|
|
|
|
|
| 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 al.; Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision . We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process . In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants . Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process . The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating. Arch Androl, 1999 Mar-Apr, 42(2), 71 - 84 Molecular cloning and characterization of a novel testis-specific nucleoporin-related gene; Wang LF et al.; A 20-kDa sperm membrane protein cDNA, designated as RSD-1, was isolated by epitope selection from a rat testis lambda gtll expression library . RSD-1 was used as a probe to screen a human testis lambda ZAPII cDNA expression library . A cDNA designated as BS-63 was isolated and found to consist of 1933 bp with an open reading frame of 1824 bp and assigned the accession number U64675 by GenBank . The deduced polypeptide consisted of 608 amino acid residues containing XFXFG or FG motifs that are characteristic of nuclear pore complex (NPC) proteins and act as potential binding sites for Ran . The N-terminal region has high homology with RanBP2/Nup358, a nucleoporin component, showing that BS-63 is a member of the NPC family . Northern blot analysis of mRNAs prepared from various human tissues shows that BS-63 is transcribed in two forms: 6.0 and 8.5 kb . The 8.5-kb transcript was present in low amounts in several somatic tissues, whereas the 6.0-kb transcript is expressed only in testis . In situ hybridization analysis of human testis sections showed that BS-63 mRNA is expressed only in germ cells at all stages of spermatogenesis . Sertoli cells did not transcribe the gene. Arch Androl, 1999 Mar-Apr, 42(2), 63 - 9 Expression and characterization of an epididymis-specific gene; Fan HY et al.; A truncated cDNA coding a rabbit epididymal protein (BE-20) was identified in a previous study . In the present study the full-length cDNA was isolated by the method of rapid amplification of cDNA ends . The BE-20 cDNA consisted of 585 bp with a poly(A) tail of 26 residues and an open reading frame composed of 369 bp encoding a deduced polypeptide containing 123 amino acid residues with a calculated molecular mass of 13 kDa . The N-terminus-contained a leucine-rich segment . BE-20 cDNA has about 76.8% homology with the HE4 gene of human epididymis . Northern blot analysis of mRNAs prepared from 17 different human tissues was performed using as probe a 0.5-kb DNA fragment corresponding to a segment of BE-20 cDNA . Positive reaction was elicited only with epididymal mRNA . A DNA fragment corresponding to a section of the open reading frame of BE-20 cDNA was cloned in Escherichia coli under the control of the T7 promoter . The cellular content of the expressed recombinant protein comprised about 55% of the total protein . The chromatographically purified bacterial product migrated as a single band with an estimated M(r) of 15 kDa on analysis by SDS-PAGE . In conclusion, BE-20 cDNA is expressed only in the epididymis . It is structurally related to the four-disulfide core family of extracellular proteinase inhibitors and may be involved in sperm maturation. J Biochem (Tokyo), 1999 Apr, 125(4), 832 - 7 Functional domain structure of human heterochromatin protein HP1(Hsalpha): involvement of internal DNA-binding and C-terminal self-association domains in the formation of discrete dots in interphase nuclei; Yamada T et al.; Human heterochromatin protein HP1(Hsalpha) possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region . Here, to examine its in vivo properties, we expressed HP1(Hsalpha) as a fusion product with green fluorescent protein in human cells . HP1(Hsalpha) was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy . Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain . However, the chromo domain alone stained nuclei homogeneously . To correlate this dot-forming activity with self-associating activity in vitro, the chromo and chromo-shadow domain peptides were independently expressed in Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde . In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer . When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively . These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation in vivo. Biochim Biophys Acta, 1999 Mar 25, 1437(3), 409 - 14 Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var . capitata); Kim DU et al.; We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage . Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa . The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively . Expression of this cDNA clone in E . coli shows a functional PLD activity similar to that of the natural PLD. Nucleic Acids Res, 1999 Apr 15, 27(8), 1906 - 11 Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II); Muth GW et al.; Studying the intricate folding of rRNA within the ribosome remains a complex problem . Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion . We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100 . This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA . It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data . The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA. Nucleic Acids Res, 1999 Apr 15, 27(8), 1795 - 801 Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA; Kooistra R et al.; The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents . A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man . The DmAGT gene is located at position 84A on the third chromosome . The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids . The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA . Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion . Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine . In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo . These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila. Biotechnol Bioeng, 1999 Feb 5, 62(3), 301 - 10 High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli; Ejima D et al.; Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use . Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E . coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6 . Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22 degrees C for 16 h . After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E . coli proteins were efficiently removed by successive steps of chromatography . The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC . These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use . Biotechnol Bioeng, 1999 Jan 20, 62(2), 208 - 15 A simple, two-component buffer enhances use of chromatofocusing for processing of therapeutic proteins; Logan KA et al.; To extend the feasibility of chromatofocusing to industrial use, we have developed a simple chromatofocusing buffer system capable of generating a smooth pH gradient without the use of an external gradient maker . Using two cationic buffering components, an internal pH gradient is produced on appropriate chromatography media over a broad pH range (9.5 to 5.0) . The utility of this buffer system is demonstrated with PBE94 and DEAE Sepharose fast flow ion-exchangers, as well as with experimental fast flow chromatofocusing gels . Using a rapid flow rate, we evaluated this buffer system for recovery of a therapeutic protein from a bacterial cell extract . The simplicity of the buffer system requiring no external gradient maker, coupled with the use of fast flow chromatographic media to produce broad-range pH gradients, improves the scalability of chromatofocusing for processing of therapeutic proteins . Biotechnol Bioeng, 1999 Jan 5, 62(1), 44 - 55 A patch coating method for preparing biocatalytic films of Escherichia coli; Lyngberg OK et al.; A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches . The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges . This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells . Large numbers of patches of immobilized E . coli can be generated on metal or polyester sheets . Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells . The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively . No leakage of cells was detected from 87% of the patches during 15 days of rehydration . Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability . E . coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days . Biotechnol Bioeng, 1998 Nov 5, 60(3), 271 - 6 Effect of post-induction nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli; Wong HH et al.; The effect of post-induction nutrient feeding strategies on the production of bioadhesive protein using an IPTG inducible expression system in Escherichia coli was investigated . Cells were cultured in an exponential fed-batch mode to the OD600 of ca . 100 (48 gDCW/L) prior to induction . Six different post-induction nutrient feeding strategies (pH-stat, exponential, constant and linear change in feeding rate with three different slopes) were then applied, and bioadhesive protein production was examined . It was found that post-induction cell growth was independent of nutrient feeding rate . However, bioadhesive protein production was significantly affected by post-induction feeding strategies . Linearly changing post-induction feeding rate with a suitable slope allowed production of bioadhesive protein up to 5.3 g/L, which was higher than that obtained by the other post-induction feeding strategies . Biotechnol Bioeng, 1998 Oct 20, 60(2), 230 - 8 Effect of Escherichia coli biomass composition on central metabolic fluxes predicted by a stoichiometric model; Pramanik J et al.; The amino acid composition of proteins and the fatty acid composition of the cell membranes were measured in Escherichia coli growing exponentially in batch culture on glucose, succinate, glycerol, pyruvate, and acetate, and growing under continuous culture conditions on glucose at dilutions rates equivalent to the growth rates of the batch cultures . Although the fatty acid composition of the membranes did change significantly with carbon source and dilution rate, the amino acid content of proteins did not change significantly under either condition . A previously developed stoichiometric model of metabolism was used to calculate the fluxes through the metabolic reactions and to determine their sensitivity to changes in fatty acid and amino acid composition . Biotechnol Bioeng, 1998 Oct 5, 60(1), 44 - 52 Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase; Kusnadi AR et al.; The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry . This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system . The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations . The accumulation level of rGUS reached 0.4% of total extractable protein . Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression . Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS . Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity . rGUS exposed to dry heat was more stable in ground than in whole kernels . The enzyme stability was correlated with the moisture loss of the samples during the heating . Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions . The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples . The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity . rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography . Similar purity and yield of rGUS were obtained from both extracts . Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E . coli GUS . Biotechnol Bioeng, 1998 Sep 20, 59(6), 666 - 72 mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system; Carrier T et al.; The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5' hairpin structures . Product formation and cell growth were evaluated under a number of inducer concentrations . The introduction of a 5' hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated . With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate . At inducer concentrations between 1 x 10(-4) and 4 x 10(-4)%, the productivity of low-copy plasmids containing the 5'-hairpin was equal to or greater than that from multicopy plasmids . Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression . Biotechnol Bioeng, 1998 Aug 5, 59(3), 294 - 301 A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme; Villaverde A et al.; The G-H loop of foot-and-mouth disease virus is a disordered protrusion of the VP1 protein exposed on the virion surface . This short stretch includes an arginine-glycine-aspartic acid tripeptide, a recognized integrin-binding motif, which is responsible for cell attachment and infection . Eight copies of a peptide reproducing the amino acid sequence of this FMDV ligand have been displayed in solvent-exposed regions on an enzymatically active recombinant beta-galactosidase . This viral peptide segment enables the chimeric enzyme to bind mammalian cell lines with different efficiencies, probably depending on the number of suitable cell receptors present on each of them . Moreover, it also promotes the internalization of the attached enzyme, which is transiently active inside the cells . These results suggest further exploration of the potential use of short adhesion peptides of viral origin as cell attachment tags to direct the targeted delivery of both genes and enzymes, instead of whole, infectious viruses . Biotechnol Bioeng, 1998 Jun 20, 58(6), 658 - 62 Activity losses among T4 lysozyme variants after adsorption to colloidal silica; Bower CK et al.; Maintaining a specific molecular conformation is essential for the proper functioning of an enzyme . A substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution . Activity may also be lost as an enzyme undergoes a conformational change during adsorption . In this study, we investigated the effect of thermostability on the activities of three T4 lysozyme variants after adsorption to 9 nm colloidal silica particles . Less-stable T4 lysozyme variants lost more activity after adsorption than did more stable variants, apparently because they experienced more extensive structural alteration . Biotechnol Bioeng, 1998 Jun 5, 58(5), 502 - 9 Genetically structured mathematical modeling of trp attenuator mechanism; Koh BT et al.; A genetically structured mathematical model of the trp attenuator in Escherichia coli based on known coupling mechanisms of the transcription of the trp leader region and translation of the trp leader peptide region is proposed . The model simulates, both qualitatively and quantitatively, the effects of tryptophan on the repression of cloned gene products . It shows that repression by attenuation mechanism alone operates over a narrow trp concentration range of 1 to 5 microM compared with 1 to 100 microM for trp repressor mechanism . This implies that attenuation by transcription termination is not relaxed until tryptophan starvation is severe . Simulation results show that the attenuator starts to derepress when the repressor is about 40% repressed, and becomes significantly derepressed only when the repressor repression decreased to about 20% . Unlike the case of repressor-operator interaction, the operating range of tryptophan concentration in the attenuator mechanism is not sensitive to plasmid copy number . Biotechnol Bioeng, 1998 Apr 5, 58(1), 92 - 100 Growth characteristics of Escherichia coli HB101{pGEc47} on defined medium; Rothen SA et al.; This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101{pGEc47} can be related to yeast extract-enriched medium rather than plasmid properties . An optimal medium for growth of E . coli HB101{pGEc47} was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1) . The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation) . Growth on defined medium after medium optimization is as rapid as on complex medium (0 . 42-0.45 h-1) . The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1 . Biotechnol Bioeng, 1998 Feb 20, 57(4), 381 - 6 Chemical treatment of Escherichia coli . II . Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells; Falconer RJ et al.; A method is presented for the direct extraction of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells . Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells . Comparable levels of Long-R3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption . However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components . The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min . Urea appears important for permeabilization of the cell and dissolution of the inclusion body . Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release . pH proved to be important with lower levels of protein release achieved at low pH values (<9) . Cell concentration also had a minor effect on Long-R3-IGF-I release and caused an observable increase in viscosity . Advantages of the direct extraction method include its speed, simplicity, and efficiency at releasing product . Can J Physiol Pharmacol, 1998 Oct-Nov, 76(10-11), 1008 - 16 Increased neural damage to global hemispheric hypoxic ischemia (GHHI) in febrile but not nonfebrile lipopolysaccharide Escherichia coli injected rats; Thornhill J et al.; Experiments were conducted to compare the impact of febrile versus nonfebrile lipopolysaccharide (LPS) induced bacterial infection at the time of global hemispheric hypoxic ischemia (GHHI) on the neural damage evoked by the GHHI insult . In the first study acute intraperitoneal (i.p.) sterile saline (SS) or LPS Escherichia coli (60 microg/kg) was given to groups of male, conscious Long Evans rats, and core (colonic, Tc) temperatures were monitored over 6 h postinjection . Peak febrile response occurred approximately 5 h after the LPS E . coli was injected . Upon sacrifice 7 days later, no hemispheric or regional brain damage occurred in the saline or LPS-injected groups of this first study . In the second study, GHHI was applied (ligation of right common carotid artery + 35 min of 12% O2) in groups of anesthetized, male Long Evans rats previously given an acute i.p . injection of sterile saline or 60 microg/kg LPS E . coli 5 h earlier . Temperatures (Tc) were monitored before, during, and 1.5 and 24 h following GHHI . The LPS-injected group was subdivided into a febrile (Tc > 38 degrees C before and (or) after GHHI) and nonfebrile (Tc < 38 degrees C before and after GHHI) subgroups . A significant correlation was found between the peak temperature rise from preinjection control values following drug administration of either saline or LPS E . coli and the resultant hemispheric damage caused by GHHI . Moreover, upon sacrifice 7 days later ipsilateral hemispheric and regional (i.e., hippocampal, thalamic) damage to GHHI of the febrile LPS E . coli group was significantly increased from respective hemispheric, hippocampal, and thalamic damage of the saline and nonfebrile, LPS groups given the same ischemic insult . Results suggest that the heightened Tc of a LPS infection at the time of global ischemia exacerbated the neural damage of GHHI, a finding similar to that reported with heightened core temperatures induced by external heating. FEBS Lett, 1999 Mar 12, 446(2-3), 343 - 50 DNA binding protein dbpA binds Cdk5 and inhibits its activity; Moorthamer M et al.; Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks) . Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons . Interestingly, the only known protein that activates Cdk5 (i.e . p35) is expressed solely in the brain . It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis . However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation . In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library . We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5 . Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase . The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme . Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A . It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other. FEBS Lett, 1999 Mar 12, 446(2-3), 331 - 7 The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells; Kedzierska S et al.; The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins . In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C . In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min) . This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ . The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent . It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins . Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates . The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system. FEBS Lett, 1999 Mar 12, 446(2-3), 287 - 91 A novel dnaK operon from Porphyromonas gingivalis; Yoshida A et al.; The nucleotide sequence of the dnaK operon cloned from Porphyromonas gingivalis revealed that the operon does not contain homologues of either dnaJ or grpE . However, there were two genes which encode small heat shock proteins immediately downstream from the dnaK and they were transcribed together with dnaK as one unit . The ATPase activity of the P . gingivalis DnaK was synergistically stimulated up to 40-fold in the simultaneous presence of Escherichia coli DnaJ and GrpE . These results suggest that the DnaK homologue of P . gingivalis, with its unique genetic structure and evolutionary features, works as a member of the DnaK chaperone system. FEBS Lett, 1999 Mar 12, 446(2-3), 278 - 82 Identification and characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase; Uchida T et al.; A second member of the parvulin family of peptidyl-prolyl cis/trans isomerases was identified in a human lung cDNA library . The gene encoded a protein named hPar14 that has 131 amino acid residues and a molecular mass of 13676 Da . Sequence comparison showed 34.5% identity to E . coli Par10 and 34% identity to human Pin1 (hPar18) within a C-terminal region of 87 or 120 amino acid residues, respectively . In comparison to the E . coli Par10, hPar14 possesses a N-terminal extension of 41 amino acid residues . This extension does not contain a polyproline II helix-binding motif typical of the known eukaryotic parvulins . The hPar14 does not accelerate the cis to trans interconversion of oligopeptides with side chain-phosphorylated Ser(Thr)-Pro moieties as hPin1 did . In contrast, it showed preference of an arginine residue adjacent N-terminal to proline . Northern blot analysis revealed expression of the gene within various human tissues like heart, placenta, liver, kidney and pancreas. FEBS Lett, 1999 Mar 12, 446(2-3), 243 - 6 Complement component C8gamma is expressed in human fetal and adult kidney independent of C8alpha; Trojer P et al.; Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily . So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging . We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma . Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney . Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney . Thus its physiological function is not restricted to a specific action in association with complement components . As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli . To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm. Clin Cancer Res, 1999 Mar, 5(3), 637 - 42 Intracerebral adenovirus-mediated p53 tumor suppressor gene therapy for experimental human glioma; Li H et al.; Malignant gliomas of astrocytic origin are good candidates for gene therapy because they have proven incurable with conventional treatments . Although mutation or inactivation of the p53 tumor suppressor gene occurs at early stages in gliomas and is associated with tumor progression, many tumors including high-grade glioblastoma multiforme carry a functionally intact p53 gene . To evaluate the effectiveness of p53-based therapy in glioma cells that contain endogenous wild-type p53, a clinically relevant model of malignant human glioma was established in athymic nu/nu mice . Intracerebral, rapidly growing tumors were produced by stereotactic injection of the human U87 MG glioma cell line that had been genetically modified for tracking purposes to express the Escherichia coli lacZ gene encoding beta-galactosidase . Overexpression of the p53 gene by adenovirus-mediated delivery into the tumor mass resulted in rapid cell death with the eradication of beta-galactosidase-expressing glioma cells through apoptosis . In long-term experiments, the survival of mice treated with the p53 adenoviral recombinant was significantly longer than that of mice that had received control adenoviral recombinant . During the observation period of 1 year, a complete cure was achieved in 27% of animals after a single injection of p53 adenoviral recombinant, and 38% of the animals were tumor free in the group receiving multiple injections of p53 adenoviral recombinant into a larger tumor mass . These experiments demonstrate that overexpression of p53 in gliomas, even in the presence of endogenous functional wildtype p53, leads to efficient elimination of tumor cells . These results point to the potential therapeutic usefulness of this approach for all astrocytic brain tumors. FEBS Lett, 1999 Mar 5, 446(1), 133 - 6 Localization of the site for the nucleotide effectors of Escherichia coli carbamoyl phosphate synthetase using site-directed mutagenesis; Mora P et al.; Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP . The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished . The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere . Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP. FEBS Lett, 1999 Mar 5, 446(1), 127 - 32 Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy; Quirk PG et al.; We have analysed 1H, 15N-HSQC spectra of the recombinant, NADP(H)-binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein . Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein . These observations reflect the structural changes resulting from hydride transfer . The interactions between the recombinant, NADP(H)-binding component and its partner, NAD(H)-binding protein, are complicated . Helix B of the recombinant, NADP(H)-binding component may play an important role in the binding process. FEBS Lett, 1999 Mar 5, 446(1), 75 - 80 Contributions of the ionization states of acidic residues to the stability of the coiled coil domain of matrilin-1; Dames SA et al.; The pKa values of eight glutamic acid residues in the homotrimeric coiled coil domain of chicken matrilin-1 have been determined from 2D H(CA)CO NMR spectra recorded as a function of the solution pH . The pKa values span a range between 4.0 and 4.7, close to or above those for glutamic acid residues in unstructured polypeptides . These results suggest only small favorable contributions to the stability of the coiled coil from the ionization of its acidic residues. FEBS Lett, 1999 Mar 5, 446(1), 30 - 4 The L2 loop peptide of RecA stiffens and restricts base motions of single-stranded DNA similar to the intact protein; Selmane T et al.; The L2 loop in the RecA protein is the catalytic center for DNA strand exchange . Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light . Both fluorescence intensity and anisotropy of an etheno-modified poly(dA) increase upon peptide binding, indicate that the base motions of single-stranded DNA are restricted in the complex . In agreement with this conclusion, the peptide-poly(dT) complex exhibits a significant linear dichroism signal . The peptide is also found to modify the structure of double-stranded DNA, but does not denature it . It is inferred that strand separation may not be required for the formation of a joint molecule. FEBS Lett, 1999 Mar 5, 446(1), 15 - 7 Effect of magnetic resonance imaging on human respiratory burst of neutrophils; Heine J et al.; It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro . We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions . Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(+/-11.4 min)) . The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry . The respiratory burst was induced either with phorbol 12-myristate 13-acetate, Escherichia coli, N-formyl-methionyl-leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation . There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent . Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils. Mol Cell Biochem, 1999 Jan, 190(1-2), 133 - 41 Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin; Kusano K et al.; Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm . The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104-M115 of the cofilin primary sequence (Yonezawa et al . 1991 ) . In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3'-region of the open reading frame . The truncated cofilin molecules produced in E . coli were purified and examined for their actin-binding and PIP2-binding ability . We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker . In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction . These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin. J Vet Diagn Invest, 1999 Mar, 11(2), 146 - 51 Prevalence of genotypes for fimbriae and enterotoxins and of O serogroups in Escherichia coli isolated from diarrheic piglets in Korea; Kwon D et al.; Polymerase chain reaction for 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa, STb), and 1 heat-labile enterotoxin (LT) were performed on 400 Escherichia coli isolates to determine their genotype prevalence among enterotoxigenic E . coli isolates from preweaned pigs with diarrhea in the Republic of Korea . A total of 200 of the 400 E . coli isolates were also selected for characterization of the O serogroup . Of these 200 isolates, serogroup could be determined in 139 (69.5%) but not in 61 isolates (30.5%) . Isolates of serogroup O101 were the most common, followed in descending order by 08, 020, 0162, 0141, and 0149 . Ninety-seven (24.3%) of the 400 E . coli isolates carried genes for at least 1 of the entertoxins or fimbrial adhesins . Of these 97 isolates, 27 carried genes for at least 1 of the fimbrial adhesins and entertoxins . Sixty-six percent of the isolates that carried fimbrial adhesin genes carried genes for at least 1 of the enterotoxins, and 71% of the isolates that carried enterotoxin genes carried genes for at least 1 of the fimbrial adhesins . Genes for the F6 fimbriae were detected in 6% of the E . coli isolates, and F4+, F41+, and F5+ genes were detected in 4.3%, 3.3%, and 2% of the isolates, respectively . Genes for STa, STb, and LT were detected in 10%, 8.5%, and 4.3% of the isolates, respectively . The 6 major genotypes observed in this study (in decreasing order) were F6+, STb+, F41+, STa+STb+, F6+STa+, and STa+. Free Radic Res, 1998 Dec, 29(6), 585 - 94 DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation; Pflaum M et al.; Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells . By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver . Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications . Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis . In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells . Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions . The percentage was dependent on the species ultimately responsible for the DNA damage and was approx . 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA. Free Radic Res, 1998 Dec, 29(6), 487 - 97 Excision repair of 8-hydroxyguanine in mammalian cells: the mouse Ogg1 protein as a model; Boiteux S et al.; 8-Hydroxyguanine (8-OH-Gua) is a major mutagenic lesion produced on DNA by the oxidative stress induced by either the endogen metabolism or the exposure to external agents . In bacteria and yeast this modified base can be removed by specific DNA glycosylases . Recently a human gene coding for an 8-OH-Gua DNA glycosylase/AP lyase has been identified by its homology to the yeast OGG1 . This gene is located in human chromosome 3p25, a region commonly rearranged in various cancers, specially in lung tumor cells . We report here the cloning, by sequence homology to the yeast OGG1, of a mouse cDNA coding for a 8-OH-Gua DNA glycosylase with 84% and 38% identity to the human and yeast relevant proteins, respectively . The Ogg1 gene is localized to the mouse chromosome 6E . The mouse Qgg1 cDNA, when expressed in Eschierichia coli, is capable of suppressing the spontaneous mutator phenotype of a DNA repair deficient fpg mutgamma strain . The mouse Ogg1 protein acts efficiently on duplexes in which the 8-OH-Gua is paired with a cytosine but is inactive on 8-OH-Gua: Ade pair, consistently with its proposed biological role in the avoidance of mutations . A comparison of the mouse enzyme with other eukaryotic Ogg1 enzymes is also presented . The isolation of this gene will allow the development of an animal model to study the effects of oxidative stress on carcinogenesis and degenerative diseases. Baillieres Clin Haematol, 1998 Jun, 11(2), 497 - 507 The haemolytic-uraemic syndrome in childhood; van de Kar NC et al.; Haemolytic-uraemic syndrome (HUS) is a clinical syndrome characterized by acute haemolytic anaemia with fragmented erythocytes, thrombocytopenia and acute renal failure . It is one of the leading causes of acute renal failure in childhood . HUS in children can be divided into the so-called typical, diarrhoea-associated HUS, and atypical HUS, which is not preceded by acute gastroenteritis . Infection with verocytotoxin-producing Escherichia coli is the main cause of diarrhoea-associated HUS . In this chapter the pathogenesis of diarrhoea-associated HUS and the role of verocytotoxin-producing Escherichia coli in this form of HUS is emphasized. Plant J, 1999 Feb, 17(3), 241 - 50 Regulation of gibberellin biosynthesis genes during flower and early fruit development of tomato; Rebers M et al.; Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization . In the GA-biosynthetic pathway, the reactions catalyzed by GA 20-oxidases have been implicated as site of regulation . To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20-oxidase cDNA clones, Le20ox-1, -2 and -3 . The three genes showed different organ-specific patterns of mRNA accumulation . Analysis of the transcript levels of the three GA 20-oxidase genes, as well as those of copalyl diphosphate synthase (LeCPS) and GA 3 beta-hydroxylase (Le3OH-2) during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation . Up until anthesis, transcripts were observed for LeCPS, Le20ox-1, -2 and Le3OH-2, with an accumulation of Le20ox-1 mRNA . In contrast to the high level of Le3OH-2 transcripts in the fully open flower, mRNA levels of Le20ox-1, -2 and LeCPS were reduced at this stage . After anthesis, LeCPS and Le20ox-1 transcripts increased again . In addition, Le20ox-3transcripts increased whereas the transcripts of Le3OH-2 decreased to an undetectable level . In situ hybridization results demonstrated that during early stages of bud development, Le20ox-2 transcripts were localized in the tapetum and placenta . The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3769 - 74 Regulation of p53 expression by thymidylate synthase; Ju J et al.; Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA . In this report, we present evidence that p53 expression is affected in a similar manner by TS . For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene . The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells . This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA . Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E . coli TS gene . These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level . In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as gamma-irradiation . These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3611 - 5 Expression of Batis maritima methyl chloride transferase in Escherichia coli; Ni X et al.; Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride . A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme . In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme . The recombinant enzyme is active and promotes the production of methyl chloride by E . coli under in vivo conditions . The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl-, Br-, and I-)-binding capacities . Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations . This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes . These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3562 - 7 Combinatorial protein engineering by incremental truncation; Ostermeier M et al.; We have developed a combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer . This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding . We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation . Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor . Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3537 - 9 Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli; Liochev SI et al.; Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH . In this paper, nitroreductase A is induced in Escherichia coli by exposure to paraquat in a manner that depends on the expression of soxR . Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gene encoding nitroreductase A, but it was approximately 3-fold induced in the parental strain . Nitroreductase A thus appears to be a member of the soxRS regulon and probably contributes to the defenses against oxidative stress by minimizing the redox cycling attendant upon the univalent reduction of nitro compounds, quinones, and dyes. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3525 - 30 Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease; Sondej M et al.; Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli . By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIAGlc . Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity . Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIAGlc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAGlc . The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIAGlc. Nat Biotechnol, 1999 Mar, 17(3), 259 - 64 Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling; Christians FC et al.; The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling . A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT) . Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT . After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK . Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations . Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine . In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine . Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity . Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations. Mol Microbiol, 1999 Feb, 31(4), 1265 - 74 Molecular genetic basis for the variable expression of Lewis Y antigen in Helicobacter pylori: analysis of the alpha (1,2) fucosyltransferase gene; Wang G et al.; Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y . The synthesis of Lewis Y involves the actions of alpha (1,3) and alpha (1,2) fucosyltransferases (FucTs) . Here, we report the molecular cloning and characterization of genes encoding H . pylori alpha (1,2) FucT (Hp fucT2) from various H . pylori strains . We constructed Hp fucT2 knock-out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy . The Hp fucT2 gene contains a hypermutable sequence {poly (C) and TAA repeats}, which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism . Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full-length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695) . In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full-length protein . The production of the full-length protein by the 26695 fucT2 gene could be attributed to translational-1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present . Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype . In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter . Based on these data, we proposed a model for the variable expression of Lewis Y by H . pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved. Mol Microbiol, 1999 Feb, 31(4), 1243 - 54 DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli; Bilcock DT et al.; In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites . The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction . To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites . The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently . Plasmids containing methylated SfI sites were not restricted . No rearrangements of the plasmids carrying SfiI sites were detected among the transformants . Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E . coli cells . The properties of the system in vivo match those of the enzyme in vitro . For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA. Mol Microbiol, 1999 Feb, 31(4), 1171 - 81 The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12; Kulasekara HD et al.; The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases . The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion . DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity . Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively . In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate . Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms . First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination . Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved . Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression. Mol Microbiol, 1999 Feb, 31(4), 1161 - 9 The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain; King GF et al.; Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE . MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites . MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles . Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88 . Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization . While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations . This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD. Mol Microbiol, 1999 Feb, 31(4), 1125 - 37 Chemotactic-like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY; Barak R et al.; We describe a chemotactic-like response of Escherichia coli strains lacking most of the known chemotaxis machinery but containing high levels of the response regulator CheY . The bacteria accumulated in aspartate-containing capillaries, they formed rings on tryptone-containing semisolid agar, and the probability of counterclockwise flagellar rotation transiently increased in response to stimulation with aspartate (10(-10)-10(-5) M; the response was inverted at > 10(-4) M) . The temporal response was partial and delayed, as was the response of a control wild-type strain having a high CheY level . alpha-Methyl-DL-aspartate, a non-metabolizable analogue of aspartate as well as other known attractants of E . Coli, glucose and, to a lesser extent, galactose, maltose and serine caused a similar response . So did low concentrations of acetate and benzoate (which, at higher concentrations, act as repellents for wild-type E . coli) . Other tested repellents such as indole, Ni2+ and CO2+ increased the clockwise bias . These observations raise the possibility that, at least when the conventional signal transduction components are missing, a non-conventional chemotactic signal transduction pathway might be functional in E . coli . Potential molecular mechanisms are discussed. Mol Microbiol, 1999 Feb, 31(4), 1025 - 38 A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in Escherichia coli; Martin-Farmer J et al.; When placed downstream of the start codon, multimers of the dinucleotide CA stimulated translation from lacZ, gusA and neo mRNAs in the presence or absence of an untranslated leader sequence . Enhanced expression in the absence of a leader and Shine-Dalgarno sequence indicated that stimulation by CA multimers was independent of translation signals contained within the untranslated leader . Multimers of CA stimulated a significantly higher level of lacZ expression than multimers of individual C or A nucleotides . Translation levels increased as the number of CA repeats increased; fewer multimers were required for enhanced expression from leadered mRNA than from mRNA that was deleted for its leader sequence . Addition of down-stream CA multimers increased the ribosome binding strength of mRNA in vitro and the amount of full-length mRNA in vivo, suggesting that the enhanced expression resulted from translation of a more abundant functional message containing a stronger ribosome binding site . The presence of downstream CA-rich sequences, occurring naturally in several Escherichia coli genes, might contribute to translation of other mRNAs . Addition of CA multimers might represent a general mechanism for increasing expression from genes of interest. Mol Microbiol, 1999 Feb, 31(4), 1013 - 24 Insertion of Escherichia coli alpha-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment; Soloaga A et al.; alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells . The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential . In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage . The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy . All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins . In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e . it is not a transmembrane protein . The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin . These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin. Genetika, 1998 Nov, 34(11), 1480 - 3 {Effect of pH on cea gene expression in cells irradiated with UV-light and unirradiated Escherichia coli cells}; Smirnova GV et al.; The effect of pH on the expression of the cea gene encoding colicin EI in Escherichia coli was investigated by measuring the beta-galactosidase activity in the UV-irradiated growing cells carrying the cea-lacZ fusion . Maximum activity was observed at pH 7, and inhibition of expression was observed at pH 6 and pH 8 . Treatment of the irradiated cells with 50-mM acetate increased inhibition at pH 6.0-7.5 . No correlation between cea expression and the rate of cell growth was observed at different pH levels . Preliminary treatment with acetate at pH 7 reduced the expression of the recA gene, which participates in the regulation of the cea gene to 33% in irradiated cells and to 25% in nonirradiated cells. Genes Cells, 1998 Dec, 3(12), 777 - 88 Hierarchical and co-operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli; Bergstrom LC et al.; BACKGROUND: OmpR is a transcription factor that regulates the expression of the porin genes ompF and ompC in Escherichia coli . The phosphorylation state of OmpR, directed by the osmosensor EnvZ, determines its ability to bind to the upstream regulatory regions of these genes, a total of 14 phospho-OmpR binding sites . While it has been possible to study the stoichiometry and hierarchy of the OmpR-DNA interaction in the upstream regions of ompF and ompC, their disunited location on the bacterial chromosome has made it difficult to compare the individual binding affinities of respective sites . RESULTS: Using 1,10-phenanthroline-Cu+ footprinting on a fused construct containing both the ompF and ompC upstream regulatory sequences, and gel shift experiments on oligomers corresponding to individual sites, we have established a comparative hierarchy for OmpR binding, as F1, C1 > F2, F3 > C2 > C3 . In addition, the binding patterns reveal an apparent co-operative relationship between OmpR molecules bound at several upstream motifs . Densitometric analyses of the footprinted regions provide support for these observations . Mutational analysis of this construct reveals that the alteration of a conserved cytidine in the F1 motif (-86) causes a loss of OmpR affinity and disrupts hierarchical OmpR-binding in the entire ompF region . CONCLUSIONS: The present results provide a unique view of the OmpR interaction with the two respective promoters, ompF and ompC, and an insight into the question of how the expression of ompF and ompC are reciprocally regulated by medium osmolarity. Eur J Biochem, 1999 Mar, 260(2), 430 - 8 Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins; Stanasila L et al.; Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP . Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein . Binding of {3H}diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively) . Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists . Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells . The effect of sodium on agonist binding to E . coli-expressed receptors was investigated . Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins . We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E . coli and in COS cells . Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E . coli as an expression system for pharmacological studies. Eur J Biochem, 1999 Mar, 260(2), 308 - 17 Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers; Baykov AA et al.; Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme . By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold) . By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer . These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed . As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface . Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris . Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site. Mutat Res, 1999 Mar, 436(2), 179 - 84 Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking; Foster PL; The levels of proteins required for methyl-directed mismatch repair appear to decline in stationary-phase and nutritionally-deprived cells of Escherichia coli . It has been hypothesized that error-correction by the system also declines, and this decline is responsible for adaptive or stationary-phase mutations . However, evidence in support of this hypothesis is lacking . The mismatch repair system is no less effective in correcting errors during prolonged selection than it is during growth . Furthermore, mismatch repair proteins supplied in excess reduce both growth-dependent and adaptive mutation . Mutat Res, 1999 Mar 15, 440(1), 59 - 74 Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P-WP3106P tester strains; Ohta T et al.; We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA . In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P . The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E . coli K-12 strains CC101-CC106 . All the tester strains show Lac- and Trp- phenotype . Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid . The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well . In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains . Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested . Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains . In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions . Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions . Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds . All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P . In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens . Biochim Biophys Acta, 1999 Mar 19, 1444(3), 326 - 36 Cloning and functional expression of the cytoplasmic form of rat aminopeptidase P; Czirjak G et al.; A rat cytoplasmic aminopeptidase P was purified from liver cytosol with a procedure including an affinity elution step with 3 microM inositol 1,3,4-trisphosphate . Proteolytic fragments were generated, sequenced and the enzyme was cloned from a rat liver cDNA library . The structure shows high (87.8% and 95.5%, respectively) sequence identity at the nucleotide and amino acid levels with the previously described human putative cytoplasmic aminopeptidase P . The cloned rat enzyme was functionally expressed in Escherichia coli and also in COS-1 cells . Western blot analysis, using an antibody generated against the recombinant protein, and Northern blot hybridization showed ubiquitous expression of the protein in different tissues with the highest expression level in the testis. Gynecol Oncol, 1999 Apr, 73(1), 27 - 34 Growth suppression of human ovarian cancer cell lines by the introduction of a p16 gene via a recombinant adenovirus; Wolf JK et al.; OBJECTIVE: The cell cycle regulatory protein p16 (CDKN2/cyclin dependent kinase 4 inhibitor/multiple tumor suppressor-1) causes cell cycle arrest at the G1 checkpoint by inhibiting activity of cyclin D-CDK4 complexes . The purpose of this study is to assess the effect of introduction of the p16 gene into two ovarian cancer cell lines via a recombinant adenoviral vector (Ad5CMV-p16) . METHODS: Cells lines used were SKOV3, which has a p16 deletion, and OVCA420, which has normal p16 . Transduction efficiency was established by infecting cells with an adenovirus containing the Escherichia coli beta-galactosidase gene (Ad5CMV-beta-gal) at multiplicity of infection from 0 to 1000 and staining for X-gal . Cells were infected with Ad5CMV-p16 and cell growth was assessed by counting cells every other day for up to 7 days . Western blotting was done to assess for p16 expression after infection . Fluorescence-activated cell sorting after staining with propidium iodide was done to assess the effect of p16 on the cell cycle . RESULTS: The SKOV3 cell line was transduced with the adenovirus at a slightly lower MOI than the OVCA420 cell line . Growth of the Ad5CMV-p16-infected cells was suppressed 75-80% by cell count in both cell lines and caused morphologic changes of the cells consistent with apoptosis . The p16 protein expression was seen to increase within 24 h after introduction of the p16 gene . G1 arrest of cells occurred beginning 24 h after introduction of the p16 gene . CONCLUSIONS: These results suggest that Ad5CMV-p16 may be further studied as a potential therapeutic agent for ovarian cancer as introduction of the p16 gene into ovarian cancer cell lines causes a G1 arrest and attenuation of growth, regardless of the endogenous p16 status of the cells . Pharmacol Res, 1999 Mar, 39(3), 247 - 51 Antioxidant status in experimental peritonitis: effects of alpha tocopherol and taurolin; Konukoglu D et al.; The role of oxidative stress and antioxidant defences in inflammation-induced organ injury is not clearly understood . We determined the effects of Escherichia coli (E . coli) peritonitis in rats on peritoneum lipid peroxidation and antioxidant defences . Tissue malondialdehyde (MDA) levels were measured to determine the free radical-induced lipid peroxidation in peritonitis . Tissue glutathione (GSH) levels, and activities of GSH-peroxidase, GSH-reductase and superoxide dismutase were examined to show antioxidant status . We also examined the effects of alpha-tocopherol (20 mg kg-1 body weight) as antioxidant and taurolin (200 mg kg-1 body weight) as chemotherapeutic agents on the oxidant stress and antioxidant defence . The treatment agents and E . coli were administrated intraperitoneally . Animals were killed at 2 h after the onset symptoms and then the peritoneum were obtained . Untreated rats with peritonitis had significantly higher MDA levels and significantly lower antioxidant activity than that of the control animals . Treatment of alpha-tocopherol and taurolin decreased the antioxidant activity and improved the antioxidant status . Pretreatment with alpha-tocopherol for 3 days prior to the induction of peritonitis (IP) and administration of taurolin at the time of the IP were more effective than treatment with alpha-tocopherol at the time of the IP and pretreatment of taurolin, respectively . These results are consistent with the idea that an oxidant/antioxidant imbalance is involved in animal peritonitis . Uses of alpha-tocopherol and taurolin in peritonitis were effective in decreasing the oxidative stress of tissue during peritonitis . Anal Biochem, 1999 Apr 10, 269(1), 105 - 12 Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry; Arnold RJ et al.; Ribosomes from the K-12 strain of Escherichia coli were analyzed with good sensitivity and high mass accuracy using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . Fifty-five of the 56 subunit proteins were observable . Mass spectral peak locations were consistent with previously reported post-translational modifications involving N-terminal methionine loss, methylation, thiomethylation, and acetylation for all but one case . The speed and accuracy of mass spectrometry make it a good candidate for phylogenetic studies of ribosomes and the observation of posttranslational modifications in other organisms . J Bacteriol, 1999 Apr, 181(7), 2225 - 35 Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus; Youderian P et al.; The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus . Mutants of M . xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency . Expression of the Escherichia coli glk (glucokinase) gene in M . xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate . Enzyme assays of M . xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities . The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation . Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M . xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo. Nature, 1999 Mar 18, 398(6724), 246 - 51 A new protease required for cell-cycle progression in yeast; Li SJ et al.; In eukaryotes, protein function can be modulated by ligation to ubiquitin or to ubiquitin-like proteins (Ubl proteins) . The vertebrate Ubl protein SUMO-1 is only 18% identical to ubiquitin but is 48% identical to the yeast protein Smt3 . Both SUMO-1 and Smt3 are ligated to cellular proteins, and protein conjugation to SUMO-1/Smt3 is involved in many physiological processes . It remained unknown, however, whether deconjugation of SUMO-1/Smt3 from proteins is also essential . Here we describe a yeast Ubl-specific protease, Ulp1, which cleaves proteins from Smt3 and SUMO-1 but not from ubiquitin . Ulp1 is unrelated to any known deubiquitinating enzyme but shows distant similarity to certain viral proteases, indicating the existence of a widely conserved protease fold . Proteins related to Ulp1 are present in many organisms, including several human pathogens . The pattern of Smt3-coupled proteins in yeast changes markedly throughout the cell cycle, and specific conjugates accumulate in ulp1 mutants . Ulp1 has several functions, including an essential role in the G2/M phase of the cell cycle. J Bacteriol, 1999 Apr, 181(7), 2279 - 85 Genetic organization of the Escherichia coli K10 capsule gene cluster: identification and characterization of two conserved regions in group III capsule gene clusters encoding polysaccharide transport functions; Clarke BR et al.; Analysis of the Escherichia coli K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters . Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins . An rfaH mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters . An IS3 element and a phiR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes. J Bacteriol, 1999 Apr, 181(7), 2236 - 43 A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity; Ebel W et al.; Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication . Little is known about the mechanism of substrate recognition by Lon . To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity . The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded) . Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded . The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine . E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation . This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon . We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon. J Bacteriol, 1999 Apr, 181(7), 2217 - 24 Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB; Summers ML et al.; Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation . Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI) . Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation . Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of orfA . The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in S . meliloti and to the consensus PHO box in Escherichia coli . Introduction of a phoB mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the ackA::lacZ fusion . Mutations in either ackA alone or both pta and ackA did not affect the nodulation or nitrogen fixation phenotype of S . meliloti. J Bacteriol, 1999 Apr, 181(7), 2148 - 57 Analysis of the role of trans-translation in the requirement of tmRNA for lambdaimmP22 growth in Escherichia coli; Withey J et al.; The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid lambdaimmP22 phages in Escherichia coli . tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins . This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins . A model describing this function of tmRNA, the trans-translation model (K . C . Keiler, P . R . Waller, and R . T . Sauer, Science 271:990-993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA . Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity . However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific . In light of this new information, we examine the effects on lambdaimmP22 growth of mutations eliminating activities postulated to be important for two different steps in the trans-translation model, alanine charging of tmRNA and degradation of tagged proteins . This mutational analysis suggests that, while charging of tmRNA with alanine is essential for lambdaimmP22 growth in E . coli, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth . Based on these results, we propose that trans-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation. J Bacteriol, 1999 Apr, 181(7), 2124 - 31 Stabilization of the relaxosome and stimulation of conjugal transfer are genetically distinct functions of the R1162 protein MobB; Perwez T et al.; MobB is a small protein encoded by the broad-host-range plasmid R1162 and required for efficient mobilization of its DNA during conjugation . The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the origin of transfer (oriT) . We have generated in-frame mobB deletions that specifically inactivate the stabilizing effect of MobB while still allowing a high rate of transfer . Thus, MobB has two genetically distinct functions in transfer . The effect of another deletion, extending into mobA, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain . The mobB mutations that specifically affected stability also resulted in poor growth of cells, due to increased transcription from the promoters adjacent to oriT . The effects of the mutations could be suppressed not only by full-length MobB provided in trans, as expected, but also by additional copies of oriT, cloned in pBR322 . In addition, in the presence of MobA both the full-length and truncated forms of MobB stimulated recombination between oriT-containing plasmids . We propose a model in which MobB regulates expression of plasmid genes by altering the stability of the relaxosome, in a manner that involves the coupling of plasmid molecules. J Bacteriol, 1999 Apr, 181(7), 2084 - 93 The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon; Suziedeliene E et al.; Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component . At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA . The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E . coli chromosome . Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa . According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein . The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system . Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium . Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant . Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes . Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start . The asr promoter DNA region was demonstrated to bind PhoB protein in vitro . We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene. Mol Microbiol, 1998 Nov, 30(4), 843 - 53 Translational control of mRNA processing in the F1845 fimbrial operon of Escherichia coli; Loomis WP et al.; Endoribonucleolytic processing followed by differential decay of the cleavage products is an increasingly recognized mechanism for achieving co-ordinate regulation of functionally related proteins encoded by bacterial polycistronic transcripts . Unlike most examples when RNases E or III initiate decay, the daa transcript encoding F1845 fimbriae, a member of the Dr family of adhesins in Escherichia coli, is processed by an as yet unidentified endoribonuclease using a unique recognition mechanism . An open reading frame (ORF) predicted to encode a 57-amino-acid polypeptide was identified flanking the daa processing site . To determine whether this ORF is involved in processing, site-directed mutagenesis was used to generate mutants with altered translational efficiencies . A mutation in the putative ribosome binding site preceding the ORF significantly inhibited processing while the introduction of a premature stop codon abolished processing . Site-directed mutagenesis was used to introduce a limited number of mutations into the ORF, designated daaP, to alter the reading frame such that a different polypeptide of a similar size was encoded . Despite the presumed presence of trafficking ribosomes, this mutant failed to be processed, suggesting that the sequence of the DaaP peptide is important . However, the failure of a wild-type copy of the daaP gene to complement these mutations in trans suggested that the presence of wild-type daaP gene product was not sufficient to promote processing . Although active translation has been found to inhibit processing by RNases E and III, our data suggest that translation of the daaP gene is required in cis to promote processing by the endonuclease, perhaps due to an interaction of the nascent peptide with the ribosome or the daaP mRNA. FEBS Lett, 1999 Feb 26, 445(2-3), 425 - 30 Purification, characterization and crystallization of ERA, an essential GTPase from Escherichia coli; Chen X et al.; ERA is an essential GTPase widely conserved in bacteria . Homologues of ERA are also present in higher eukaryotic cells . ERA is involved in bacterial cell cycle control at a point preceding cell division . In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein . Here, we report the purification and crystallization procedures and results . The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity . ERA is one of the very few multi-domain GTPases crystallized to date. FEBS Lett, 1999 Feb 26, 445(2-3), 389 - 94 Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide; Kim SO et al.; The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress . Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein . NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem . Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised . These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp . The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions. Mol Cell Biochem, 1999 Jan, 191(1-2), 181 - 6 A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII; Sheu GT et al.; EF-1alpha binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the betagammadelta complex facilitates the exchange of GDP for GTP to initiate another round of elongation . To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant beta, gamma, and delta subunits from rabbit were expressed in E . coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration . To determine the availability of the beta and delta subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII . Formation of the nucleotide exchange complex increased the rate of phosphorylation of the beta subunit and reduced the Km, while addition of alpha to beta or the betagammacomplex inhibited phosphorylation by CKII . However, alpha had little effect on phosphorylation of delta . Thus, the beta and delta subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of beta was altered by association with other subunits, while the site on delta was always available for phosphorylation by CKII . From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of(alpha2betagamma2delta)2 is proposed and discussed. Mol Cell Biochem, 1999 Jan, 191(1-2), 169 - 80 A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity; Lin R et al.; The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes . The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment . In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity . During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E . coli extracts . The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro . Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified . Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII) . Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein . Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231 . Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function . Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha . The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity. RNA, 1999 Mar, 5(3), 409 - 19 Cloning and characterization of a mammalian pseudouridine synthase; Chen J et al.; This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p) . The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected . The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p) . The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates . The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA . A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus . The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame. RNA, 1999 Mar, 5(3), 344 - 59 Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA; Hunt SL et al.; Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for "internal ribosome entry site") . Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes . This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES . There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic . One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor . Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES. Hum Gene Ther, 1999 Mar 1, 10(4), 649 - 57 Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy; Puhlmann M et al.; Tumor-directed gene therapy faces many obstacles . Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome . A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models . Purine nucleoside phosphorylase (PNP), from E . coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine . In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP) . In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV {365 nm}) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7% . At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days . Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin . The potency of such a system was similar among all histologies tested . Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system . When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate . In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy. Oral Microbiol Immunol, 1998 Aug, 13(4), 246 - 52 Immunochemical detection of CD14 on human gingival fibroblasts in vitro; Hiraoka T et al.; The activation of monocytes and macrophages induced by lipopolysaccharide has been shown to contribute to the binding of lipopolysaccharide and lipopolysaccharide-binding protein complex to the cell surface CD14 molecule . To clarify the mechanism of the lipopolysaccharide-induced modulation of the function of gingival fibroblasts, we investigated the effect of anti-CD14 on interleukin 6 (IL-6) production on human gingival fibroblasts in vitro . Immunochemical staining revealed weak positivity for CD14 on fibroblasts from healthy gingiva, while strong positivity for CD14 was found on fibroblasts from inflamed gingiva . Western blot profiles of the fibroblasts and monocytes showed a CD14-positive reaction at 55 kDa . Fluorescein isothiocyanate-conjugated Escherichia coli lipopolysaccharide bound to fibroblasts more strongly in the presence of 10% fetal bovine serum than without serum . This binding, as well as IL-6 production, was blocked by anti-CD14 monoclonal antibody . The results showed that CD14 was present on human gingival fibroblasts, which suggests that lipopolysaccharide modulation of gingival fibroblast function depends on CD14. Eur J Clin Invest, 1999 Jan, 29(1), 63 - 7 Direct in vivo gene transfer to healing rat patellar ligament by intra-arterial delivery of haemagglutinating virus of Japan liposomes; Ozkan I et al.; BACKGROUND: Manipulation of ligament healing has been a major focus of orthopaedic research . In recent years, gene transfer to healing ligament appears to be a feasible method for manipulating the healing process . In this study, we investigated the feasibility of gene transfer to healing rat patellar ligament by intra-arterial delivery . METHODS: An attempt was made to transfer a reporter gene (Escherichia coli, beta-galactosidase gene) to healing rat patellar ligament using the haemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer method . Three days after cutting the patellar tendons of 25 14-week-old male Wistar rats, HVJ-liposome complexes containing beta-galactosidase (beta-gal) cDNA were injected into the femoral artery of 15 Wistar rats as the experimental group . HVJ liposomes without DNA were injected into the femoral artery of 10 Wistar rats as the control group . Three rats from the experimental group and two control rats were killed 3, 7, 14, 28 and 56 days after the injection . RESULTS: After X-gal staining, the rate of transfection in the experimental group (mean +/- SEM) was found to be 12.1% +/- 0.590%, 8.7% +/- 0.217%, 10.2% +/- 0.227%, 3.2% +/- 0.247% and 0.7% +/- 0.060% at post-injection days 3, 7, 14, 28 and 56 respectively . In control sections the number of blue-stained cells were very few at any point . CONCLUSION: We succeeded in introducing a reporter gene into healing rat patellar ligament by infra-arterial delivery of HVJ-liposome complexes . This method appears to have the potential to be applicable for soft-tissue healing studies and also healing studies of other tissues and organs. Eur J Biochem, 1999 Feb, 259(3), 851 - 8 ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5'-nucleotidase . Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity; Spychala J et al.; IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate . The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus . To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties . As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate . Although the S0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar . The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa . Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa . ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa . In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP . Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP . These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits. Eur J Biochem, 1999 Feb, 259(3), 789 - 94 Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata; Guerrero SA et al.; Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata {Nogoceke et al . (1997) Biol . Chem . 378, 827-836} where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione . Here we report on the full-length DNA sequence of the TXN previously isolated from C . fasciculata (TXN1) . The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1 . It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function . This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions . Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins . The TXNs thus form a distinct molecular clade within the thioredoxin superfamily . TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally . The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay . The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents. Eur J Biochem, 1999 Feb, 259(3), 770 - 5 Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli; Seto NO et al.; The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively . Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase . A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli . The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates . The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively . The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs . B donor specificity. Eur J Biochem, 1999 Feb, 259(3), 731 - 8 Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli; Sakaki T et al.; Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109 . As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM . Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase . In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3 . However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 . These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity . Another remarkable finding is that living recombinant E . coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E . coli cells. Eur J Biochem, 1999 Feb, 259(3), 618 - 25 Homologous plasminogen N-terminal and plasminogen-related gene A and B peptides . Characterization of cDNAs and recombinant fusion proteins; Lewis VO et al.; The cDNA corresponding to exons 2-4 of the processed human plasminogen (Pgn) gene, encoding the N-terminal peptide domain (NTP), has been cloned, expressed in Escherichia coli as a recombinant protein (r-NTP) containing a hexahistidine tag, and refolded to the native structure that contains two internal cystine bridges . RNA expression of the two Pgn-related genes, PRG A and PRG B, that potentially encode 9-kDa polypeptides having extensive similarity to the NTP has been investigated . Using RNA-based PCR with liver RNA as template, we demonstrate that PRG A encodes a detectable mRNA species . PRG A and PRG B have been found to be transcribed in the liver and yield virtually identical mRNAs . Neither of the PRGs are expressed in a variety of other normal tissues, as determined by Northern blot analysis . Factor-Xa digestion of the tagged r-NTP yields cleavage products which indicates that the expressed r-NTP domain of Pgn is endowed with a flexible conformation . Recombinant PRG B protein (r-PRG B) fused to a hexahistidine tag was purified and analyzed for structural integrity . Preliminary 1H-NMR spectroscopic data for r-NTP and r-PRG B indicate relatively fast amide 1H-2H exchange in 2H2O and close conformational characteristics for the two homologous polypeptides . Far ultraviolet-CD spectra for r-NTP and r-PRG B at pH 7.0 indicate similar defined secondary structure content for both domains, with 13-17% alpha-helix and 24-27% antiparallel beta-sheet . The fact that two transcriptionally active genes encode almost identical polypeptides supports the hypothesis that the Pgn NTP, together with the putative polypeptides encoded by the PRGs, may serve an important function, such as controlling the conformation of Pgn and thus its susceptibility to tissue activators. J Biol Chem, 1999 Apr 2, 274(14), 9677 - 85 Effect of cold shock on lipid A biosynthesis in Escherichia coli . Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein; Carty SM et al.; Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C . The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5% . We now report that wild-type E . coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA . The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C . The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear . Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J . J., and Raetz, C . R . H . (1996) J . Biol . Chem . 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase . Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E . coli . This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase . Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid . The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA . The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C. J Biol Chem, 1999 Apr 2, 274(14), 9665 - 72 A structure-function study of the C2 domain of cytosolic phospholipase A2 . Identification of essential calcium ligands and hydrophobic membrane binding residues; Bittova L et al.; The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein . To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration . The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles . The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane . Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted . Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding . In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W . (1998) J . Biol . Chem . 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins. J Biol Chem, 1999 Apr 2, 274(14), 9524 - 30 Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems; Ness LS et al.; KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization . The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles . By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins . A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC . Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB . Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity . The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB . We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations. J Biol Chem, 1999 Apr 2, 274(14), 9482 - 8 Probing the unfolding pathway of alpha1-antitrypsin; James EL et al.; Protein misfolding plays a role in the pathogenesis of many diseases . alpha1-Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease . In order to understand the misfolding process, it is necessary to examine the folding of alpha1-antitrypsin through the different structures involved in this process . In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into alpha1-antitrypsin and fluorescently labeled with N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine . The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding . The studies found that alpha1-antitrypsin unfolded through a series of intermediate structures . From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of alpha1-antitrypsin occurs via disruption of the A and C beta-sheets followed by the B beta-sheet . The implications of these data on both alpha1-antitrypsin function and polymerization are discussed. J Biol Chem, 1999 Apr 2, 274(14), 9479 - 81 Induction of the soxRS regulon of Escherichia coli by superoxide; Liochev SI et al.; The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of approximately 15 genes . The induction of this regulon by redox agents, known to mediate O-2 production, led to the view that O-2 is one signal to which it responds . However, redox cycling agents deplete cellular reductants while producing O-2, and one may question whether the regulon responds to the depletion of some cytoplasmic reductant or to O-2, or both . We demonstrate that raising {O-2} by mutational deletion of superoxide dismutases and/or by addition of paraquat, both under aerobic conditions, causes induction of a member of the soxRS regulon and that a mutational defect in soxRS eliminates that induction . This establishes that O-2, directly or indirectly, can cause induction of this defensive regulon. Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 199 - 205 Expression of the human hepatocyte growth factor cDNA in primary cultures of rat hepatocytes; Runge DM et al.; Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are primary mitogens for hepatocytes in culture . hepatocytes express the HGF-receptor MET but not HGF itself . To investigate the influence of autocrine HGF expression on the proliferative potential of hepatocytes, primary cultures were submitted to retrovirus-mediated transduction of the human hgf (huHGF) cDNA . Expression of the transduced cDNA revealed a minimum 2-fold increase in HGF-mRNA, whereas expression of the Escherichia coli beta-galactosidase gene remained even . Estimation of huHGF copy numbers showed there was a minimum 4-fold increase, suggesting an increase in the population of transduced cells . Immunoprecipitation of excreted huHGF and growth bioassays proofed that HGF was present and functional . HGF is excreted into the medium and therefore, by diffusion, available to transduced and non-transduced cells . The increase in huHGF-transduced cells suggests that the autocrine pathway as opposed to the paracrine pathway, which are both present at the same time, confers a growth advantage to these cells . Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 156 - 62 Multiple alternative transcripts of the human homologue of the mouse TRAD/R51H3/RAD51D gene, a member of the rec A/RAD51 gene family; Kawabata M et al.; Yeast RAD51, a homologue of Escherichia coli recA, plays a crucial role in mitotic and/or meiotic recombination and in the repair of double-strand DNA breaks . We have identified unique multiple alternative transcripts of a human TRAD/R51H3/RAD51D gene, a member of the recA/RAD51 gene family . One of the transcripts encoded a 328-amino-acid protein with 83.0% overall amino acid identity and 98 . 2% similarity with the mouse TRAD gene and had two nucleotide binding consensus sequences, motif A and motif B, conserved among members of this family . Other transcripts encoded truncated proteins with a partial N-terminal region of the orthologue or short proteins lacking internal sequences which contain nucleotide binding motifs . Northern blot analysis revealed that multiple transcripts of the human TRAD gene were expressed in various tissues and their distribution was not ubiquitous . Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 79 - 83 Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity; Arita Y et al.; We isolated the human adipose-specific and most abundant gene transcript, apM1 (Maeda, K., et al., Biochem . Biophys . Res . Commun . 221, 286-289, 1996) . The apM1 gene product was a kind of soluble matrix protein, which we named adiponectin . To quantitate the plasma adiponectin concentration, we have produced monoclonal and polyclonal antibodies for human adiponectin and developed an enzyme-linked immunosorbent assay (ELISA) system . Adiponectin was abundantly present in the plasma of healthy volunteers in the range from 1.9 to 17.0 mg/ml . Plasma concentrations of adiponectin in obese subjects were significantly lower than those in non-obese subjects, although adiponectin is secreted only from adipose tissue . The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of adiponectin in humans . Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 63 - 6 Association and dissociation of the calcium-binding domains of calpain by Ca2+; Suo S et al.; The calmodulin-like domain of calpain is important for the association of the calpain large and small subunits . We expressed the calmodulin-like domains of the large subunits of rabbit mu- and m-calpains and their small subunits in E . coli and purified them to homogeneity . Unlike the full-length subunits, the calmodulin-like domains are soluble in buffer containing Ca2+ . We performed gel filtration chromatography of the purified proteins and found that all three calmodulin-like domains exist as homodimers in the absence of Ca2+ and dissociate into monomers upon the addition of Ca2+ . Protein Expr Purif, 1999 Apr, 15(3), 389 - 400 Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases; Pedersen J et al.; We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins . The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP) . In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain . Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion . For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone . When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein . A protein with a HisTag-Gln extension is incubated with both DPPI and GCT . As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented . The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP . All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes . Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC . The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin . Protein Expr Purif, 1999 Apr, 15(3), 370 - 80 Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides; Guergova-Kuras M et al.; The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex . Constructions were made in vitro in a pUC-derived background using PCR amplification . The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17 . The transformants showed normal rates of growth . Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type . Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin . A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold . Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration . The purified enzyme contained all four subunits in an active dimeric complex . This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies . Protein Expr Purif, 1999 Apr, 15(3), 265 - 70 Expression and purification of human calbindin D28k; Thulin E et al.; Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport . Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli . The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA . The protein was then supplemented with calcium and dialyzed against neutral water . The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture . The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy . The entire expression and purification protocol takes only 3 days and is easy to scale up and down . It was designed to minimize degradation and deamidation . Protein Expr Purif, 1999 Apr, 15(3), 258 - 64 Overexpression and purification of an immunologically reactive His-BIV capsid fusion protein; Betemps D et al.; The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein . The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside . Purification was based on interaction of the hexa-histidine polypeptide with metal ions . Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture . The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein . The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples . J Mol Biol, 1999 Apr 2, 287(3), 627 - 44 Exploring the kinetic requirements for enhancement of protein folding rates in the GroEL cavity; Betancourt MR et al.; The chaperonin system, GroEL and GroES of Escherichia coli enable certain proteins to fold under conditions when spontaneous folding is prohibitively slow as to compete with other non-productive channels such as aggregation . We investigated the plausible mechanisms of GroEL-mediated folding using simple lattice models . In particular, we have investigated protein folding in a confined environment, such as those offered by the GroEL, to decipher whether rate and yield enhancement can occur when the substrate protein is allowed to fold within the cavity of the chaperonins . The GroEL cavity is modeled as a cubic box and a simple bead model is used to represent the substrate chain . We consider three distinct characteristic of the confining environment . First, the cavity is taken to be a passive Anfinsen cage in which the walls merely reduce the available conformation space . We find that at temperatures when the native conformation is stable, the folding rate is retarded in the Anfinsen cage . We then assumed that the interior of the wall is hydrophobic . In this case the folding times exhibit a complex behavior . When the strength of the interaction between the polypeptide chain and the cavity is too strong or too weak we find that the rates of folding are retarded compared to spontaneous folding . There is an optimum range of the interaction strength that enhances the rates . Thus, above this value there is an inverse correlation between the folding rates and the strength of the substrate-cavity interactions . The optimal hydrophobic walls essentially pull the kinetically trapped states which leads to a smoother the energy landscape . It is known that upon addition of ATP and GroES the interior cavity of GroEL offers a hydrophilic-like environment to the substrate protein . In order to mimic this within the context of the dynamic Anfinsen cage model, we allow for changes in the hydrophobicity of the walls of the cavity . The duration for which the walls remain hydrophobic during one cycle of ATP hydrolysis is allowed to vary . These calculations show that frequent cycling of the wall hydrophobicity can dramatically reduce the folding times and increase the yield as well under non-permissive conditions . Examination of the structures of the substrate proteins before and after the change in hydrophobicity indicates that there is global unfolding involved . In addition, it is found that a fraction of the molecules kinetically partition to the native state in accordabce with the iterative annealing mechanism . Thus, frequent "unfoldase" activity of chaperonins leading to global unfolding of the polypeptide chain results in enhancement of the folding rates and yield of the folded protein . We suggest that chaperonin efficiency can be greatly enhanced if the cycling time is reduced . The calculations are used to interpret a few experiments on chaperonin-mediated protein folding . J Mol Biol, 1999 Apr 2, 287(3), 539 - 54 Probing the physical basis for trp repressor-operator recognition; Grillo AO et al.; The bacterial repressor protein, trp repressor, is one of the best studied transcriptional regulatory proteins in terms of function, structure, dynamics and stability . Despite these significant advances, the structural and energetic basis for the specific recognition of its operator sites by trp repressor remains poorly understood . In fact, recognition in this system is controled by the binding of the co-repressor ligand, l-tryptophan, as well as by conformational and dynamic properties of the operator targets, DNA sequence-dependent control of the oligomerization properties of the repressor, water-mediated interactions, and specific interactions involving the peptide backbone and phosphate moieties . Moreover, only one direct contact between the protein and the DNA is evident from the crystallographically determined structure of the complex . In an attempt to better define how the various sequence elements in the operator target contribute to this complex control of affinity and cooperativity of trp repressor binding, we have studied the binding of trp repressor to a series of mutated operator targets using fluorescence anisotropy, which provides very high quality data allowing fairly precise estimations of the affinities involved . We conclude from these studies that even on very small (25 bp) targets, the repressor binds slightly cooperatively, populating a 2:1 dimer/DNA complex, and then at higher concentrations a third dimer is bound with significantly lower affinity, revealing an inherent asymmetry in the trpEDCBA-derived target . Investigation of the basis for the asymmetry implicates the identity of the second base in the so-called structural half-site GNACT, which apparently influences the switch between tandem and simple binding . Mutation of the C or the T bases in the structural half-site abolishes all specificity in binding, and alteration of the single direct contact, the G of the structural half-site, or the central TTAA significantly lowers the affinity of the dimer for its site, without modifying the apparent cooperativity . Finally, we note that the order of affinity is conserved in the absence of the co-repressor, and moreover, it is in all cases significantly higher than that observed for holo-repressor binding to non-specific DNA, indicating that one cannot simply equate apo-repressor and non-specific binding . J Mol Biol, 1999 Apr 2, 287(3), 485 - 97 Differential binding of the Escherichia coli HU, homodimeric forms and heterodimeric form to linear, gapped and cruciform DNA; Pinson V et al.; We have shown recently that the relative abundance of the three dimeric forms (alpha2, alphabeta and beta2) of the HU protein from Escherichia coli varies during growth and in response to environmental changes . Using gel retardation assays we have compared the DNA binding properties of the three dimers with different DNA substrates . The determination of their DNA binding parameters shows that the relative affinities of HUalphabeta and HUalpha2 are comparable . Both recognize, with a high degree of affinity under stringent conditions, cruciform structures or DNA molecules with a nick or a gap, whereas they bind to linear DNA only at low salt . DNA containing a gap of two nucleotides is in fact the substrate recognized with the highest degree of affinity by these two forms under all conditions . Conversely, HUbeta2 binds very poorly to duplex DNA and shows a much lower affinity for nicked or gapped DNAs . However, HUbeta2 binds to cruciform DNA structures almost as well as HUalphabeta and HUalpha2 . This almost exclusive binding of HUbeta2 to a unique substrate is surprising in regards of the quasi identity, in the three forms, of the flexible arms considered as the DNA-binding domains of the three forms of HU . Cruciform DNA may stabilize HUbeta2 structure which could be structurally defective . J Mol Biol, 1999 Apr 2, 287(3), 449 - 57 Isolation of chloroform-resistant mutants of filamentous phage: localization in models of phage structure; Oh JS et al.; Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms) . To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8 . 2 and 4 degrees C . DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T . All 26 M13 isolates were I37T . These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4 degrees C . They all contracted like wild-type phage at 37 degrees C . Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance . In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside . The M28L and I37T loci are buried in a mostly hydrophobic region in the middle . Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model . The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression . These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage . These results add weight to suggestions that the sequential contraction of filaments-->I-forms-->S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly . Environ Res, 1999 Feb, 80(2 Pt 1), 122 - 6 Genotoxicity of fumes from heated cooking oils produced in Taiwan; Wu PF et al.; Epidemiologic investigations of lung cancer among Taiwanese nonsmoking women have found that exposure to fumes from cooking oils may be an important risk factor . Fume samples from three different commercial cooking oils (lard, soybean, and peanut oils) often used in Taiwan for preparing Chinese meals were collected for genotoxicity analysis in SOS chromotest and sister chromatid exchange (SCE) assays . The induction factors of the SOS chromotest in Escherichia coli PQ 37 were dependent on the concentrations of lard and soybean cooking oil extracts without S9 mix . In addition, when CHO-K1 cells were exposed to condensates of cooking oil fumes for 12 h, SCEs showed a dose-related increase in extracts of lard and soybean oil fumes . This result provides experimental evidence and is in accordance with the findings of epidemiologic studies that women exposed to the emitted fumes of cooking oils are at an increase risk of contracting lung cancer . J Surg Res, 1999 Apr, 82(2), 216 - 21 Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NF kappa B activity; Lo CJ et al.; BACKGROUND . Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown . This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MO) . METHODS . RAW 264.7 cells, a mouse MO cell line, were grown in EPA-rich media for 24-48 h . MO were washed and exposed to Escherichia coli LPS (1 microg/ml) for 2 h . TNF mRNA expression was measured by Northern blot assays . Total nuclear extracts were harvested for the measurement of NF kappa B with electrophoretic mobility shift assays . Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NF kappa B dimers . TNF production was determined by L929 bioassays . RESULTS . LPS stimulated RAW cell TNF mRNA expression and NF kappa B activity . In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NF kappa B subunits (P65/P50 dimers) in the presence of LPS . TNF production by LPS-stimulated MO was reduced by EPA . CONCLUSIONS . The inhibitory effect of EPA on LPS-stimulated MO TNF gene transcription and protein elaboration is, in part, mediated through altering NF kappa B activation by reducing the P65/P50 dimers . J Surg Res, 1999 Apr, 82(2), 188 - 93 Different role of antioxidants in endotoxin-induced late myocardial protection; Wang S et al.; Previous studies have proposed that endogenous antioxidants play a protective role against cardiac ischemia-reperfusion injury in endotoxin pretreatment . However, the mechanism underlying this effect remains elusive . We therefore evaluated the role of endogenous antioxidants in delayed myocardial protection after different doses of endotoxin administration using cultured rat neonatal cardiomyocytes . Myocytes were treated with normal saline (control) or lipopolysaccharide (Escherichia coli, serotype O111) at doses of 40 and 80 microg/ml (ET40 and ET80) . Also, antisense oligodeoxyribonucleotide (1.5 micromol/L) to manganese superoxide dismutase (Mn-SOD) and 3-amino-1,2,4-triazole (25 mg/ml) were added along with a 40 or 80 microg/ml endotoxin pretreatment in the IET40 and IET80 groups . Twenty-four hours later, Cells were subjected to hypoxia (pO2 < 1 kPa, 3 h) and reoxygenation (pO2: 19 kPa, 1 h) . Compared with controls, cell viability enhanced significantly (65.3 +/- 5.9, 63.8 +/- 4.6, and 69.7 +/- 5.2% vs 47.2 +/- 4.3%, P < 0.05) and creatine kinase release decreased (7.34 +/- 1.76, 7.11 +/- 1.49, and 6.27 +/- 1.24 U/mg protein vs 11.23 +/- 2.49 U/mg protein, P < 0 . 