sequence covariants) and accessory information require deviation from A-form geometry . The structures of regions containing more complex base-pairing arrangements including the central pseudoknot, the 530 region, and the pseudoknot involving base-pairing between G570-U571/A865-C866 and G861-C862/G867-C868 were predicted by this approach . These molecular models provide insight into the connection between patterns of H-bonding, the presence of unpaired nucleotides, and the overall geometry of each element. J Biol Chem, 2001 Nov 9, 276(45), 41938 - 44 Epub 2001 Sep 10. Action of RuvAB at replication fork structures; McGlynn P et al.; The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery . In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease . This fork regression may be catalyzed by RecG but is thought to occur even in its absence . Here we test whether RuvAB helicase can also catalyze the unwinding of forked DNA to form Holliday junctions . We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm . If the binding of RuvB is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction . This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer . These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation. J Biol Chem, 2001 Nov 30, 276(48), 44993 - 5000 Epub 2001 Sep 10. Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution; Harrop SJ et al.; CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms . Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo . The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution . The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin . The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues . Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site . The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes. J Biol Chem, 2001 Nov 30, 276(48), 44919 - 25 Epub 2001 Sep 10. Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin; Carr KM et al.; Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border . The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork . We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis . DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA . In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid . That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC. J Biol Chem, 2001 Nov 30, 276(48), 44541 - 50 Epub 2001 Sep 10. A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment; Tieman BC et al.; Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds . Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C . Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization . Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested . Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms . Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C) . Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s) . The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction . Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished . Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement. J Biol Chem, 2001 Nov 9, 276(45), 41588 - 93 Epub 2001 Sep 10. Substitution of aspartate and glutamate for active center histidines in the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system maintain phosphotransfer potential; Napper S et al.; The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIA(Glc) were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation . The mutants {H189E}enzyme I, {H15D}HPr, and {H90E}enzyme IIA(Glc) retained ability for phosphorylation as indicated by {(32)P}phosphoenolpyruvate labeling . As the active center histidines of both enzyme I and enzyme IIA(Glc) undergo phosphorylation of the N(epsilon2) atom, while HPr is phosphorylated at the N(delta1) atom, a pattern of successful substitution of glutamates for N(epsilon2) phosphorylations and aspartates for N(delta1) phosphorylations emerges . Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes. Bioorg Med Chem Lett, 2001 Oct 8, 11(19), 2623 - 6 RNA-DNA hybrids containing damaged DNA are substrates for RNase H; Shiels JC et al.; During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H . Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal . The rates and sites of digestion by E . coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined . The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex . There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs . Cleavage is partially redirected to the position adjacent to the damaged sites . The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex . These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand. Virus Res, 2001 Nov 5, 79(1-2), 153 - 64 Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor; Chisholm J et al.; The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) is responsible for the processing of the RNA1-encoded (P1) and RNA2-encoded (P2) polyproteins . Cleavage between the VPg and Pro domains is inefficient in vitro and in E . coli, resulting in the accumulation of the VPg-Pro . In this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors . Recombinant Pro and VPg-Pro were partially purified using an E . coli expression system . A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of the Pro . The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains) . Neither the Pro nor the VPg-Pro could cleave in trans P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains . These results are discussed in light of the possible regulation of the proteinase activity during virus replication. Virus Res, 2001 Nov 5, 79(1-2), 137 - 44 The structure and function of a gene encoding a basic peptide from prawn white spot syndrome virus; Zhang X et al.; Prawn white spot syndrome virus (WSSV) is the major pathogen responsible for the high mortality of cultured prawns . A gene (termed as p6.8) encoding a basic peptide was found by screening the cDNA and DNA libraries of WSSV . The peptide was highly homologous with proteins rich in arginine and lysine . A fusion protein containing the p6.8 and green fluorescent protein (GFP) genes was cloned into pBV220 and expressed in E . coli . Gel mobility shift assay indicated that the peptide encoded by