Biochemistry, 1999 Apr 6, 38(14), 4586 - 94 Subunit affinities and stoichiometries of the human papillomavirus type 11 E1:E2:DNA complex; Chao SF et al.; The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication . Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium . For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared . The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site . HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM . Binding was saturable and consistent with a single class of noninteracting sites . The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication . Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM) . E1 binding to Fl-E1E2BS was of very low affinity . Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM . This effect was not dependent upon ATP or magnesium ion . Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur . In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each . The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA. Biochemistry, 1999 Apr 6, 38(14), 4526 - 32 Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli; Osborne JP et al.; Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli . Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases . Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane . The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown . Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump . This would imply lability of the ligands to CuB . In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination . The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water) . Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population . These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping. Biochemistry, 1999 Apr 6, 38(14), 4448 - 54 Site-directed mutants of charged residues in the active site of tyrosine hydroxylase; Daubner SC et al.; The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom . Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases . We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues . Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme . Arginine 316 is therefore critical for the binding of tyrosine . Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin . The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold . These data suggest that glutamate 326 is not directly involved in catalysis . Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation . These data suggest that aspartate 328 has a role in tyrosine binding . Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation . These data suggest that glutamate 332 is required for productive tetrahydropterin binding. Biochemistry, 1999 Apr 6, 38(14), 4441 - 7 5-lipoxygenase binds calcium; Hammarberg T et al.; 5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of leukotrienes and lipoxins and has therefore become an important target for pharmacological treatment of inflammatory disorders . Binding of calcium to 5LO was shown using several different approaches . Human recombinant enzyme was expressed in E . coli and purified . Association of Ca2+ to 5LO was demonstrated by a calcium-induced mobility shift in gel electrophoresis, by calcium overlay, by gel filtration in the presence of calcium, and by equilibrium dialysis . The two latter methods also showed that calcium binds reversibly to 5LO . Equilibrium dialysis gave a Kd close to 6 microM; the stoichiometry of maximum calcium binding seemed to average around two Ca2+ per 5LO . Similar results were obtained when 5LO was inactivated during equilibrium dialysis, indicating that the calcium binding site(s) is (are) different from the active site . By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO. Biochemistry, 1999 Apr 6, 38(14), 4313 - 8 Identification of residues of Escherichia coli phosphofructokinase that contribute to nucleotide binding and specificity; Wang X et al.; The apparent affinity of phosphofructo-1-kinase (PFK) of Escherichia coli for ATP is at least 10 times higher than for other nucleotides . Mutagenesis was directed toward five residues that may interact with ATP: Y41, F76, R77, R82, and R111 . Alanine at position 41 or 76 increased the apparent Km by 49- and 62-fold, respectively . Position 41 requires the presence of a large hydrophobic residue and is not restricted to aromatic rings . Tryptophan and, to a lesser extent, phenylalanine could substitute at position 76 . None of the mutants at 41 or 76 showed a change in the preference for alternative purines, although F76W used CTP 3 times better than the wild type enzyme . Mutations of R77 suggested that the interaction was hydrophobic with no influence on nucleotide preference . Mutation of R82 to alanine or glutamic acid increased the apparent Km for ATP by more than 20-fold and lowered the kcat/Km with ATP more than 30-fold . However, these mutants had a higher kcat/Km than wild type for both GTP and CTP, reflecting a loss of substrate preference . A loss in preference is seen as well with R111A where the kcat/Km for ATP decreases by only 68%, but the kcat/Km with GTP increases more than 10-fold . Activities with ITP, CTP, and UTP are also higher than with the wild type enzyme . Arginine residues at positions 82 and 111 are important dictators of nucleoside triphosphate preference. Biochemistry, 1999 Apr 6, 38(14), 4303 - 12 Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+; Cody V et al.; Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR . These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution . Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes . In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts . The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex . Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states . The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR. Biochemistry, 1999 Apr 6, 38(14), 4266 - 76 X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution; Erskine PT et al.; 5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen . ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm . Pairs of monomers associate with their arms wrapped around each other . Four of these dimers interact, principally via their arm regions, to form octamers in which each active site is located on the surface . The active site contains two lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid . Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds . The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312 . In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule . We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety . Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate . Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions. Biochemistry, 1999 Apr 6, 38(14), 4259 - 65 Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase; Traviglia SL et al.; Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-{p-(bromoacetamido)benzyl}ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency . The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein . Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds . Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward . We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase . The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H . The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G . No cut sites on the alpha subunit were observed . These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants {Owens, J . T., Miyake, R., Murakami, K., Chmura, A . J., Fujita, N., Ishihama, A., and Meares, C . F . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 6021-6026} . This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis. Biochemistry, 1999 Mar 30, 38(13), 4177 - 87 Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time; Bilsel O et al.; Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli . Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively {Higgins, W., Fairwell, T., and Miles, E . W . (1979) Biochemistry 18, 4827-4835} . By use of a double-kinetic approach {Jones, B . E., Beechem, J . M., and Matthews, C . R . (1995) Biochemistry 34, 1867-1877}, the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques . Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale . If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A . The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s) . Viewed within the context of the kinetic folding model of alphaTS {Bilsel, O., Zitzewitz, J . A., Bowers, K . E . , and Matthews, C . R . (1999) Biochemistry 38, 1018-1029}, the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit . The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation . The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure. Biochemistry, 1999 Mar 30, 38(13), 4165 - 76 DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates; Russell R et al.; Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action . We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK . DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis . At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min-1 at 25 degrees C and 1-1.5 min-1 at 5 degrees C . By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is approximately 15 000-fold at 5 degrees C . The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of approximately 20 microM at 5 degrees C, and weaker still at 25 degrees C . In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK.ATP . This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK.ADP . Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides. Biochemistry, 1999 Mar 30, 38(13), 4053 - 7 Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli; Gu XR et al.; The fmu gene product has been proposed to be an RNA methyltransferase {Koonin, E . V . (1994) Nucleic Acids Res . 22, 2476-2478} . Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized . The enzyme catalyzed specific methylation of C967 of unmodified 16S rRNA transcripts . A 16mer stem-loop structure containing C967 (nt 960-975) was also a good substrate for the enzyme in vitro . Methylation of C967 was confirmed by several methods including analysis of RNase T1 digests and nearest-neighbor analysis . Fmu did not catalyze methylation of transcripts of 23S rRNA . E . coli cells that contained kanr-disrupted fmu produced 16S rRNA that could be specifically methylated by Fmu in vitro at C967 but not C1407 . Further, fmu disruption did not significantly alter the growth rate of E . coli in rich or minimal media . We propose renaming this ORF "rrmB" and the enzyme "RrmB" for rRNA methyltransferase. Biochemistry, 1999 Mar 30, 38(13), 3910 - 7 Photoaffinity labeling with the activator IMP and site-directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP; Bueso J et al.; Photoaffinity labeling with IMP was used to attach covalently this activator to its binding site of Escherichia coli carbamoyl phosphate synthetase . We now identify histidine 995 of the large enzyme subunit as the amino acid that is cross-linked with IMP . The identification was carried out by comparative peptide mapping in two chromatographic systems of peptides differentially labeled with {3H}IMP and with the labeled inhibitor {14C}UMP, followed by automated Edman degradation and radiosequence analysis . Site-directed substitution of His995 by alanine confirmed His995 to be the only amino acid in the protein forming a covalent adduct with IMP . The His995Ala mutant protein was soluble and active and exhibited normal kinetics for the activator ornithine and for the substrates in the presence of ornithine . However, the mutation selectively induced changes in the activation by IMP and the inhibition by UMP, and it abolished the photolabeling of the enzyme by IMP without affecting the photolabeling by the inhibitor UMP . Since UMP is cross-linked to Lys993 {Cervera, J., et al . (1996) Biochemistry 35, 7247-7255} only two residues upstream of the site of IMP labeling, the results provide structural evidence for earlier proposals which suggested that UMP and IMP bind in a single or overlapping site . The two residues are within the region previously proposed as the binding fold for the nucleotide effectors . In the crystal structure of the enzyme, Lys993 and His995 are exposed and line a crevice where a Pi molecule was found {Thoden, J . B., et al . (1997) Biochemistry 36, 6305-6316} . UMP and IMP appear to bind in this crevice, possibly toward the C-side of the beta-sheet in a Rossman fold . Their binding in this site is consistent with the selectivity of adduct formation of UMP with Lys993 and of IMP with His995 . It is also consistent with the nonessentiality of His995 for the binding, since the interactions with other residues that line the crevice must contribute a large part of the binding energy . The lack of an effect of the mutation on the activation by ornithine is consistent with the binding of this activator in a separate site in the protein. Biochemistry, 1999 Mar 30, 38(13), 3895 - 901 Catalase HPII from Escherichia coli exhibits enhanced resistance to denaturation; Switala J et al.; Catalase HPII from Escherichia coli is a homotetramer of 753 residue subunits . The multimer displays a number of unusual structural features, including interwoven subunits and a covalent bond between Tyr415 and His392, that would contribute to its rigidity and stability . As the temperature of a solution of HPII in 50 mM potassium phosphate buffer (pH 7) is raised from 50 to 92 degrees C, the enzyme begins to lose activity at 78 degrees C and 50% inactivation has occurred at 83 degrees C . The inactivation is accompanied by absorbance changes at 280 and 407 nm and by changes in the CD spectrum consistent with small changes in secondary structure . The subunits in the dimer structure remain associated at 95 degrees C and show a significant level of dissociation only at 100 degrees C . The exceptional stability of the dimer association is consistent with the interwoven nature of the subunits and provides an explanation for the resistance to inactivation of the enzyme . For comparison, catalase-peroxidase HPI of E . coli and bovine liver catalase are 50% inactivated at 53 and 56 degrees C, respectively . In 5.6 M urea, HPII exhibits a coincidence of inactivation, CD spectral change, and dissociation of the dimer structure with a midpoint of 65 degrees C . The inactive mutant variants of HPII which fold poorly during synthesis and which lack the Tyr-His covalent bond undergo spectral changes in the 78 to 84 degrees C range, revealing that the extra covalent linkage is not important in the enhanced resistance to denaturation and that problems in the folding pathway do not affect the ultimate stability of the folded structure. Biochemistry, 1999 Mar 30, 38(13), 3857 - 66 Origin of the transient electron paramagnetic resonance signals in DNA photolyase; Gindt YM et al.; DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer . During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical . In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue . This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR . By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor . Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms . The fast component appears to correlate with a dismutation reaction occurring with the flavin . The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan . We also report a new procedure for the rapid isolation of DNA photolyase. Am J Respir Crit Care Med, 1999 Apr, 159(4 Pt 1), 1308 - 15 LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock; Mignon A et al.; Experimental models of sepsis using endotoxin challenges, including studies with sensitized animals with D-galactosamine, have largely contributed to the basic rationale for innovative clinical trials in human septic shock, which have, to date, failed . The ability of these models to reproduce human disease has been highly discussed . We report here that the widely used D-galactosamine/LPS model does not account for septic shock . Treatment with YVAD-CMK, a potent tetrapeptide inhibitor of caspases of the interleukin (IL)-1beta converting enzyme (ICE) family, protects from LPS-induced liver apoptosis and mortality in D-galactosamine-sensitized mice when administered either before or up to 2 h after the lethal challenge . This curative effect is related to complete inhibition of caspase-3 activity in the liver . However, YVAD-CMK does not affect LPS-induced release of IL-1beta and does not protect from a lethal dose of LPS in unsensitized mice . These experiments demonstrate the difference between these two widely recognized experimental models of sepsis . LPS toxicity in D-galactosamine-treated mice, leading to blocked gene transcription, results from tumor necrosis factor (TNF)-alpha-induced caspase-3-dependent liver injury, not from the systemic inflammatory response . These results provide evidence that inhibitors of the ICE caspase family can prevent or even overcome the ongoing hepatic injury induced by TNF-alpha during sepsis, ischemia-reperfusion, or severe hepatitis. Clin Infect Dis, 1999 Mar, 28(3), 451 - 5 Interactions between enteropathogenic Escherichia coli and epithelial cells; Donnenberg MS; Enteropathogenic Escherichia coli (EPEC) may be considered a paradigm for a multistage interaction between pathogen and host cell . EPEC strains produce a type IV pilus that is associated with initial adherence to host cells, and these strains possess a type III secretion apparatus that is necessary for transducing signals to host cells . Secretion of three Esp proteins is required for activation of a phosphotyrosine-containing receptor that allows EPEC to bind intimately to host cells via the bacterial outer membrane protein intimin . Intimately attached bacteria rest upon a pedestal composed of host cytoskeletal proteins in an arrangement recognized as the attaching and effacing phenotype . The precise molecular interactions that lead to these dramatic alterations in the host cell cytoskeleton remain to be elucidated. Clin Infect Dis, 1999 Mar, 28(3), 442 - 50 Cystalysin, a 46-kDa L-cysteine desulfhydrase from Treponema denticola: biochemical and biophysical characterization; Chu L et al.; A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67 . Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate . With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics . Cystathionine and s-aminoethyl-L-cysteine were also substrates . Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin . The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide . We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase) . Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T . denticola may also function in vivo as an important virulence molecule. Clin Exp Immunol, 1999 Mar, 115(3), 397 - 403 Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16; Fernando GJ et al.; E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16) . This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein . E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography . After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial . This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line . Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer . This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 427 - 9 fcsA29 mutation is an allele of polA gene of Escherichia coli; Nagano K et al.; The cold-sensitive fcsA29 mutation of Escherichia coli was found to be a new type of cold-sensitive allele of the polA gene encoding DNA polymerase I, caused by an Asp(116)-->Asn change in the 5'-->3' exonuclease domain . The fcsA29 mutant showed typical polA mutant phenotypes such as UV sensitivity and unacceptability of recA mutation . Cold-sensitive growth of the mutant was suppressed by introduction of a sulA mutation, indicating that cell filamentation was due to the SOS response. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 408 - 14 EnvZ-independent phosphotransfer signaling pathway of the OmpR-mediated osmoregulatory expression of OmpC and OmpF in Escherichia coli; Matsubara M et al.; The Escherichia coli EnvZ-OmpR regulatory system is a paradigm of intracellular signal transduction mediated by the well-documented phosphotransfer mechanism, by which the expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity . Although it is clear that the EnvZ histidine(His)-kinase is the major player in the phosphorylation of OmpR, it has been assumed for some time that there may be an alternative phospho-donor(s) that can phosphorylate OmpR under certain in vitro and in vivo conditions . In this study, to address this long-standing issue, extensive genetic studies were done with certain mutant alleles, including delta envZ, delta(ackA-pta), and delta sixA, as well as delta ompR . Here, for the first time, genetic evidence is provided that, in addition to EnvZ, acetyl phosphate and an as yet unidentified sensor His-kinase can serve as alternative in vivo phospho-donors for OmpR, even in the envZ+ background . A model for the alternative phosphotransfer signaling pathway involved in the phosphorylation of OmpR is proposed. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 302 - 8 Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.); Kaminaka H et al.; We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.) . The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues . This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs . The metal analysis and SOD assays of the partial purified recombinant protein expressed in E . coli showed that this cDNA encodes an iron-containing SOD . However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity . mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction . These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs. Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 299 - 302 An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase; Brautaset T et al.; Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source . In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation . By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein . Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required . Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 296 - 8 Rational design of an improved induction scheme for recombinant Escherichia coli; Mattanovich D et al.; In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred . Aiming at a reduction of very high product formation rates, we engineered E . coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters . The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations . J Infect Dis, 1999 May, 179(5), 1278 - 82 Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin; Suffredini AF et al.; The administration of reference endotoxin (Escherichia coli O:113, Lot EC-5) to humans has been an important means to study inflammation in vivo; however, the supply of Lot EC-5 is depleted . A new lot of reference endotoxin (Clinical Center reference endotoxin {CCRE}), derived from the original bulk material extracted from E . coli O:113, was processed . The effects of 0-, 1-, 2-, and 4-ng/kg doses of intravenous CCRE and EC-5 were studied in 20 male subjects . CCRE resulted in dose-related increases in symptoms, temperature (P= . 016), total leukocyte count (P=.014), tumor necrosis factor-alpha (P=.004), interleukin (IL)-1 receptor antagonist (P=.004), IL-6 (P= . 005), IL-8 (P=.011), cortisol (P<.05), and C-reactive protein (P= . 04) . These responses were attenuated (all P<.012) in subjects given Lot EC-5 (4 ng/kg) in comparison with those in subjects given CCRE, showing that, over several years, EC-5 had lost potency . Thus, in healthy subjects, the magnitude of exposure to CCRE results in a graded dose response of major components of innate immunity. Antisense Nucleic Acid Drug Dev, 1999 Feb, 9(1), 53 - 60 Boranophosphates support the RNase H cleavage of polyribonucleotides; Rait VK et al.; Modification of the phosphodiester linkages in DNA by replacing one of the nonbridging oxygens with borane, BH3, produces an isoelectronic mimic of DNA called boranophosphates . Nonstereoregular oligodeoxyribonucleoside all-boranophosphates are shown here for the first time to elicit the RNase H hydrolysis of polyribonucleotides . We compared the ability of three types of dodecamers (dodecathymidine phosphate, phosphorothioate, and boranophosphate) to mediate the cleavage of poly(A) by Escherichia coli RNase H1 . The rates of poly(A) hydrolysis induced by boranophosphates were 76-fold (at 20 degrees C) and 18-fold (at 30 degrees C) greater than the rates induced by dodecathymidine phosphate . In conjunction with the measured melting temperatures for each heteroduplex, carried out under the same conditions as the RNAse H cleavage experiments, the data establish an inverse relationship between the heteroduplex thermostability and the rate of poly(A) hydrolysis . Chromatographic analysis revealed another correlation: the higher the heteroduplex Tm, the higher the pApA:pApApA ratio in the corresponding hydrolysates . The specific content of these final products provides insight into the relative contribution of RNase H1 exonucleolytic/endonucleolytic mechanisms, with a low ratio for the lower melting heteroduplexes reflecting more endonucleolytic-type hydrolysis . In total, our data support the concept that antisense molecules with a weakened hybridization potential enhance the rate of hydrolysis of RNA in RNA-DNA hybrids. Pharmazie, 1999 Mar, 54(3), 191 - 4 A spermine-deoxycholic acid conjugate based lipid as a transfecting agent; Ajmani PS et al.; Deoxycholic acid-spermine conjugate (DAS), which is composed of natural components (deoxycholic acid and spermine), was incorporated in liposomes and evaluated for its interaction with plasmid DNA (pDNA) and in vitro transfection efficiency . Electromicrographs demonstrated that DAS-pDNA complexes are spherical, compact and electronically dense compared to the toroidal shapes formed by the monovalent lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and pDNA . In comparison to the singly charged, non-cholesterol based lipid (DOTAP), the multivalent lipid DAS had similar transfection efficiency in two cell lines . The monovalent sterol, deoxycholic acid propyldiamine conjugate (DAP) was not effective as a transfecting agent . This suggests that multivalent facial amphiphiles such as DAS may serve as excellent candidates for non-viral gene transfer and warrant further study. J Microbiol Methods, 1999 Mar, 35(2), 129 - 41 Rapid isolation of genes from an indexed genomic library of C . cinereus in a novel pab1+ cosmid; Bottoli AP et al.; In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a novel cosmid carrying pab1+ as a selectable Coprinus marker . The average insert size per cosmid comprises 41 kb . We screened the library and detected copies of known (a1-2, beta-tub, cgl1, ras, trp1) and of new Coprinus genes (cac, lac1, lac2, lac3) . Screening was performed either by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification . We successfully applied PCR with specific and with degenerate primers, multiplex PCR and colony PCR in library screening . Our results suggest a new, more efficient pooling strategy for future high throughput screenings to be used in PCR with pooled cosmid DNAs, or in a less laborious approach using pooled Escherichia coli colonies for PCR. J Membr Biol, 1999 Apr 1, 168(3), 229 - 39 Cystic fibrosis transmembrane conductance regulator (CFTR) confers glibenclamide sensitivity to outwardly rectifying chloride channel (ORCC) in Hi-5 insect cells; Julien M et al.; Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC) . We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, DeltaF508-CFTR or E . coli beta galactosidase cDNA and in control cells either infected with virus alone or uninfected . Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 +/- 1.6 pS) only in cells expressing CFTR . In contrast, we identified 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 +/- 2.5 pS at -80 mV; 55 +/- 4.1 pS at +80 mV) and properties . In the presence of 500 microm SITS, channel open probability (Po) of ORCC was reversibly reduced to 0.05 +/- 0.01 in CFTR-cells, to 0.07 +/- 0.02 in non-CFTR expressing cells and to 0.05 +/- 0.02 in DeltaF508-cells . In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 microm), whereas 500 microm SITS reversibly inhibited ORCC . In contrast in cells expressing CFTR or DeltaF508, glibenclamide dose dependently (IC50 = 17 microm, Hill coefficient 1.2) and reversibly inhibited ORCC . Cytoplasmic application of 100 microm glibenclamide reversibly reduced Po from 0.88 +/- 0.03 to 0.09 +/- 0.02 (wash: Po = 0.85 +/- 0.1) in CFTR cells and from 0.89 +/- 0.05 to 0.08 +/- 0.05 (wash: Po = 0.87 +/- 0.1) in DeltaF508 cells . In non-CFTR expressing cells, glibenclamide (100 microm) was without effect on Po (control: Po = 0 . 89 +/- 0.09, glib.: Po = 0.86 +/- 0.02; wash: Po = 0.87 +/- 0.05) . These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells. Pediatr Dev Pathol, 1999 May-Jun, 2(3), 286 - 91 Isovaleric acidemia with promyelocytic myeloproliferative syndrome; Gilbert-Barness E et al.; Isovaleric acidemia, an autosomal recessive disorder, is due to isovaleryl-coenzyme A dehydrogenase deficiency and is one of the branched-chain aminoacidopathies . Isovaleric acidemia may present in the neonatal period with an acute episode of severe metabolic acidosis, ketosis, and vomiting and may lead to coma and death in the first 2 months of life . This report concerns an infant who presented at 10 days of age because of lethargy, poor feeding, hypothermia, cholestasis, and thrombocytopenia, leukopenia, and profound pancytopenia . Death occurred at 19 days of age . Autopsy showed mild fatty change in the liver and extramedullary hematopoiesis, generalized Escherichia coli sepsis, and myelodysplasia of the bone marrow with arrest of the myeloid series at the promyelocytic stage . The appearance resembled promyelocytic leukemia, but the diagnostic 15:17 translocation was not present . The maturation arrest in granulopoiesis in isovaleric acidemia appears to be most likely due to a direct metabolic effect on granulocyte precursor cells. J Lipid Res, 1999 Apr, 40(4), 708 - 14 Phytanic acid is ligand and transcriptional activator of murine liver fatty acid binding protein; Wolfrum C et al.; Branched-chain phytanic acid is metabolized in liver peroxisomes . Sterol carrier protein 2/sterol carrier protein x (SCP2/SCPx) knockout mice, which develop a phenotype with a deficiency in phytanic acid degradation, accumulate dramatically high concentrations of this fatty acid in serum (Seedorf at al . 1998 . Genes Dev . 12: 1189-1201) and liver . Concomitantly, a 6.9-fold induction of liver fatty acid binding protein (L-FABP) expression is observed in comparison to wild-type animals fed standard chow, possibly mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha) . Cytosolic transport of phytanic acid to either peroxisomal membranes or to the nucleus for activation of PPARalpha may be mediated by L-FABP, which gives rise to the question whether phytanic acid is a transactivator of this protein . Here we show first that phytanic acid binds to recombinant L-FABP with high affinity . Then the increase of the in vivo phytanic acid concentration by phytol feeding to mice results in a 4-fold induction of L-FABP expression in liver, which is in the order of that attained with bezafibrate, a known peroxisome proliferator . Finally to test in vitro whether this induction is conferred by phytanic acid, we cotransfected HepG2 cells with an expression plasmid for murine PPARalpha and a CAT-reporter gene with 176 bp of the murine L-FABP promoter, containing the peroxisome proliferator responsive element (PPRE) . After incubation with phytanic acid, we observed a 3.2-fold induction of CAT expression . These findings, both in vivo and in vitro, demonstrate that phytanic acid is a transcriptional activator of L-FABP expression and that this effect is mediated via PPARalpha. Biochem J, 1999 Apr 15, 339 ( Pt 2), 261 - 7 Protein structure and gene cloning of Syncephalastrum racemosum nuclease; Ho HC et al.; The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides . The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop . On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I . Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases . Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site . The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence . Subsequently, specific primers were synthesized for use in the 3' and 5' rapid amplification of cDNA ends (RACE) . Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease . The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein . Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities . The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA. J Mol Biol, 1999 Apr 9, 287(4), 761 - 71 Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts; Kopp J et al.; Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway . The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized . The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution . The enzyme is a homohexamer with D3 symmetry . The subunit chain fold is a (beta alpha)8-barrel . A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism . The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system . A mutation confirmed the crucial role of one of these carboxylates . The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization . Arch Biochem Biophys, 1999 Apr 15, 364(2), 219 - 27 Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer; Eyschen J et al.; Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate . The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation . GAPDH is classified as a B-specific oxidoreductase . The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridinium si face requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring orientation . In previous studies carried out on Escherichia coli GAPDH (C . Corbier, A . Mougin, Y . Mely, H . W . Adolph, M . Zeppezauer, D . Gerard, A . Wonacott, and G . Branlant, Biochimie 72, 545-554, 1990; J . Eyschen, C . Corbier, B . Vitoux, G . Branlant, and M . T . Cung, Protein Pept . Lett . 1, 19-24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors the syn orientation of the nicotinamide ring, was examined . Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity . Two single {Gly317} and {Ala317} GAPDH mutants and one double {Thr313-Gly317} GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH from E . coli (E . Duee, L . Olivier-Deyris, E . Fanchon, C . Corbier, G . Branlant, and O . Dideberg, J . Mol . Biol . 257, 814-838, 1996) . The Kd constants of {Ala317}, {Gly317}, and {Thr313-Gly317} GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH . Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of bound nicotinamide remains unchanged in the {Gly317} and {Ala317} mutants, whereas a conformational equilibrium between the syn and anti forms occurs in the {Thr313-Gly317} double mutant with a preference for the anti conformer . Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereospecificity . Yet, the catalytic efficiency is much less than that of the wild type . This indicates that the holo GAPDH mutant fraction with an anti orientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process . The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases . J Hepatol, 1999 Mar, 30(3), 366 - 75 Epitope mapping of cytochrome P4502D6 autoantigen in patients with chronic hepatitis C during alpha-interferon treatment; Dalekos GN et al.; BACKGROUND/AIMS: Cytochrome P450 2D6 (CYP2D6) has been documented as the major target antigen of liver kidney microsomal autoantibodies type-1 (anti-LKM-1) in both autoimmune hepatitis type-2 (AIH-2) and hepatitis C (HCV) . In HCV/anti-LKM-1-positive patients, the choice between alpha-interferon (alpha-IFN) or immunosuppression may be difficult . This study was conducted to evaluate the course and outcome of alpha-IFN therapy in HCV/anti-LKM-1-positive and -negative patients and the alterations in these autoantibody titers by the indirect immunofluorescence and a novel radioligand assay . Epitope mapping was also performed to screen for a potential shift in anti-LKM-1 binding towards small linear epitopes, which are more often detected in AIH-2 patients . METHODS: Twenty-one patients with HCV infection received alpha-IFN . Seven patients were anti-LKM-1 positive (study group) and 14 patients were anti-LKM-1 negative (disease control group) . Anti-CYP2D6 detection was based on immunoprecipitation of {35S}-methionine-labeled CYP2D6 recombinant protein (rCYP2D6) produced by in vitro transcription/translation . RESULTS: Four out of seven (57%) patients in the study group and 5/14 (36%) in the disease control group initially responded, but subsequently relapsed . During follow-up, alanine aminotransferase significantly increased in the study group compared to the disease control group (p<0.01) . A slight increase, followed by a plateau of autoantibody titers was recorded by the radioligand assay and by indirect immunofluorescence during therapy and follow-up in most cases . In one patient, however, gamma-globulins and anti-LKM-1 titers increased, reaching very high levels (1:40 960) . alpha-IFN was interrupted and immunosuppression was started . HCV/anti-CYP2D6 positive sera recognized CYP2D6 expressed in E . coli and two truncated proteins (aa 250-494 and 321-494) . Two out of seven sera, in addition reacted with a small linear epitope of aa 257-269 (one of which also reacted with a C-terminal domain of aa 350-494) . CONCLUSIONS: A rather mild deterioration in liver disease was observed in only 1/7 HCV/anti-LKM-1-positive patients during alpha-IFN treatment . This patient showed high anti-CYP2D6 titers before the initiation of therapy, a sharp increase in anti-LKM-1 titers during treatment, and reactivities to a small linear epitope and an infrequently recognized C-terminal domain of CYP2D6 . After switching to immunosuppressive treatment, a complete and sustained response was recorded . Further prospective studies from many centers are needed to define whether these features have general, clinical significance or not. Vopr Virusol, 1999 Jan-Feb, 44(1), 12 - 5 {Preparation of single-chained antibodies to surface glycoprotein E from tick-borne encephalitis virus}; Tikunova NV et al.; A single-stranded antibody gene scFv was designed on the base of cDNA fragments of genes coding for variable domains of heavy and light chains of MAb E6B to tick-borne encephalitis glycoprotein E . High production of stable soluble scFv was reproduced in Escherichia coli cells . Recombinant antibodies bound to antiidiotypical antibodies to initial MAb E6B and to recombinant virus protein E . Competitive analysis showed that single-stranded antibodies inhibited reaction between MAb E6B and protein E . These results confirm the formation of scFv with the original antigen-binding specificity towards tick-borne encephalitis virus glycoprotein E. Gut, 1998 Aug, 43(2), 196 - 202 Bromelain protects piglets from diarrhoea caused by oral challenge with K88 positive enterotoxigenic Escherichia coli; Chandler DS et al.; BACKGROUND: K88 positive enterotoxigenic Escherichia coli (K88+ ETEC) is an important cause of diarrhoea in young piglets . K88+ ETEC pathogenesis relies on attachment to specific glycoprotein receptors located on the intestinal mucosa . Proteolytic treatment of these receptors in vitro and in vivo prevents attachment of K88+ ETEC to piglet small intestines and may be of clinical use to prevent K88+ ETEC pathogenesis . AIMS: To determine whether bromelain, a proteolytic extract obtained from pineapple stems, would protect piglets against K88+ ETEC diarrhoea and to confirm and extend earlier findings on the effects of bromelain on K88+ ETEC receptors in vivo . METHODS: Bromelain (0, 12.5, or 125 mg) was orally administered to just weaned piglets for 10 days . One day following commencement of bromelain treatment, piglets were challenged with K88+ ETEC (5 x 10(10) K88ac:0149) for seven days . Intestinal contents from unchallenged piglets were obtained via an intestinal fistula, and tested for their ability to bind K88+ ETEC before and after bromelain treatment . RESULTS: Both doses of bromelain were successful in reducing the incidence of K88+ ETEC diarrhoea and protected piglets from life threatening disease . Bromelain treated pigs also had significantly increased weight gain compared with untreated pigs . Bromelain only temporarily inhibited K88+ ETEC receptor activity, with receptor activity being regenerated 30 hours following treatment, consistent with the regeneration of new enterocytes . CONCLUSION: Results show that bromelain can temporarily inactivate ETEC receptors in vivo and protect against ETEC induced diarrhoea . Bromelain may therefore be an effective prophylaxis against ETEC infection. Trends Biotechnol, 1999 Mar, 17(3), 115 - 21 The selection of intracellular antibodies; Cattaneo A et al.; The intracellular expression of antibodies in mammalian cells is a strategy to inhibit the in vivo function of selected molecules but is limited by the unpredictable behaviour of antibodies when intracellularly expressed . Recent advances in the field of antibody expression in Escherichia coli show that the introduction of mutations can improve the properties of some antibody domains, but the general applicability of this approach to intracellular antibodies remains to be proved . As a complement to rational approaches, we describe selection schemes in which antibodies are selected on the basis of their performance in vivo as intracellular antibodies. Syst Appl Microbiol, 1999 Feb, 22(1), 1 - 8 In situ detection of Escherichia coli cells containing ColE1-related plasmids by hybridization to regulatory RNA II; Juretschko S et al.; A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids . It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes . Various methods for signal amplification were compared . Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells. FEMS Microbiol Lett, 1999 Mar 15, 172(2), 145 - 51 Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups; Martinez MB et al.; The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis . The invasive strains were isolated in different parts of the world and most of them recovered from dysentery . Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages . These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes . The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains. Biochemistry (Mosc), 1999 Feb, 64(2), 169 - 74 Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions; Avaeva SM et al.; Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E . coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis . The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly . This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration. J Biol Chem, 1999 Apr 9, 274(15), 10609 - 17 CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts; Umayahara Y et al.; Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation . Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter . We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site . We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D . C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta . C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting . C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies . As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity . We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid . Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species. J Biol Chem, 1999 Apr 9, 274(15), 10405 - 12 Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro; Huang YS et al.; Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins . Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding . In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex . The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1 . Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1 . Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively . This explained the occurrence of an MBP-alpha . GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates . The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold . Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans) . In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration . These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES. J Biol Chem, 1999 Apr 9, 274(15), 10395 - 404 GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase . Obligatory interaction of chaperonins with an alpha beta dimeric intermediate; Chuang JL et al.; The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits . In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP . Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1 . Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates . However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL . The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP . In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent . Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter . The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex . Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1 . Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete . Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers. J Biol Chem, 1999 Apr 9, 274(15), 10356 - 62 In vitro analysis of the interaction between the FinO protein and FinP antisense RNA of F-like conjugative plasmids; Jerome LJ et al.; The FinO protein regulates the transfer potential of F-like conjugative plasmids through its interaction with FinP antisense RNA and its target, traJ mRNA . FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA in vitro . The FinP secondary structure consists of two stem-loop domains separated by a 4-base spacer and terminated by a 6-base tail . Previous studies suggested FinO bound to the smooth 14-base pair helix of stem-loop II . In this investigation, RNA mobility shift analysis was used to study the interaction between a glutathione S-transferase (GST)-FinO fusion protein and a series of synthetic FinP and traJ mRNA variants . Mutations in 16 of the 28 bases in stem II of FinP that are predicted to disrupt base pairing did not significantly alter the GST-FinO binding affinity . Removal of the single-stranded regions on either side of stem-loop II led to a dramatic decrease in GST-FinO binding to FinP and to the complementary region of the traJ mRNA leader . While no evidence for sequence-specific contacts was found, the results suggest that FinO recognizes the overall shape of the RNA and is influenced by the length of the single-stranded regions flanking the stem-loop. J Biol Chem, 1999 Apr 9, 274(15), 10244 - 8 Identification of a surface of FNR overlapping activating region 1 that is required for repression of gene expression; Green J et al.; A library of Escherichia coli fnr mutants has been screened to identify FNR (regulator of fumarate and nitrate reduction) variants that are defective repressors, but competent activators . All but one of seventeen