p6.8 had the capability of binding DNA and might be involved in DNA packaging. Virus Res, 2001 Nov 5, 79(1-2), 27 - 37 Expression of the simian varicella virus glycoprotein E; Gray WL et al.; Simian varicella virus (SVV) is closely related to human varicella-zoster virus (VZV) and induces a varicella-like disease in nonhuman primates . The SVV genome encodes a glycoprotein E (gE) which is homologous to the gE of VZV and other alphaherpesviruses . The SVV gE was expressed in Escherichia coli and rabbits were immunized with the recombinant gE fusion proteins to generate polyclonal gE antiserum . Immunofluorescence and immunoprecipitation analyses demonstrated that the SVV gE is expressed on the surface and within SVV-infected cells . The gE is also expressed on SVV virions as indicated by serum neutralization assay . The mature SVV gE is glycosylated and is similar in size ( approximately 100 kd) to the mature VZV gE . Immunohistochemical analysis detected gE within skin vesicles and lung tissue of SVV-infected monkeys . Analysis of the humoral immune response to gE in an SVV-infected monkey determined that anti-gE antibody is induced as early as day 9 postinfection and persists at high titer for longer than 4 months . The simian varicella model offers an opportunity to investigate the role of gE in viral pathogenesis and immunity and to evaluate its potential as a varicella vaccine. Chem Phys Lipids, 2001 Aug, 112(2), 109 - 19 The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding; de Brouwer AP et al.; Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein . In addition, active PC-TP was obtained from inclusion bodies . An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer . PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS) . By incubation with microsomal membranes the endogenous PE and PG were replaced by PC . Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound . PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat . Struct . Biol . 7 (2000) 408) . Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein . This suggests that Lys(55) be involved in the binding of the PC molecule. Cell, 2001 Sep 7, 106(5), 585 - 94 Crystal structure of LexA: a conformational switch for regulation of self-cleavage; Luo Y et al.; LexA repressor undergoes a self-cleavage reaction . In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH . Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus . We have solved the crystal structures of several mutant forms of LexA . Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center . Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction . We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation. Mutat Res, 2001 Sep 20, 496(1-2), 97 - 104 Exposure of Escherichia coli to intestinal myoelectrical activity-related electric field induces resistance against subsequent UV(254 nm) (UVC) irradiation; Wojcik-Sikora A et al.; Survival of Escherichia coli K-12 AB1157 irradiated with UVC (UV(254 nm)) was enhanced after pre-treatment with a low-tension electric field (EF) . The EF used was identical to the electrical field generated by the small intestine (myoelectrical migrating complex--MMC), registered in a healthy calf and transmitted into the memory of an EF generator . The EF emitted by the generator was transmitted via electrodes placed in shaken bacterial cultures . The protective effects of the EF on the E . coli survival after exposure to UV were: (i) observed only for the dnaJ(+)dnaK(+) strain, and not for the DeltadnaJdnaK heat shock mutant; (ii) strictly dependent on the temperature at which the bacteria were grown; (iii) most obvious when the bacteria were incubated at 37 degrees C . Moreover, the MMC-related EF and a higher temperature (40 degrees C) show a similar protective effect against UV-irradiation . The results point to the involvement of the heat shock response in the low-tension EF-induced protection of bacterial cells against UVC-irradiation . Additionally, treatment with the MMC-related EF affects total protein contents and their pattern in E . coli cells . The EF-treatment did not show any influence on the level of the argE3(ochre) --> Arg(+) reversions. Mutat Res, 2001 Sep 20, 496(1-2), 89 - 95 Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12; Vasilieva S; PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979 . In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU) . Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment . NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics . PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation . PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude . PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E . coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda . Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity . The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E . coli . PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response. Mutat Res, 2001 Sep 20, 496(1-2), 33 - 8 Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli; Melo SF et al.; Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences . Much is known about stannous chloride (SnCl(2)) toxicity, although, there is no general agreement regarding its genotoxicity . Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine . We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl(2) treatment . Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl(2) . E . coli culture was treated simultaneously with SnCl(2) and a specific extract . Our results showed a reduction of the SnCl(2) effect on the survival of the cultures in presence of the crude extracts . The extract of M . ilicifolia showed the highest level of protection action against the SnCl(2) effect in comparison with the other extracts . This protector effect could due to the redox properties of these crude extracts . The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl(2) oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl(2) effect. J Inorg Biochem, 2001 Jul, 85(4), 301 - 7 Human catalytic antibodies with glutathione peroxidase activity; Fang F et al.; In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens . Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library . The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25 . In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E . coli . After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively . With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site . Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied. J Helminthol, 2001 Sep, 75(3), 273 - 8 Molecular cloning and characterization of a 21 kDa protein secreted from Trichinella pseudospiralis; Nagano I et al.; Recombinant protein was produced from the cDNA library of Trichinella pseudospiralis, which seemed to form part of the excretory-secretory (ES) products . The library was constructed from cDNA of muscle larvae at 1 month post-infection, and immunoscreened with antibody against T . pseudospiralis ES products . A clone, designated Tp21-3, contained a cDNA transcript of 657 bp in length with a single open reading frame, which encoded 172 amino acids (19617 Da in the estimated molecular mass) . The predicted amino acid sequence of clone Tp21-3 had a similarity of 76% to that of clone ORF 17.20 (GenBank under accession number U88239) from T . spiralis . The recombinant fusion proteins encoded by clone Tp21-3 were produced in an Escherichia coli expression system and affinity purified . On Western blotting analysis, Tp21-3 recombinant proteins migrated at 40 kDa and reacted to antibody against T . pseudospiralis ES products and T . pseudospiralis-infected sera . Sera were developed against Tp 21-3 recombinant proteins, which reacted to a single band migrating at 21 kDa in crude worm extract and ES products from T . pseudospiralis on Western blotting analysis, and reacted with stichocytes of T . pseudospiralis on immunohistochemical staining. Biochemistry, 2001 Sep 18, 40(37), 11234 - 45 Enzyme electrokinetics: energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase; Leger C et al.; Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB) . The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension . Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD . Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting . The sequence of binding and protonation events over the whole catalytic cycle is deduced . Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II. Biochemistry, 2001 Sep 18, 40(37), 11219 - 26 Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability; Dirnbach E et al.; The in vitro reactivation of unfolded Escherichia coli alkaline phosphatase (AP) in the presence of the two natively bound metals Zn2+ and Mg2+ produces two protein species, characterized by different guanidine hydrochloride denaturation kinetics . The high-lability AP form slowly converts to the low-lability form in a first-order reaction with a characteristic lifetime (inverse rate constant) of approximately 300 h at pH 8.0 and 25 degrees C . Addition of Zn2+ and Mg2+ ligands to (folded) apo-AP also produces two protein species, with denaturation kinetics and a long conversion lifetime similar to those found in refolding AP . In contrast, adding Zn2+ alone to apo-AP produces only the high-lability species with no subsequent structural change, suggesting that Mg2+ binding is the event which is responsible for the production of the low-lability AP . The rate of conversion from high- to low-lability AP was found to be linearly dependent on Mg2+ concentration, indicating that Mg2+ binding is rate limiting for this reaction . Experiments where either Zn2+ or Mg2+ was added first, with the second metal added later, show that Mg2+ binding is slowed by the prior presence of bound Zn2+ . Mg2+ binding to Zn-AP also slightly increases the enzymatic activity; however, the extent of formation of the low-lability species is related to the square of the Mg2+-induced activity increase . Thus the binding of two Mg2+ to AP produces the dramatic reduction in the rate of denaturation that characterizes the low-lability species . The data suggest the possibility of long distance intersubunit interactions and a role for Mg2+ in providing "kinetic stability" for AP. Biochemistry, 2001 Sep 18, 40(37), 11184 - 92 Probing the structure and stability of a hybrid protein: the human-E . coli thioredoxin chimera; Louis JM et al.; The structure and stability of a hybrid protein composed of N-terminal human and C-terminal E . coli thioredoxin domains were investigated by NMR, fluorescence, and circular dichroism spectroscopy . We demonstrate that the chimeric protein is correctly folded and exhibits the common thioredoxin architecture . However, the stability of the hybrid protein toward thermal and chemical denaturation is clearly reduced when compared with both parent proteins . Altogether, our data indicate that the interface between the two folding units of thioredoxin is tolerant toward changes in exact interdigitation of side chains, allowing for the formation of the unique overall thioredoxin fold . Further, the gene encoding the human-E . coli chimera was tested in vivo whether it supports the assembly of filamentous phages . No complementation of a thioredoxin-deficient E . coli mutant for the replication of the phages M13 or fd was observed, suggesting that parts of the overall protein structure in the N-terminal domain are crucial for this activity. Biochemistry, 2001 Sep 18, 40(37), 11168 - 75 Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B; Boehlein SK et al.; Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate . Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme . Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding . Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E . coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle . While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor . A sulfoximine reported recently by Koizumi et al . as a competitive inhibitor of the ammonia-dependent E . coli asparagine synthetase A (AS-A) {Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J . (1999) J . Am . Chem . Soc . 121, 5799-5800} can be regarded as such a species . We found that this sulfoximine also inhibited AS-B, effectively irreversibly . Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine . Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B. Biochemistry, 2001 Sep 18, 40(37), 11106 - 13 Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor; Melnyk RA et al.; Biophysical study of the transmembrane (TM) domains of integral membrane proteins has traditionally been impeded by their hydrophobic nature . As a result, an understanding of the details of protein-protein interactions within membranes is often lacking . We have demonstrated previously that model TM segments with flanking cationic residues spontaneously fold into alpha-helices upon insertion into membrane-mimetic environments . Here, we extend these studies to investigate whether such constructs consisting of TM helices from biological systems retain their native secondary structures and oligomeric states . Single-spanning TM domains from the epidermal growth factor receptor (EGFR), glycophorin A (GPA), and the influenza A virus M2 ion channel (M2) were designed and synthesized with three to four lysine residues at both N- and C-termini . Each construct was shown to adopt an alpha-helical conformation upon insertion into sodium dodecyl sulfate micelles . Furthermore, micelle-inserted TM segments associated on SDS-PAGE gels according to their respective native-like oligomeric states: EGFR was monomeric, GPA was dimeric, and M2 was tetrameric . This approach was then used to investigate whether one or both of the TM segments (Tar-1 and Tar-2) from the Escherichia coli aspartate receptor were responsible for its homodimeric nature . Our results showed that Tar-1 formed SDS-resistant homodimers, while Tar-2 was monomeric . Furthermore, no heterooligomerization between Tar-1 and Tar-2 was detected, implicating the Tar-1 helix as the oligomeric determinant for the Tar protein . The overall results indicate that this approach can be used to elucidate the details of TM domain folding for both single-spanning and multispanning membrane proteins. Biochemistry, 2001 Sep 18, 40(37), 11073 - 81 Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts; Nazimiec M et al.; The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair . The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex . Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E . coli cells and overproducing cells . UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl . In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct . They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1 . UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut) . Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII . DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site . Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding . Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG- . These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage. Biochemistry, 2001 Sep 18, 40(37), 11060 - 4 Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system; Hohsaka T et al.; Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research . Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins . In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons . The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system . The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein . Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU . This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility. Biochemistry, 2001 Sep 18, 40(37), 11030 - 6 Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli; Kim J et al.; Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP . Ornithine and IMP function as activators, whereas UMP is an inhibitor . CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors . Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer . The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units . Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains . There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains . Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units . When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP . In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors . These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains . Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed . The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer . In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain . No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another. Biochemistry, 2001 Sep 18, 40(37), 11013 - 21 Differential activity and structure of highly similar peroxidases . Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2; Nielsen KL et al.; Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2 . The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron {Ostergaard, L., et al . (2000) Plant Mol . Biol . 44, 231-243}, whereas spectroscopic studies of HRP A2 in solution at room temperature {Feis, A., et al . (1998) J . Raman Spectrosc . 29, 933-938} showed five-coordinated heme iron, which is common in peroxidases . Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant . Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases . Ostergaard et al . (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates . In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases . We confirm these predictions for ATP A2, HRP A2, and HRP C . The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1) . The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively . The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions . The results are of general importance to the use of homologous models and structure determination at low temperatures. J Struct Biol, 2001 May-Jun, 134(2-3), 191 - 203 On the evolution of protein folds: are similar motifs in different protein folds the result of convergence, insertion, or relics of an ancient peptide world? Lupas AN, Ponting CP, Russell RB. This paper presents and discusses evidence suggesting how the diversity of domain folds in existence today might have evolved from peptide ancestors . We apply a structure similarity detection method to detect instances where localized regions of different protein folds contain highly similar sequences and structures . Results of performing an all-on-all comparison of known structures are described and compared with other recently published findings . The numerous instances of local sequence and structure similarities within different protein folds, together with evidence from proteins containing sequence and structure repeats, argues in favor of the evolution of modern single polypeptide domains from ancient short peptide ancestors (antecedent domain segments (ADSs)) . In this model, ancient protein structures were formed by self-assembling aggregates of short polypeptides . Subsequently, and perhaps concomitantly with the evolution of higher fidelity DNA replication and repair systems, single polypeptide domains arose from the fusion of ADSs genes . Thus modern protein domains may have a polyphyletic origin . Gen Comp Endocrinol, 2001 Jul, 123(1), 38 - 50 Production, in vitro characterisation, in vivo clearance, and tissue localisation of recombinant barramundi (Lates calcarifer) insulin-like growth factor II; Degger B et al.; Recombinant barramundi insulin-like growth-factor-II (bIGF-II) has been produced in Escherichia coli after modification of an expression plasmid that coded for a chicken IGF-II fusion protein . The bIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, and refolded . The protein was then released from the fusion protein by cleavage with subtilisin BPN' . Finally the protein was purified to homogeneity with a number of HPLC steps . In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis when compared to human and barramundi IGF-I (bIGF-I) . The in vivo distribution of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake of radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calcarifer) . Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that bIGF-II was degraded faster than bIGF-I . Moreover, neutral gel chromatography of these samples suggested this difference may be due to reduced affinity of bIGF-II, compared to blGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi circulation . Based on these results, it would appear that elements important in the function of IGFs have been well conserved during vertebrate evolution . However, to clearly define the IGF system in fish it will be necessary to characterise the IGFBPs present and to determine how they influence the biological actions of native IGFs. Genome, 2001 Aug, 44(4), 651 - 7 Functional analysis of the Arabidopsis thaliana mismatch repair gene MSH2; Ade J et al.; The Arabidopsis thaliana MSH2 (AtMSH2) gene encodes a protein that belongs to a family of highly conserved proteins (MutS homologues (MSH)) involved in DNA mismatch repair . Sequence analysis strongly suggests that this single copy gene is indeed a homologue of MSH2, a gene known to play a central role in eukaryotic mismatch repair . In this report, we show that the AtMSH2 protein has functional attributes characteristic of previously described mismatch repair proteins . First, over-expression of this protein in Escherichia coli leads to a mutator phenotype similar to that reported previously for known functional homologues . Second, gel retardation assays revealed that the AtMSH2 protein has a 10-fold greater affinity for DNA containing a single pair of mismatched nucleotides versus perfectly matched DNA . These results provide experimental evidence that AtMSH2 is indeed a functional homologue of MutS. Prog Nucleic Acid Res Mol Biol, 2001, 69, 317 - 49 Domain-domain communication in aminoacyl-tRNA synthetases; Alexander RW et al.; Aminoacyl-tRNA synthetases are modular proteins, with domains that have distinct roles in the aminoacylation reaction . The catalytic core is responsible for aminoacyl adenylate formation and transfer of the amino acid to the 3' end of the bound transfer RNA (tRNA) . Appended and inserted domains contact portions of the tRNA outside the acceptor site and contribute to the efficiency and specificity of aminoacylation . Some aminoacyl-tRNA synthetases also have distinct editing activities that are localized to unique domains . Efficient aminoacylation and editing require communication between RNA-binding and catalytic domains, and can be considered as a signal transduction system . Here, evidence for domain-domain communication in aminoacyl-tRNA synthetases is summarized, together with insights from structural analysis. Cancer, 2001 Aug 15, 92(4), 822 - 9 Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma; Bauer C et al.; BACKGROUND: Recently, the authors reported identification and cloning of several novel immunogenic antigens in squamous cell lung carcinoma . Of 14 corresponding genes, 9 mapped in an amplified chromosomal region with the gene for eIF-4G that was amplified most frequently . METHODS: Recombinant eIF-4G was expressed in E . coli and screened with sera from patients with squamous cell carcinoma of the lung and of the head and neck . Protein extracts from squamous cell carcinoma tissues were analyzed for eIF-4G expression by Western blot analysis . Mutation analysis was performed using an automatic DNA sequencer . RESULTS: The authors screened a spectrum of 33 heterologous sera from lung carcinoma patients and detected antibodies against eIF-4G in 5 of these sera (15%) . They found no immune response to eIF-4G in 17 sera from squamous cell carcinoma tissues derived from the head and neck . In addition, no antibodies were found to eIF-4G in a group of 17 control sera from individuals without known tumor formation . Sequence analysis of the eIF-4G gave no indication of mutations . To analyze the expression of eIF-4G, a monoclonal antibody was used . Western blot analysis clearly showed overexpression in the tumor tissue compared with the corresponding normal lung tissues . CONCLUSIONS: The translation initiation factor eIF-4G is the first protein in which the gene shows amplification, has increased expression in a human tumor, and induces an immune response in patients . eIF-4G lends itself as a marker for the diagnosis of and possibly as a future therapeutic target in patients with squamous cell lung carcinoma .
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