variants had substitutions close to or within the face of FNR that contains activating region 1 (AR1) . Activating region 1 is known to contact the alpha subunit of RNA polymerase to facilitate transcription activation . It is now evident that this face also has a role in FNR-mediated repression . Single amino acid substitutions at Lys54, Gly74, Ala95, Met147, Leu193, Arg197, or Leu239, and double substitutions at Ser13 and Ser145, Cys16 and Ile45, Tyr69 and Ser133, or Lys164 and Phe191, impaired FNR-mediated repression of ndh without greatly affecting activation from model Class I (FNR site at -71.5) and Class II (FNR site at -41.5) FNR-activated promoters . Although repression was impaired in a second group of FNR variants with substitutions at Leu34, Arg72 and Leu193, Phe92, or Ser178, transcription activation from the simple FNR-dependent promoters was severely reduced . However, expression from pyfiD (FNR sites at -40.5 and -93.5) and a derivative lacking the site at -93.5, pyfiD-/+, remained relatively high indicating that this second group have a context-dependent activation defect as well as a repression defect . The prediction that the substitutions affecting repression were likely to be in solvent exposed regions of FNR was supported by analysis of peptides produced by partial proteolysis of FNR . Thus, FNR-mediated repression at promoters with multiple FNR sites requires regions of FNR that are different from, but overlap, AR1. J Biol Chem, 1999 Apr 9, 274(15), 10235 - 43 Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence; Pingoud V et al.; PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases . According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding . To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA . Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm) . The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand . The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif . With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction . Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI . This inactivation can be suppressed by substrate binding . This result further supports the finding that at least one His residue is in close contact to the DNA . Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI. J Biol Chem, 1999 Apr 9, 274(15), 10119 - 28 The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli; Messner KR et al.; The fitness of organisms depends upon the rate at which they generate superoxide (O-2) and hydrogen peroxide (H2O2) as toxic by-products of aerobic metabolism . In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain . Inverted vesicles that were incubated with NADH generated O-2 and H2O2 at accelerated rates either when treated with cyanide or when devoid of quinones, implicating an NADH dehydrogenase as their source . Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, and its overproduction accelerated it . Thus NADH dehydrogenase II but not NADH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed substantial O-2 and H2O2 . NADH dehydrogenase II that was purified from both wild-type and quinone-deficient cells generated approximately 130 H2O2 and 15 O-2 min-1 by autoxidation of its reduced FAD cofactor . Sulfite reductase is a second autoxidizable electron transport chain of E . coli, containing FAD, FMN, {4Fe-4S}, and siroheme moieties . Purified flavoprotein that contained only the FAD and FMN cofactors had about the same oxidation turnover number as did the holoenzyme, 7 min-1 FAD-1 . Oxidase activity was largely lost upon FMN removal . Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor . Great variability in the oxidation turnover numbers of these and other flavoproteins suggests that endogenous oxidants will be predominantly formed by only a few oxidizable enzymes . Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types . Furthermore, the chemical nature of these reactions was manifested by their acceleration at high temperatures and oxygen concentrations . Thus these environmental parameters may also directly affect the O-2 and H2O2 loads that organisms must bear. J Biol Chem, 1999 Apr 9, 274(15), 10079 - 85 Translational enhancement by an element downstream of the initiation codon in Escherichia coli; Etchegaray JP et al.; The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence . This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs . It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive . Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD. J Biol Chem, 1999 Apr 9, 274(15), 10039 - 46 Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR; Shi L et al.; The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned . The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M . aeruginosa UTEX 2063 . By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms . PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity . Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers . This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M . aeruginosa . Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A . PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro . Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases . The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism. J Biol Chem, 1999 Apr 9, 274(15), 9937 - 45 Biochemical characterization of the small heat shock protein IbpB from Escherichia coli; Shearstone JR et al.; Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomal ibp operon and purified by refolding . Under our experimental conditions, IbpB exhibited pronounced size heterogeneity . Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100-200-nm range, which themselves associated to yield loose aggregates of micrometer size . IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly beta-pleated secondary structure . Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into approximately 600-kDa oligomers, and an increase in surface hydrophobicity . Structural changes were reversible between 37 and 55 degrees C, and, up to 55 degrees C, hydrophobic sites were reburied upon cooling . IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition . However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features . By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures . Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB. Appl Environ Microbiol, 1999 Apr, 65(4), 1801 - 5 Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme; Izumi M et al.; The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli . Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit) . The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P . kodakaraensis) in E . coli . The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ . Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins . The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys . Effect of the mutation on the ATPase activity and CobQ solubilization was examined . Neither mutant exhibited ATPase activity in vitro . Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB . These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E . coli without requiring energy from ATP hydrolysis. Antimicrob Agents Chemother, 1999 Apr, 43(4), 969 - 71 Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases; Arlet G et al.; The sequences of the blaTEM genes encoding TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases were determined . Analysis of the deduced amino acid sequences indicated that TEM-20 and TEM-29 were derived from TEM-1 and that TEM-21 and TEM-22 were derived from TEM-2 . The substitutions involved were Ser-238 and Thr-182 for TEM-20; His-164 for TEM-29; Lys-104, Arg-153, and Ser-238 for TEM-21; and Lys-104, Gly-237, and Ser-238 for TEM-22 . The promoter region of the blaTEM-22 gene was identical to that of blaTEM-3 . High-level production of TEM-20 could result from a 135-bp deletion which combined the -35 region of the Pa promoter with the -10 region of the P3 promoter and a G-->T transition in the latter motif. Antimicrob Agents Chemother, 1999 Apr, 43(4), 957 - 9 Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli; Nelson EC et al.; DNA sequencing data showed that five clinical isolates of Escherichia coli with reduced susceptibility to ceftazidime, ceftriaxone, and cefotaxime contain an ampC gene that is preceded by a strong promoter . Transcription from the strong promoter was 8- to 18-fold higher than that from the promoter from a susceptible isolate . RNA studies showed that mRNA stability does not play a role in the control of AmpC synthesis. Antimicrob Agents Chemother, 1999 Apr, 43(4), 789 - 93 Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26; Horii T et al.; We evaluated the susceptibilities of 129 Shiga-like toxin-producing Escherichia coli (STEC) isolates to various antibiotics . The numbers of isolates for which MICs were high (> or = 128 micrograms/ml) were as follows: 5 for fosfomycin, 14 for ampicillin, 1 for cefaclor, 6 for kanamycin, 22 for tetracycline, and 2 for doxycycline . For two isolates of STEC O26 MICs of fosfomycin were high (1,024 and 512 micrograms/ml, respectively) . Conjugation experiments and glutathione S-transferase assays suggested that the fosfomycin resistance in these isolates was determined not by a plasmid but chromosomally . The amount of active intracellular fosfomycin in these STEC isolates was 100- to 200-fold less than that in E . coli C600 harboring pREFTT47B408 in the presence of either L-alpha-glycerophosphate or glucose-6-phosphate . Cloning, sequencing, and Northern blot analysis demonstrated that the transcriptional level of the murA gene encoding UDP-N-acetylglucosamine enolpyruvoyl transferase in these isolates was greater than that in E . coli C600 . Our results suggest that the fosfomycin resistance in these STEC isolates is due to concurrent effects of alteration of the glpT and/or uhp transport systems and of the enhanced transcription of the murA gene. Eur J Biochem, 1999 Apr, 261(1), 261 - 8 Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases; Dupureur CM et al.; The activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA . While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes . Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation . The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease . Replacement of 74% (+/- 10%) of the Tyr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme . Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies . 1H- and 19F-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift . Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II) . Coupled with earlier mutagenesis studies that place Ca(II) in the active site {Nastri, H . G., Evans, P.D., Walker, I.H . & Riggs, P.D . (1997) J . Biol . Chem . 272, 25761-25767}, these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes. Eur J Biochem, 1999 Apr, 261(1), 197 - 207 Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme . The effects on folding, stability and enzymatic properties; Janssen MJ et al.; Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges . These PLA2s have been classified into several groups based on the disulphide bond patterns found {Dennis, E . A . (1997) Trends Biochem . Sci . 22, 1-2} . To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2 . Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues . Introduction of a C-terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability . In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability . Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stability of the enzyme . Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2. Eur J Biochem, 1999 Apr, 261(1), 115 - 23 Pen c 1, a novel enzymic allergen protein from Penicillium citrinum . Purification, characterization, cloning and expression; Su NY et al.; A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus . The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies . Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1 . The caseinolytic enzyme activity of this protein was studied . The protein binds to serum IgE from patients allergic to Penicillium citrinum . The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide . Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein . Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family . The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved . Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture . The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag . The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium . Recombinant Pen c 1 can therefore be expressed in E . coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders . In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein. Eur J Biochem, 1999 Apr, 261(1), 57 - 65 NMR studies on the 46-kDa dimeric protein, 3,4-dihydroxy-2-butanone 4-phosphate synthase, using 2H, 13C, and 15N-labelling; Richter G et al.; 3,4-Dihydroxy-2-butanone 4-phosphate synthase catalyses the release of C-4 from the substrate, ribulose phosphate, via a complex series of rearrangement reactions . The cognate ribB gene of Escherichia coli was hyperexpressed in a recombinant E . coli strain . The protein was shown to be a 46-kDa homodimer by hydrodynamic analysis . A variety of protein samples labelled with different grades of 13C, 15N and 2H, i.e . one with 100% 2H and 15N, one with 75% 2H, 99% 13C, 15N, and one with 100% 2H, 99% 13C,15N were prepared . Despite the large molecular size, 2- and 3-dimensional NMR spectra of reasonable quality were obtained . Attempts at the assignment of individual 13C, 15N and 1H signals show, in principle, the feasibility of structure determination . The number of NMR signals shows unequivocally that the homodimeric protein obeys strict C2 symmetry. Eur J Biochem, 1999 Apr, 261(1), 40 - 7 Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C; Reiffert S et al.; Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation . Four different phosphorylation states can be formed . To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated . These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy . Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation . Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes . In all cases a functional holotroponin complex was obtained . Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI . However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution . Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex . Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified. Eur J Biochem, 1999 Apr, 261(1), 10 - 8 Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up; Maier G et al.; The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66 . A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed . In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions . We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively . The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis . The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination . Truncated forms of p51 are efficiently removed . Mobility-shift assay revealed that the preparations are free of p66 homodimer . In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays . This indicates the p51 subunit has an active role in DNA binding Eur J Biochem, 1999 Apr, 261(1), 1 - 9 Cobalt proteins; Kobayashi M et al.; In the form of vitamin B12, cobalt plays a number of crucial roles in many biological functions . However, recent studies have provided information on the biochemistry and bioinorganic chemistry of several proteins containing cobalt in a form other than that in the corrin ring of vitamin B12 . To date, eight noncorrin-cobalt-containing enzymes (methionine aminopeptidase, prolidase, nitrile hydratase, glucose isomerase, methylmalonyl-CoA carboxytransferase, aldehyde decarbonylase, lysine-2,3-aminomutase, and bromoperoxidase) have been isolated and characterized . A cobalt transporter is involved in the metallocenter biosynthesis of the host cobalt-containing enzyme, nitrile hydratase . Understanding the differences between cobalt and nickel transporters might lead to drug development for gastritis and peptic ulceration. Eur J Biochem, 1999 Mar, 260(3), 923 - 7 Tissue expression and amino acid sequence of murine UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase; Horstkorte R et al.; Neuraminic acids are widely expressed as terminal carbohydrates on glycoconjugates and are involved in a variety of biological functions . The key enzyme of N-acetylneuraminic acid synthesis is UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, which catalyses the first two steps of neuraminic acid biosynthesis in the cytosol . In this study we report the complete amino acid sequence of the mouse UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase . The ORF of 2166 bp encodes 722 amino acids and a protein with a predicted molecular mass of 79.2 kDa . Northern blot analysis and in situ hybridization revealed that UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase is expressed at early stages during development and in all tissues investigated with a maximal expression in the liver. Eur J Biochem, 1999 Mar, 260(3), 896 - 903 Structural characterization of l-aspartate oxidase and identification of an interdomain loop by limited proteolysis; Tedeschi G et al.; l-Aspartate oxidase is the first enzyme in the de novo biosynthesis of pyridinic coenzymes in facultative aerobic organisms . The enzyme is FAD dependent and it shares common features with both the oxidase and the fumarate reductase classes of flavoproteins . In this report we focused our attention on the supersecondary structure of the molecule by means of limited proteolysis studies . Moreover the polymerization state of the protein at different pH and the interactions with NAD and its analogues are described . The results suggest that l-aspartate oxidase is a monomer at pH values lower than 4.5 and a dimer at pH values higher than 6.5 . The protein is organized in two major domains connected by a flexible loop located in the 120-140 region . The data obtained by limited proteolysis of the holo and the apo form in the presence and in the absence of substrates (fumarate and menadione), inhibitors (succinate) and NAD allows the proposition that both domains are involved in the binding of the flavin coenzyme . Moreover the data reported in this manuscript suggest that NAD inhibits l-aspartate oxidase activity by competing with the flavin for the binding to the enzyme. Eur J Biochem, 1999 Mar, 260(3), 861 - 8 Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat; Lullien-Pellerin V et al.; The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b . The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them . In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays . Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized . No significant changes in structure or activity were found for three of the mutants . By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1 . Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data. Eur J Biochem, 1999 Mar, 260(3), 818 - 24 Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function; Kleivdal H et al.; FomA porin is the major outer-membrane protein of Fusobacterium nucleatum . It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins . According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting of 16 transmembrane segments with eight surface-exposed loops . Several conserved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure . The goal of our study was to identify particular basic residues contributing to such an electric field in FomA . Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines . The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of beta-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes . Some of the point mutations had a significant impact on the channel properties . The substitution R92A produced a 130% increased permeability of the zwitterionic beta-lactam cephaloridine, and the cation selectivity of R92E increased by 70% . The effects of the R90E substitution on channel properties were similar . Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity . The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine . The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well. Eur J Biochem, 1999 Mar, 260(3), 761 - 7 Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene; Fujiwara K et al.; Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes . We isolated human lipoyltransferase cDNA and genomic DNA . The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids . Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively . Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb . mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group . The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA . Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon . The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization. Eur J Biochem, 1999 Mar, 260(3), 692 - 700 Domain-domain interactions in high mobility group 1 protein (HMG1); Ramstein J et al.; The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C . The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown . By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B . In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature tm = 43 degrees C and 41 degrees C, denaturation enthalpies DeltaH = 46 kcal.mol-1) . The thermal melting of protein A-B presents two nearly identical transitions (tm = 40 degrees C and 41 degrees C, DeltaH = 44 kcal.mol-1 and 46 kcal.mol-1, respectively) . We conclude that the two domains A and B within protein A-B behave as independent domains . The thermal melting of HMG1 is biphasic . The two transitions have a different value of tm (38 degrees C and 55 degrees C) and corresponding values of DeltaH around 40 kcal.mol-1 . We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other . At 37 degrees C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native . The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal.mol-1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy. Eur J Biochem, 1999 Mar, 260(3), 649 - 60 Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences; Chi SW et al.; The interactions of the second RNA-binding domain of the Drosophila melanogaster Sex-lethal protein (Sxl RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3'-splicing site of the transformer pre-mRNA (GU8CUAGUG), were studied using heteronuclear two-dimensional NMR techniques . The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sxl RBD2 with each of these RNAs were recorded . It was found that Sxl RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3' splice-site sequence, CUAGUG, with similar affinities . In contrast, a nonspecific sequence, C8, did not bind to Sxl RBD2 . This result is consistent with previous in vitro RNA-selection and UV-cross-linking results which indicated that the Sex-lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AUnNnAGU . In both cases, the chemical-shift perturbations were significant for almost the same amino acid residues, including the two central beta-strands formed by the RNP2-motif and RNP1-motif with the two highly conserved aromatic residues (Y214 and F256) in the middle . As the first RNA-binding domain of Sex-lethal (Sxl RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sxl RBD2 was replaced by Ile using site-directed mutagenesis . On the one hand, the 1H and 15N chemical-shift perturbations indicated that GU8 binds to the same interface of mutant Sxl RBD2 as of wild-type Sxl RBD2, although its binding affinity was decreased significantly . On the other hand, the specific binding of Sxl RBD2 to CUAGUG was abolished almost completely by the Y-->I mutation . Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences. Eur J Biochem, 1999 Mar, 260(3), 609 - 18 Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor; Zerovnik E et al.; Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry . The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence . All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule . The pH transition is not two-state as the CD data at 222 nm deviated from all other probes . Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration . However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation . CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding . Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states . The kinetics were multiphasic with at least two folding intermediates . The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1. Science, 1999 Apr 2, 284(5411), 159 - 62 Phenotypic change caused by transcriptional bypass of uracil in nondividing cells; Viswanathan A et al.; Cytosine deamination to uracil occurs frequently in cellular DNA . In vitro, RNA polymerase efficiently inserts adenine opposite to uracil, resulting in G to A base substitutions . In vivo, uracil could potentially alter transcriptional fidelity, resulting in production of mutant proteins . This study demonstrates that in nondividing Escherichia coli cells, a DNA template base replaced with uracil in a stop codon in the firefly luciferase gene results in conversion of inactive to active luciferase . The level of transcriptional base substitution is dependent on the capacity to repair uracil . These results provide evidence for a DNA damage-dependent, transcription-driven pathway for generating mutant proteins in nondividing cells. Mol Cells, 1999 Feb 28, 9(1), 110 - 4 Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants; Eum WS et al.; Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS) . We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al . 1998a; 1998b) . Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms . We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E . coli cells, purified the protein, and studied its enzymic activities . Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity . However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A) . These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A) . The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules . Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme. Mol Gen Genet, 1999 Mar, 261(2), 374 - 80 The reversed SoxS-binding site upstream of the ribA promoter in Escherichia coli; Koh YS et al.; The ribA gene, encoding GTP cyclohydrolase II in Escherichia coli, is a member of the soxRS regulon, which is induced by superoxide-generating agents . By evaluating lacZ expression driven by the ribA promoter carrying different lengths of upstream region in a monolysogen, we found that the superoxide-responsive element resides between 56 and 94 nucleotides upstream of the transcriptional start site . Purified SoxS protein bound to this region and protected nucleotides between positions -80 and -58 from degradation by DNase I . This region contains a putative SoxS-binding sequence (soxbox) in reverse orientation . The SoxS protein interacted specifically with four guanine residues within the soxbox sequence, as demonstrated by methylation interference analysis . These results clearly indicate that SoxS binds to the reversed soxbox sequence in the ribA gene, while in other known genes of the soxRS regulon it binds to the normally oriented soxbox . Possible modes of interaction between SoxS and RNA polymerase are discussed. Mol Gen Genet, 1999 Mar, 261(2), 307 - 16 Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum; Miyazaki S et al.; Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant) . Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1 . Transcript expression is tissue specific . Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9) . Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers . Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems . Mpc7 is restricted to meristematic tissues . Mpc10 is only present in mature flowers . Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress . Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots . Four full-length transcripts have been obtained . In each case, after over-expression in E . coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs . Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism. Mutat Res, 1999 Mar 10, 433(2), 99 - 107 RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli; Nagel R et a