05) in ET40, IET40, and ET80 groups following reoxygenation . No statistically significant difference was found between the control and the IET80 groups . Furthermore, the levels of Mn-SOD (1.12 +/- 0 . 31 vs 0.75 +/- 0.15 U/mg . protein, P < 0.05) and catalase activity (1265 +/- 109 vs 996 +/- 85 U/mg . protein, P < 0.05) were higher only in the ET80 group . The results suggest that at a dose of 40 microg/ml, cells were protected by mechanisms other than the augmentation of endogenous antioxidant activity which were more evident at a dose of 80 microg/ml . It seems that different doses of endotoxin pretreatment may induce delayed myocardial protection through various mechanisms . J Surg Res, 1999 Apr, 82(2), 156 - 62 Inhibition of endothelial cell migration by gene transfer of tissue inhibitor of metalloproteinases-1; Fernandez HA et al.; BACKGROUND . Angiogenesis requires degradation of the vessel's basal lamina and endothelial cell migration into the tissue stroma . Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles in this process . MMP activity is tightly regulated during vessel growth . This work was designed to characterize the effect of TIMP-1 upregulation on endothelial cell invasion of the extracellular matrix . METHODS . We constructed replication-deficient recombinant adenoviruses that encode either TIMP-1 (Ad.TIMP-1) or Escherichia coli lac Z (Ad.beta gal) cDNA . Bovine aortic endothelial (BAE) cells were infected with 100 infectious particles/cell . Gene expression was assessed by Northern and Western blotting . TIMP-1 activity in cell-conditioned media was measured by a resorufin-labeled casein protease assay . BAE cell migration was measured by Boyden chamber assays with 0.2% gelatin-coated, 8 . 0-mcm polycarbonate membranes . RESULTS . TIMP-1 was overexpressed by Ad.TIMP-1-infected BAE cells relative to control, Ad . beta gal-infected or uninfected cells . TIMP-1 activity in Ad.TIMP-1 cell-conditioned medium was 2.8-fold higher than in control cells . By Boyden chamber assays with gelatin-coated membranes, Ad . TIMP-1-infected BAE cells showed 89.97 +/-1.64% (mean +/- SEM) reduction in migration relative to Ad.beta gal-infected cells (P < 0 . 02) and 90.53 +/- 1.12% relative to uninfected cells (P < 0.02) . Without gelatin coating, migration was equivalent in all groups . CONCLUSION . The replication-deficient recombinant adenovirus we constructed affords rapid and efficient upregulation of functional TIMP-1 in endothelial cells . Infection results in a dramatic decrease in cell migration and invasion of extracellular matrix . Thus, such a recombinant vector may provide a useful tool for the gene therapy of vascular remodeling and inhibition of angiogenesis . Gastroenterology, 1999 Apr, 116(4), 936 - 42 A recombinant bovine gallbladder mucin polypeptide binds biliary lipids and accelerates cholesterol crystal appearance time; Nunes DP et al.; BACKGROUND & AIMS: Mucin has a central role in the pathogenesis of cholesterol gallstones, in part because of its ability to bind biliary lipids and accelerate cholesterol crystal appearance time . Previous studies have localized these properties to nonglycosylated mucin domains, and we have recently shown that these domains contain a series of 127-amino acid, cysteine-rich repeats . The aim of this study was to express a recombinant mucin polypeptide containing these repeats and investigate its lipid-binding and pronucleating properties . METHODS: A recombinant mucin polypeptide was expressed as a glutathione S-transferase fusion protein in Escherichia coli, purified by affinity chromatography, and compared with native bovine gallbladder mucin in lipid-binding and cholesterol crystal appearance time assays . RESULTS: The recombinant mucin polypeptide bound a hydrophobic fluorescent probe and cholesterol in a concentration-dependent manner . It accelerated the appearance of cholesterol crystals from lithogenic model bile, an effect that was both time and concentration dependent . CONCLUSIONS: The cysteine-rich repeats in the recombinant mucin polypeptide correspond to the protease-sensitive hydrophobic domains identified in earlier biochemical studies . Further delineation of the lipid-binding site(s) in these repeats will provide new insights into the mechanism of cholesterol crystal nucleation and stone growth. Gastroenterology, 1999 Apr, 116(4), 804 - 12 Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults; Michetti P et al.; Background & Aims: Oral immunization with Helicobacter pylori urease can cure Helicobacter infection in animals . As a step toward therapeutic immunization in humans, the safety and immunogenicity of oral immunization with recombinant H . pylori urease were tested in H . pylori-infected adults . Methods: Twenty-six H . pylori-infected volunteers were randomized in a double-blind study to four weekly oral doses of 180, 60, or 20 mg of urease with 5 microg heat-labile enterotoxin of Escherichia coli (LT), LT alone, or placebo . Side effects and immune responses were evaluated weekly after immunization, and gastric biopsy specimens were obtained after 1 month and 6 months for histology and quantitative cultures . Results: Diarrhea was noted in 16 of 24 (66%) of the volunteers who completed the study . Antiurease serum immunoglobulin A titers increased 1 . 58-fold +/- 0.37-fold and 3.66-fold +/- 1.5-fold (mean +/- SEM) after immunization with 60 and 180 mg urease, respectively, whereas no change occurred in the placebo +/- LT groups (P = 0.005) . Circulating antiurease immunoglobulin A-producing cells increased in volunteers exposed to urease compared with placebo (38.9 +/- 13 . 6/10(6) vs . 5.4 +/- 3.1; P = 0.018) . Eradication of H . pylori infection was not observed, but urease immunization induced a significant decrease in gastric H . pylori density . Conclusions: H . pylori urease with LT is well tolerated and immunogenic in H . pylori-infected individuals . An improved vaccine formulation may induce curative immunity. Science, 1999 Mar 26, 283(5410), 2097 - 100 A cytotoxic ribonuclease targeting specific transfer RNA anticodons; Ogawa T et al.; The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli . Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon . The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide . Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5. Plant Mol Biol, 1999 Feb, 39(3), 593 - 605 Higher plant tyrosine-specific protein phosphatases (PTPs) contain novel amino-terminal domains: expression during embryogenesis; Fordham-Skelton AP et al.; Sequences encoding proteins with homology to protein tyrosine phosphatases have been identified in Arabidopsis, soybean and pea . Each contains a predicted catalytic domain containing sequence motifs characteristic of tyrosine-specific protein phosphatases (PTPs) which play an important role in signal transduction in other eukaryotes and are distinct from dual-specificity, cdc25 or low-molecular-weight protein tyrosine phosphatases . Their identity as PTPs was confirmed by characterising the soybean PTP expressed as a recombinant His-tagged fusion protein . The enzyme had phosphatase activity towards p-nitrophenolphosphate (pNPP) and phosphotyrosine, but did not hydrolyse phosphoserine or phosphothreonine at a measureable rate . Phosphotyrosine containing peptides also served as substrates, with Km values in the micromolar range . Activity was abolished by inhibitors specific for tyrosine phosphatases (vanadate, dephostatin) but was unaffected by inhibitors of serine/threonine protein phosphatases (fluoride, cantharidin, metal-chelating agents) . Gel filtration chromatography showed that the recombinant enzyme was a monomer . The Arabidopsis PTP sequence was isolated both as a genomic clone and as a partial EST, whereas the pea and soybean sequences were isolated as cDNAs . Southern analysis suggested a single gene in Arabidopsis and a small gene family in pea and soybean . In pea, PTP transcripts were present in embryos, and decreased in level with development; transcripts were also detectable in other tissues . The plant PTPs all contain a similar N-terminal domain which shows no similarity to any known protein sequence . This domain may be involved in PTP functions unique to plants. J Gen Virol, 1999 Mar, 80 ( Pt 3), 799 - 809 Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase; Wang A et al.; Tomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding protein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-dependent RNA polymerase (Pol) and a serine-like protease (Pro), which have been suggested to be involved in viral RNA replication . Proteolytic processing of protease precursors containing these proteins was studied in Escherichia coli and in vitro . The TomRSV protease could cleave the precursor proteins and release the predicted mature proteins or intermediate precursors . Although processing was detected at all three predicted cleavage sites (NTB-VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inefficient, resulting in accumulation of the VPg-Pro intermediate precursor in E . coli and in vitro . In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pol cleavage site in E . coli and in vitro . Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E . coli extracts and of radiolabelled mature polymerase purified from in vitro translation products revealed the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage sites to be Q/S, Q/G and Q/S, respectively . In vitro processing at the NTB-VPg and Pro-Pol cleavage sites was not detected upon mutation or deletion of the conserved glutamine at the -1 position of the cleavage site . These results are discussed in light of the cleavage site specificity of the TomRSV protease. J Gen Virol, 1999 Mar, 80 ( Pt 3), 701 - 9 Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein; Wardell AD et al.; The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus . Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases . Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E . coli, and make comparisons with published data of NS3 helicase domain alone . The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase . Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations . NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities . NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect . Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity . These results are in accord with a recently proposed model for NS3 helicase activity. J Gen Virol, 1999 Mar, 80 ( Pt 3), 563 - 70 Development of porcine adenovirus-3 as an expression vector; Reddy PS et al.; Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183 . As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid . The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus . The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants . Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome . The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3 . A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies . In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter . CAT gene expression was observed irrespective of the orientation of the CAT gene . These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs. J Gen Virol, 1999 Mar, 80 ( Pt 3), 537 - 41 Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B; Zaripov MM et al.; In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E . coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB . Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126 . One continuous epitope mapped between residues 214 and 279 . The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734 . The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646 . The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses. Protein Sci, 1999 Mar, 8(3), 614 - 24 Functional insights from structural predictions: analysis of the Escherichia coli genome; Rychlewski L et al.; Fold assignments for proteins from the Escherichia coli genome are carried out using BASIC, a profile-profile alignment algorithm, recently tested on fold recognition benchmarks and on the Mycoplasma genitalium genome and PSI BLAST, the newest generation of the de facto standard in homology search algorithms . The fold assignments are followed by automated modeling and the resulting three-dimensional models are analyzed for possible function prediction . Close to 30% of the proteins encoded in the E . coli genome can be recognized as homologous to a protein family with known structure . Most of these homologies (23% of the entire genome) can be recognized both by PSI BLAST and BASIC algorithms, but the latter recognizes an additional 260 homologies . Previous estimates suggested that only 10-15% of E . coli proteins can be characterized this way . This dramatic increase in the number of recognized homologies between E . coli proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms . Knowing protein structure adds a new dimension to our understanding of its function and the predictions presented here can be used to predict function for uncharacterized proteins . Several examples, analyzed in more detail in this paper, include the DPS protein protecting DNA from oxidative damage (predicted to be homologous to ferritin with iron ion acting as a reducing agent) and the ahpC/tsa family of proteins, which provides resistance to various oxidating agents (predicted to be homologous to glutathione peroxidase). Protein Sci, 1999 Mar, 8(3), 518 - 28 Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases; Baichoo N et al.; CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli . Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein . We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP {Baichoo N, Heyduk T . 1997 . Biochemistry 36:10830-10836} . In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ({Fe-DTPA}2-), iron complexed to ethylenediaminediacetic acid ({Fe-EDDA}), iron complexed to desferrioxamine mesylate ({Fe-HDFO}+), and copper complexed to o-phenanthroline ({(OP)2Cu}+) as proteases . These chemical proteases differ in size, charge, and hydrophobicity . Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases . Cleavage by {Fe-EDDA} and {Fe-DTPA}2- of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, the interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions . {Fe-EDDA} and {Fe-DTPA}2- also detected reductions in cleavage in the D and E alpha-helices, which are involved in DNA recognition . Cleavage by {Fe-HDFO}+ of CRP-cAMP detected hypersensitivities in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices . {Fe-HDFO}+ also detected protections from cleavage in beta-strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices . Cleavage by {(OP)2Cu}+ of CRP-cAMP detected hypersensitivities in beta-strands 9 and 11 as well as in the D and E alpha-helices . {(OP)2Cu}+ also detected protections in the C alpha-helix , the interdomain hinge, and beta-strands 2-7 . Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by {(OP)2Cu}+, which detected minor protections in beta-strands 3-7, the interdomain hinge, and the C alpha-helix . These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction . Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction . Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone. Protein Sci, 1999 Mar, 8(3), 490 - 8 Role of the lateral channel in catalase HPII of Escherichia coli; Sevinc MS et al.; The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase . HPII does not bind NADPH, a cofactor often found in catalases . In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase . Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII . Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding . The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels . One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide . In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270 . When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme . Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme . The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products. Eur J Biochem, 1999 Feb, 260(1), 50 - 6 Stopped-flow studies of the binding of 2-n-heptyl-4-hydroxyquinoline-N-oxide to fumarate reductase of Escherichia coli; Zhao Z et al.; We have studied the kinetics of binding of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) by fumarate reductase (FrdABCD) using the stopped-flow method . The results show that the fluorescence of HOQNO is quenched when HOQNO binds to FrdABCD . The observed quenching of HOQNO fluorescence has two phases and it can be best fitted to a double exponential equation . A two-step equilibrium model is applied to describe the binding process in which HOQNO associates with FrdABCD by a fast bimolecular step to form a loosely bound complex; this is subsequently converted into a tightly bound complex by a slow unimolecular step . The rates of the forward and the reverse reactions for the first equilibrium (k1 and k2) are determined to be k1 = (1.1 +/- 0.1) x 10(7) M-1.s-1, and k2 = 6.0 +/- 0.6 s-1, respectively . The dissociation constants of the first equilibrium (Kd1 = k2/k1) is calculated to be about 550 nM . The overall dissociation constant for the two-step equilibrium, Kd overall = Kd1/{1+ (1/Kd2)}, is estimated to be < or = 7 nM . Comparison of the kinetic parameters of HOQNO binding by FrdABCD and by dimethyl sulfoxide reductase provides important information on menaquinol binding by these two enzymes. Eur J Biochem, 1999 Feb, 260(1), 9 - 14 Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase; Tinguely JN et al.; Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site . This residue is also close to a site which binds glutathione . Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family . In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis . The mutant enzymes were expressed in Escherichia coli and purified to homogeneity . Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold . The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m) . NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled . Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme . Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate . Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme . Conversely, the binding of free glutathione to both mutants was reduced . Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. Biotechniques, 1999 Mar, 26(3), 502 - 8 Novel cloning method for recombinant adenovirus construction in Escherichia coli; Souza DW et al.; pAd(vantage) is a rapid cloning system for generating recombinant adenoviruses . The system is based on manipulating the full-length adenovirus genome as a stable plasmid in E . coli using intron-encoded endonucleases . These intron-encoded endonucleases cut their recognition sequences, which range from 15-39 bp, with high specificity . Their unusual long homing sequence makes them rare-cutting and ideal for use as cloning sites . We report how transgenes can easily be cloned directly into the E1 region of an adenoviral plasmid, followed by transfection into a mammalian packaging cell line, to produce homogeneous recombinant viruses without the need for plaque purification. Biochemistry, 1999 Mar 23, 38(12), 3677 - 82 Formation and isolation of a covalent intermediate during the glutaminase reaction of a class II amidotransferase; Schnizer HG et al.; Incubation of Escherichia coli asparagine synthetase B (AS-B) with {14C}-L-glutamine gives a covalent adduct that can be isolated . Radiolabeled protein is not observed (i) when the wild-type enzyme is incubated with 6-diazo-5-oxo-L-norleucine (DON) prior to reaction with {14C}glutamine or (ii) when the C1A AS-B mutant is incubated with {14C}-L-glutamine . Both of these alterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis . Formation of the covalent adduct therefore depends on the presence of the N-terminal active site cysteine, which has been shown to be essential for glutamine-dependent activity in this and other class II amidotransferases . The amount of covalent adduct exhibits saturation behavior with increasing concentrations of L-glutamine . The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis . The chemical properties of the isolable covalent adduct are consistent with those anticipated for the gamma-glutamyl thioester that has been proposed as an intermediate in the AS-B-catalyzed conversion of glutamine to glutamate . The covalent adduct is acid-stable but is labile under alkaline conditions . On the basis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanism . These studies represent the first description of a thioester intermediate for any class II amidotransferase and represent an important step in gaining further insight into the kinetic and chemical mechanisms of AS-B. Biochemistry, 1999 Mar 23, 38(12), 3615 - 23 Characterization of amino acid substitutions that severely alter the DNA repair functions of Escherichia coli endonuclease IV; Yang X et al.; Escherichia coli endo IV is a bifunctional DNA repair protein, i.e., possessing both apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities . The former activity cleaves AP sites, whereas the latter one removes a variety of 3'-blocking groups present at single-strand breaks in damaged DNA . However, the precise reaction mechanism by which endo IV cleaves DNA lesions is unknown . To probe this mechanism, we have identified eight amino acid substitutions that alter endo IV function in vivo . Seven of these mutant proteins are variably expressed in E . coli and, when purified, show a 10-60-fold reduction in both AP endonuclease and 3'-diesterase activities . The most severe defect was observed with the one remaining mutant (E145G) that showed normal protein expression . This mutant has lost the ability to bind double-stranded DNA and showed a dramatic 150-fold reduction in enzymatic activities . We conclude that the AP endonuclease and the 3'-diesterase activities of endo IV are associated with a single active site, that is perhaps remote from the DNA binding domain. Biochemistry, 1999 Mar 23, 38(12), 3579 - 90 Substitutions at histidine 74 and aspartate 278 alter ligand binding and allostery in lactose repressor protein; Barry JK et al.; In the inducer-bound structure of the lac repressor protein, the side chains of H74 and D278 are positioned to form an ion pair between monomers that appears to be disrupted upon operator binding (Lewis, M., Chang, G., Horton, N . C., Kercher, M . A., Pace, H . C., Schumacher, M . A., Brennan, R . G., and Lu, P . (1996) Science 271, 1247-1254) . A series of single substitutions at H74 and D278 and a double mutant, H74D-D278H, were generated to determine the influence of this interaction on ligand binding and allostery in lac repressor . Introduction of apolar amino acids at H74 resulted in distinct effects on ligand binding . Alanine and leucine substitutions decreased operator binding, while tryptophan and phenylalanine increased affinity for operator DNA . Introduction of a negatively charged residue at position 74 in H74D had minimal effects, and "inverting" the side chains in H74D/D278H did not significantly alter inducer or operator binding at neutral pH . In contrast, all substitutions of D278 increased affinity for operator DNA and diminished inducer binding . These observations can be interpreted in the context of the Monod-Wyman-Changeux model . If a salt bridge were essential for stabilizing or destabilizing the inducer-bound conformation, a mutation at either residue that interrupts this interaction should have a similar effect on allostery . Because the type and degree of alteration in ligand binding properties depended on the nature of the substitution at these residues, the individual roles played by H74 and D278 in lac repressor allostery appear more important than their direct contact across the monomer-monomer interface. Biochemistry, 1999 Mar 23, 38(12), 3508 - 18 Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion; Bystrom CE et al.; Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers . To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site {Ormo, M., Bystrom, C., and Remington, S . J . (1998) Biochemistry 37, 16565-16572} . The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition . Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures . The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily . Two of the ATP analogues bound in nonproductive conformations in both subunits . However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol . This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion. Proteins, 1999 Apr 1, 35(1), 1 - 12 The crystal structure of recombinant rat pancreatic RNase A; Gupta V et al.; The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined . The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart . However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family . The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates . The most significant difference in the backbone conformation was observed in the loop 15-25 . This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme . The rat enzyme does not get cleaved under identical conditions . Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site . It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin. Microsc Res Tech, 1999 Mar 1, 44(5), 368 - 77 Investigation of protein partnerships using atomic force microscopy; Ellis DJ et al.; The origin of contrast in atomic force microscopy (AFM) lies in the probe's response to forces between itself and the sample . These forces most commonly result from changes in height as the tip is scanned over the surface, but can also originate in properties inherent in the sample . These have been exploited as further means of contrast and have spawned an array of similar imaging techniques, such as chemical force microscopy, magnetic force microscopy, and frictional force microscopy . All of these techniques use AFM as an extremely sensitive gauge to map forces at discrete sites on the surface . A natural extension of this approach is to map forces in an array, in order to create a force map . AFM can be used in aqueous or fluid environments, thus allowing the exploration of forces in biological systems under physiologically relevant conditions . By immobilizing one half of an interacting pair of proteins onto the tip and the other half onto the substrate, it is possible to investigate the electrostatic and hydrophobic interactions between them . We employed these techniques to examine the interaction between a pair of proteins of known affinity that are involved in exocytosis (NSF and alpha-SNAP) and separately to demonstrate how two-dimensional force mapping can be applied to the nuclear envelope to identify nuclear pore complexes. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1471 - 4 Expression, crystallization and preliminary X-ray analysis of the E2 transactivation domain from papillomavirus type 16; Burns JE et al.; The N-terminal transactivation domain of the E2 protein from human papillomavirus type 16 has been crystallized by vapour diffusion . Crystals belong to the space group P3121 (or P3221) with unit-cell dimensions a = b = 54.3, c = 155.5 A . There is one molecule per asymmetric unit with a solvent content of 55% . Crystals diffract to at least 2.5 A resolution and complete X-ray data to 3.4 A have been collected on a conventional laboratory source . This 201 amino-acid domain of the E2 protein has been shown to interact functionally with both the HPV E1 protein and at least three cellular transcription factors, to fulfil its role in the control of viral transcription and replication . A knowledge of the structural basis of these multiple interactions should lead to a fuller understanding of the mechanism of action of this key regulator of the HPV life cycle. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1460 - 3 Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the receiver domain of PhoB; Sol M et al.; PhoB is the response regulator of the E . coli two-component signal transduction system for phosphate regulation . It is a transcription factor that activates more than 30 genes of the pho regulon . Crystals of the receiver domain of PhoB were obtained by applying the hanging-drop vapour-diffusion method . X-ray diffraction data have been collected using synchrotron radiation to 1.88 A resolution . The crystals belong to the orthorhombic space group P212121 with unit-cell constants a = 34.11, b = 60.42, c = 119.97 A . The Matthews parameter suggests that PhoB crystallizes with two molecules per asymmetric unit, suggesting that activating dimerization occurs in the crystal. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1456 - 9 Production, crystallization and diffraction to atomic resolution of an antibody Fv specific for the blood-group A oligosaccharide antigen; Patenaude SI et al.; The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas . A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli . The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms . The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM) . AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution . This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1442 - 5 Crystallization and preliminary X-ray studies of a recombinant calcium-binding protein from Entamoeba histolytica; Gopal B et al.; A calcium-binding protein (CaBP) of Entamoeba histolytica was purified from an E . coli recombinant clone carrying the CaBP gene in a pET-3c expression vector using anion-exchange and size-exclusion chromatography . Examination of the amino-acid sequence of the recombinant protein suggested that it has four independent EF-hand motifs . The protein dissolved in cacodylate buffer was crystallized using the hanging-drop method with 2-methylpentane-2,4-diol (MPD) as the precipitant . X-ray diffraction data have been collected on these crystals using a MAR Research imaging-plate detector system attached to a Rigaku RU200 rotating-anode X-ray generator . The crystals belong to the hexagonal space group P6122 with unit-cell dimensions of a = b = 96.21, c = 65.48 A . Preliminary molecular-replacement computations suggest that the structure of this protein is likely to be similar to that of calmodulin (CAM). Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1435 - 6 Crystallization and preliminary crystallographic analysis of the archaeal intron-encoded endonuclease I-DmoI; Dalgaard JZ et al.; Two forms of the archaeal intron-encoded site-specific endonuclease I-DmoI, namely I-DmoIc and I-DmoIl, have been purified and crystallized . Crystals of I-DmoIc are rod-shaped and diffract to 3.0 A resolution, but further analysis was hampered by twinning . Crystals of I-DmoIl, which is a six-amino-acid C-terminal truncation of I-DmoIc, are plate shaped and belong to space group C2 with cell parameters a = 93.72, b = 37.03, c = 55.56 A, beta = 113.4 degrees, with one molecule per asymmetric unit (Vm = 2.01 A3 Da-1) . The crystals diffract to at least 2.3 A resolution . A complete native data set has been measured and structure determination is on-going. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1419 - 21 Preliminary crystallographic studies of triosephosphate isomerase (TIM) from the hyperthermophilic Archaeon Pyrococcus woesei; Bell GS et al.; Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized . Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal . The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A . The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A . Diffraction from the hexagonal form showed extensive disorder . The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering . The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form . Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1401 - 4 Crystallization and preliminary X-ray analysis of the unliganded recombinant catalytic subunit of cAMP-dependent protein kinase; Narayana N et al.; X-ray diffraction-quality crystals of the unliganded mouse recombinant catalytic subunit of cAMP-dependent protein kinase were grown by the hanging-drop vapour-diffusion technique using 2-methyl-2,4-pentanediol as precipitant . The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 48.9, b = 147.4, c = 54.2 A, beta = 110.2 degrees . A data set to 3.0 A resolution with 92% completeness has been collected using synchrotron radiation . The unit cell contains four molecules of molecular weight 40 kDa with a corresponding volume solvent content of 45%. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1397 - 8 Purification and preliminary X-ray crystallographic studies of recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli; Kack H et al.; Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent aminotransferase, has been crystallized in space groups P21 and C2 . Both crystal forms were obtained at pH 7.3 with 21% polyethylene glycol and 10% 2-propanol as precipitants . The cell dimensions were a = 130, b = 57.5, c = 117 A, beta = 110 degrees for the C2 crystals, and a = 58.4, b = 55.6, c = 121 A, beta = 96.9 degrees for the P21 crystals, which diffract to at least 2.6 and 2.0 A resolution, respectively. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 717 - 20 Separation and crystallization of T = 3 and T = 4 icosahedral complexes of the hepatitis B virus core protein; Zlotnick A et al.; The icosahedral nucleocapsid of human hepatitis B virus is a homopolymer of the dimeric capsid protein also known as hepatitis B core antigen or HBcAg . Purified capsid protein obtained from an Escherichia coli expression system was reassembled into a mixture of T = 3 and T = 4 icosahedral particles consisting of 90 and 120 dimers, respectively . The two types of capsid were separated on a preparative scale by centrifugation through a sucrose gradient . In addition to this heterogeneity, the capsid protein has three cysteines, one of which has a great propensity for forming disulfide bonds between the two subunits, forming a dimer . To eliminate heterogeneity arising from oxidation, alanines were substituted for the cysteines . T = 3 and T = 4 capsids crystallized under similar conditions . Crystals of T = 3 capsids diffracted to approximately 8 A resolution; crystals of T = 4 capsids diffracted to 4 A resolution. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 679 - 82 Cloning, overexpression, crystallization and preliminary X-ray analysis of a family 1 beta--glucosidase from Streptomyces; Guasch A et al.; An intracellular beta-glucosidase (Bgl3) from Streptomyces sp . has been cloned and overexpressed in Escherichia coli . The introduction of a His tag at the N-terminal end of the protein has allowed its purification to homogeneity by a single chromatographic step, with yields of 150-200 mg of pure protein per litre of E . coli culture . The enzyme (52.6 kDa) is a retaining glycosidase able to hydrolyze a wide range of disaccharides and oligosaccharides and to perform transglycosylation . Crystals of recombinant Bgl3 have been grown from an ammonium sulfate solution using the hanging-drop vapour-diffusion method at 293 K . The crystals belong to the orthorhombic space group I222 with unit-cell dimensions a = 101.6, b = 113.4 and c = 187.5 A at room temperature and contain two molecules per asymmetric unit . A full 1.69 A resolution diffraction data set (97.7% completeness) has been collected from frozen crystals in a solution containing 30% sucrose, using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 631 - 43 Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion; Hadener A et al.; The enzyme hydroxymethylbilane synthase (HMBS, E.C . 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles . The enzyme is found in all organisms, except viruses . The crystal structure of the selenomethionine-labelled enzyme ({SeMet}HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5 . In addition, {SeMet}HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector . The structure at ambient temperature has been refined, the R factor being 16.8% at 2 . 4 A resolution . The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes . Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle . Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate . Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 610 - 24 Structure of dethiobiotin synthetase at 0.97 A resolution; Sandalova T et al.; The crystal structure of the 224-residue protein dethiobiotin synthetase from Escherichia coli has been refined using X-ray diffraction data at 0.97 A resolution at 100 K . The model, consisting of 4143 protein atoms including 1859 H atoms and 436 solvent sites, was refined to a final R factor of 11.6% for all reflections, and has an estimated mean standard uncertainty for the atomic positions of 0.022 A, derived from inversion of the blocked matrix . The structure was refined with a full anisotropic model for the atomic displacement parameters using SHELX97 . Stereochemical restraints were applied throughout the refinement . In the last cycles, the planarity of the peptide bonds was not restrained, resulting in a mean omega value of 179.6 degrees . Analysis of the most anisotropic regions of the protein shows that they form four clusters of residues . Alternate conformations for the side chains of 15 residues and for the main-chain atoms of six residues from three loops were included in the model . An analysis of C-HcO hydrogen bonds shows that such interactions occur rather frequently in DTBS; in total, 16 such hydrogen bonds were found . In the central beta-sheet, 13 C-HcO bonds between carbonyl O and Calpha H atoms were found . Other interactions of this type involve main-chain-side-chain and side-chain-side-chain C-HcO bonds . The model includes 436 water sites, of which 233 molecules form the first hydration shell . Analysis of the protein-solvent interactions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent . No difference in propensity for ordered solvent close to hydrophilic or hydrophobic surface areas was found . The comparison of the 100 K structure with the structure of the enzyme determined at room temperature shows several regions with different conformation, including areas in the active site, suggesting that structural transitions can occur during flash freezing . This observation questions one of the basic assumptions in the analysis of enzymatic reaction mechanisms using cryocrystallography. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 689 - 90 Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit; Willows RD et al.; The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis . The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli . The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution . The crystals diffract to a minimum Bragg spacing of 2.1 A . The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 256 - 9 Epub 1999 Jan 01. Preliminary X-ray crystallographic analysis of the complex between the DNAase domain of colicin E9 and its cognate immunity protein; Kuhlmann UC et al.; We have crystallized and performed preliminary X-ray characterization of the complex between the DNAase domain of the E9 colicin and its cognate immunity protein Im9 . The dissociation constant for this complex, Kd = 1 x 10(-16) M, reveals it to be one of the highest affinity protein-protein interactions known . Single crystals of the 1:1 complex were grown from microseeding experiments using PEG 4K as precipitant . The space group is P212121 with one molecule of complex in the asymmetric unit, and crystals contain approximately 43% solvent . These crystals are inherently non-isomorphous and so selenomethionine-derivatized protein has been prepared and crystals grown for MAD phasing experiments. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 347 - 9 Epub 1999 Jan 01. Expression, purification, crystallization and preliminary X-ray diffraction analysis of uracil phosphoribosyltransferase of Toxoplasma gondii; Barchue J et al.; Recombinant uracil phosphoribosyltransferase (UPRT) enzyme of Toxoplasma gondii was expressed in Escherichia coli and purified from the cell-free extract by a combination of chromatographic steps . The recombinant protein was enzymatically active when tested in an in vitro UPRT assay . The purified protein was crystallized using the hanging-drop vapor-diffusion technique with ammonium phosphate as precipitant . The crystallized protein also exhibited UPRT activity . Crystals diffract to 2.4 A resolution and belong to space group P3121 or P3221 with unit-cell dimensions a = b = 119.9, c = 70.8 A and two molecules per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 338 - 40 Epub 1999 Jan 01. Expression, purification and preliminary X-ray analysis of a fibrillarin homolog from Methanococcus jannaschii, a hyperthermophile; Wang H et al.; Fibrillarin plays a central role in ribosome biogenesis as a ribosomal RNA-processing protein . A Methanococcus jannaschii homolog of fibrillarin has been overexpressed, purified and crystallized . Crystals belong to the C2 space group with unit-cell parameters a = 121.4, b = 43.2, c = 55.3 A, beta = 96.9 degrees . Under flash-frozen conditions and using synchrotron radiation, the crystals diffract to 1.8 A resolution . For structural determination, a selenomethionine derivative of the protein has also been crystallized. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 332 - 4 Epub 1999 Jan 01. Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNAMet(f) formyltransferase complexed with formyl-methionyl-tRNAMet(f); Schmitt E et al.; The structure of methionyl-tRNAfMet(f) formyltransferase from E . coli, a monomeric protein of 34 kDa, has previously been determined at 2.0 A resolution . In the present work, this enzyme was crystallized as a complex with its macromolecular product, the initiator formyl-methionyl-tRNAfMet(f) (25 kDa) . Polyethylene glycol 5000 monomethylether was used as a precipitating agent . The crystals are orthorhombic and have unit-cell parameters a = 201.7, b = 68.1, c = 86.4 A . They belong to space group P21212 and diffract to 2.8 A resolution . The structure is being solved with the help of a mercury derivative. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 302 - 4 Epub 1999 Jan 01. Crystallization and characterization of a fragment of pseudouridine synthase RluC from Escherichia coli; Corollo D et al.; RluC from E . coli is the enzyme responsible for catalyzing the isomerization of uridines 955, 2504 and 2580 in 23S rRNA to pseudouridine . Histidine-tagged RluC was cloned, overexpressed and purified by nickel-affinity chromatography . A proteolytically derived fragment of the enzyme consisting of residues 89-319 has been shown to retain catalytic activity . Crystals of this fragment, grown by precipitation with sodium acetate at pH 8.0, belong to space group P321, with unit-cell dimensions a = b = 97.1, c = 86.3 A and have two molecules in the crystallographic asymmetric unit . The flash-frozen crystals diffract X-rays to at least 2.3 A resolution and appear suitable for crystal structure determination. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 294 - 6 Epub 1999 Jan 01. A thermostable xylose isomerase from Thermus caldophilus: biochemical characterization, crystallization and preliminary X-ray analysis; Chang C et al.; A highly thermostable xylose isomerase from Thermus caldophilus has been expressed in Escherichia coli . The purified enzyme has an optimum temperature of 363 K . It has been crystallized at room temperature using ammonium sulfate as a precipitant . The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 84.35, b = 123.60, c = 140.24 A . The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.1 A3 Da-1 and a solvent content of 41% by volume . The crystals initially showed diffraction to 1.7 A Bragg spacing with synchrotron X-rays, and a set of native data extending to 2.3 A resolution has been collected. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 291 - 3 Epub 1999 Jan 01. Purification, crystallization and preliminary X-ray diffraction analysis of the yeast phosphorelay protein YPD1; Xu Q et al.; YPD1 is a yeast osmoregulatory protein that functions in a phosphorelay signal-transduction pathway . YPD1 has been expressed in Escherichia coli, purified to homogeneity and crystallized . The crystals were obtained by hanging-drop vapor-diffusion using PEG 4000 as a precipitant . Preliminary X-ray diffraction analysis indicates that the crystals belong to tetragonal space group P43212 or P41212 with unit-cell dimensions a = b = 52.71, c = 244.02 A . X-ray data to 2.7 and 3.0 A have been collected from native crystals and a heavy-atom derivative, respectively . Positions for two Hg atoms have been located by analysis of difference Patterson maps. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 272 - 5 Epub 1999 Jan 01. Crystallization and preliminary X-ray analysis of human RCC1, the regulator of chromosome condensation; Renault L et al.; RCC1, the regulator of chromosome condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran . Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation . Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant . The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, alpha = 113.0, beta = 103.9,gamma = 103.3 degrees . The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 269 - 71 Epub 1999 Jan 01. Crystallographic characterization of a novel protein SixA which exhibits phospho-histidine phosphatase activity in the multistep His-Asp phosphorelay; Hamada K et al.; SixA has been isolated from Escherichia coli as the first protein to exhibit phospho-histidine phosphatase activity . Recent biochemical studies have shown that SixA is involved in the signal transduction of the His-Asp phosphorelay through the dephosphorylation of the histidine-containing phosphotransfer (HPt) domain of the anaerobic sensor kinase ArcB . Crystals of SixA were obtained using a hanging-drop vapour-diffusion method with polyethylene glycol and calcium ions . Preliminary X-ray crystallographic analysis revealed that the crystals belonged to space group P212121 with unit-cell dimensions a = 39.26, b = 48.62 and c = 83.18 A, having one molecule in the crystallographic asymmetric unit . The intensity data were collected up to 1.5 A resolution using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 263 - 5 Epub 1999 Jan 01. Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday junction; Hargreaves D et al.; During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday junction formed by the action of RecA to promote the strand-exchange reaction . RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E . coli, purified and co-crystallized with a synthetic Holliday junction substrate made from four 18-base deoxyoligonucleotides . Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant . The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and beta = 123 degrees . The X-ray analysis of these crystals should reveal the structure of the Holliday junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 260 - 2 Epub 1999 Jan 01. Murine class I major histocompatibility complex H-2Dd: expression, refolding and crystallization; Achour A et al.; A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system . It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein . Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions . The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 8 - 24 Epub 1999 Jan 01. The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution; Thoden JB et al.; Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation . As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events . Here the three-dimensional structure of carbamoyl phosphate synthetase from E . coli refined to 2.1 A resolution with an R factor of 17.9% is described . The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains . As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn . The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain . Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP . In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299 . Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP . In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215 . The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 557 - 60 Crystallization of hepatitis B virus core protein shells: determination of cryoprotectant conditions and preliminary X-ray characterization; Wynne SA et al.; Hepatitis B virus causes liver cirrhosis and hepatocellular cancer and is a major cause of death, particularly in Asia and sub-Saharan Africa . The virus consists of an inner core or nucleocapsid, which encloses the viral nucleic acid, with an outer lipid envelope containing surface-antigen proteins . The core protein, when expressed in E . coli, assembles into spherical shells containing 180 or 240 subunits, arranged with T = 3 or T = 4 icosahedral symmetry . The C-terminal region of the protein is involved in nucleic acid binding, and deletion of this region does not prevent capsid formation . C-terminally deleted hepatitis B core shells containing 240 subunits have been crystallized and data has been collected to 3 . 6 A resolution from frozen crystals, using butanediol as a cryoprotectant . The crystals have C2 symmetry, with unit-cell parameters a = 538.0, b = 353.0, c = 369.6 A, beta = 132.3 degrees. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 547 - 8 Crystallization and preliminary X-ray diffraction studies of blasticidin S deaminase from Aspergillus terreus; Nakasako M et al.; Blasticidin S deaminase from Aspergillus terreus was crystallized with polyethylene glycol 8000 . Two types of crystals were grown under the same crystallization conditions . One type grew as thin plates, while the other had a rhombic shape . The rhombic shaped crystal was suitable for high-resolution crystal structure analysis . Precession photographs and diffraction data showed that the crystal belonged to orthorhombic space group P212121, with unit-cell dimensions a = 70.33, b = 146.56 and c = 56.48 A . The calculated Vm value was acceptable when a tetramer of the enzyme was contained in an asymmetric unit . Preliminary diffraction data were collected to a resolution of 2.0 A with good statistics. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 542 - 3 Two crystal forms of ModE, the molybdate-dependent transcriptional regulator from Escherichia coli; Hall DR et al.; The molybdenum-responsive ModE regulatory protein from Escherichia coli has been purified and used in crystallization trials . Two crystal forms have been observed . Form I is tetragonal, P41212 (or enantiomorph), with a = b = 72.3, c = 246.2 A and diffracts to medium resolution . Form II is orthorhombic, P21212, with a = 82.8, b = 127.9, c = 64.0 A and diffraction has been observed beyond 2.8 A resolution . Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationship in regulating the uptake of molybdate in bacteria. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 531 - 3 Purification and crystallization of a proteolytic fragment of Escherichia coli pyruvate formate-lyase; Leppanen VM et al.; Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyase (PFL) is the first reaction after the production of pyruvate in the glycolytic pathway . Crystallization trials with Escherichia coli PFL were unsuccessful and therefore limited proteolysis was used to produce a stable crystallizable N--terminal protein fragment by trypsin cleavage . The molecular weight of this cleavage product was found to be 69.6 kDa by MALDI MS analysis, and the DNA sequence corresponding to this fragment was cloned . The recombinant protein fragment was crystallized by sitting-drop vapour diffusion using polyethylene glycol 1000 as precipitant . The crystals, which grew to 2 mm in length and 0.2 mm in cross section, belong to the hexagonal space group P61 or P65 with cell dimensions a = b = 140.4, c = 215.3 A and two molecules per asymmetric unit . X--ray diffraction data were collected from 20 to 3.2 A resolution from a single frozen crystal on a synchrotron-radiation beamline. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 522 - 4 X-ray analysis of crystals of rat phosphatidylinositol-transfer protein with bound phosphatidylcholine; Oliver RL et al.; Phosphatidylinositol-transfer protein (PITP) is a soluble, ubiquitously expressed, highly conserved protein encoded by two genes in humans, rodents and other mammals . A cDNA encoding the alpha isoform of the rat gene was expressed to high levels in Escherichia coli, the protein purified and the homogeneous protein used for crystallization studies . Crystals of rat PITP-alpha were obtained by vapor-diffusion techniques using the sitting-drop method . Crystals grow within two weeks by vapor-diffusion techniques in the presence of polyethylene glycol 4000 . Both crystal forms pack in the monoclinic space group P21 . Crystal form I has unit-cell parameters a = 44.75, b = 74.25, c = 48.32 A and beta = 114.14 degrees . Unit-cell parameters for crystal form II are a = 47.86, b = 73.59, c = 80.49 A and beta = 98.54 degrees . Crystal form I has a Vm of 2.295 A3 Da-1 and an estimated solvent content of 46.4% with one molecule per asymmetric unit, while crystal form II has a Vm of 2.196 A3 Da-1 and an estimated solvent content of 44.0%, assuming two molecules per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 518 - 21 Purification, crystallization and preliminary X-ray diffraction data from selenomethionine glycinamide ribonucleotide synthetase; Weaver TM et al.; In this study, the overexpression, purification and crystallization of selenomethionine (SeMet) incorporated glycinamide ribonucleotide synthetase (GAR-syn) from Escherichia coli are reported . The overexpression of SeMet GAR-syn was placed under the control of the isopropylthio-beta-galactoside (IPTG) inducible T7 RNA-polymerase system . The newly developed construct contained a removable histidine tag on the amino terminus of GAR-syn, which allowed rapid purification using metal-chelate chromatography techniques . The SeMet GAR-syn crystals were grown by hanging-drop vapor diffusion and belong to the space group P212121 with unit-cell parameters a = 56.2, b = 62.4 and c = 129.8 A and a single monomer in the asymmetric unit . The crystals diffract to 1.6 A resolution and have led to the determination of multiple-wavelength anomalous diffraction phases to 2.2 A resolution. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 399 - 402 Initiating a crystallographic study of UDP-galactopyranose mutase from Escherichia coli; McMahon SA et al.; UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been crystallized in a form suitable for X-ray diffraction studies . UDP-galactofuranose is a key component of mycobacterial cell walls . Crystals of both the native protein and a selenomethionine variant have been grown by the vapour-diffusion method in hanging drops, and diffract to beyond 3.0 A using synchrotron radiation . Equilibration was against a solution of 20%(w/v) polyethylene glycol (4K), 12%(v/v) 2--propanol, 0.1 M HEPES pH 7.6 at 293.5 K . Crystals grow as thin plates of dimensions 0.4 x 0.2 x approximately 0.02 mm . They are monoclinic {corrected}, space group P21, with unit-cell dimensions a = 71 . 12, b = 58.42, c = 96.38 A, beta = 96.38 degrees . 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively . The Matthews coefficient is 2.35 A3 Da-1 for a dimer in the asymmetric unit, the solvent content being 47% . Diffraction data have also been recorded on a putative platinum derivative to 3.5 A. Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 921 - 4 Crystallization and preliminary x-ray analysis of the catalytic subunit of the ATP-dependent arsenite pump encoded by the Escherichia coli plasmid R773; Zhou T et al.; The arsenical resistance (ars) operon of the Escherichia coli plasmid R773 encodes a system for the active extrusion from cells of the toxic oxyanions arsenite (AsIIIO2-) and antimonite (SbIIIO2-) via an ATP-driven pump . The arsA and arsB genes of the operon encode the catalytic subunit (ATPase) and the membrane subunit of the pump, respectively . The arsC gene codes for a reductase that converts arsenate (AsVO43-) to arsenite, thus extending bacterial resistance to the pentavalent state of arsenic . Crystals diffracting beyond 2.0 A were obtained for the catalytic subunit of the pump (ArsA) . These crystals belong to space group I222, with unit-cell parameters a approximately 73, b approximately 76, c approximately 223 A . A single molecule of ArsA, composed of two homologous halves, occupies the asymmetric unit of the I222 crystals with a predicted solvent content of 46% . Self-rotation function analysis suggests, however, that ArsA adopts a molecular packing corresponding to point group 422 . One possible explanation of this result is that the two homologous halves of ArsA are related by a twofold axis of local symmetry and that the two halves of a 'pseudo'-tetramer are related by a crystallographic twofold axis. Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 918 - 20 Expression, purification, crystallization and preliminary x-ray analysis of the kappa-carrageenase from Pseudoalteromonas carrageenovora; Michel G et al.; A recombinant form of His-tagged kappa-carrageenase from Pseudoalteromonas carrageenovora has been expressed, purified and crystallized . Crystals have been obtained by the vapour-diffusion method using polyethylene glycol (Mr = 4000) as a precipitant . These crystals belong to the space group P212121, with unit-cell parameters a = 58.2, b = 62.8, c = 77.9 A, and diffract to 2.2 A resolution on a rotating-anode X-ray source. Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 895 - 7 Crystallization and preliminary x-ray diffraction analysis of the regulatory subunit of human protein kinase CK2; Chantalat L et al.; Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits . A C-terminal truncated form of the beta subunit has been overproduced in Escherichia coli and purified to homogeneity . Two crystal forms of the truncated protein which diffract to at least 2 A resolution have been obtained . Form I belongs to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 92.9, c = 53.7 A, beta = 96.3 degrees, and yields plate-like crystals . Form II belongs to the tetragonal space group P42212, with unit-cell parameters a = 132.19, b = 132.19, c = 63.79 A, and produces rod-shaped crystals . Both crystal forms have a functional dimer in the crystal asymmetric unit. Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 888 - 90 Overproduction, purification, crystallization and preliminary x-ray diffraction studies of the human spliceosomal protein U5-15kD; Reuter K et al.; The gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized . Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding . The crystals belong to the orthorhombic space group P21212 with a = 62.3, b = 65.7, c = 37.1 A . They diffract to at least 3.0 A and contain one molecule in the asymmetric unit . A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection. Microb Pathog, 1999 Apr, 26(4), 195 - 206 In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TrcR/TrcS proteins; Haydel SE et al.; Two-component regulatory proteins, histidine kinases and response regulators, function in bacteria as sensing and adaptive factors in response to a wide range of environmental stimuli . Conserved histidine and glycine regions of histidine kinase sensor proteins were used to design degenerate oligonucleotide primers for amplification of DNA fragments from Mycobacterium tuberculosis . Two adjacent genes, trcR and trcS, which encode a response regulator and a histidine kinase, respectively, have been identified . Full-length and truncated TrcR and TrcS proteins have been expressed in Escherichia coli . Difficulties in expressing recombinant full-length TrcS and a truncated N -terminal form of TrcS reveal that the transmembrane domains are toxic to E . coli . Overexpressed truncated C-terminal transmitter domains of TrcS have been autophosphorylated in vitro and have transphosphorylated both the full-length recombinant TrcR protein and the N -terminal receiver/regulator domain of TrcR . In vitro autophosphorylation of TrcS requires the presence of Mn2+or Ca2+as a divalent cation cofactor and subsequent transphosphorylation of TrcR is evident in the presence of TrcS-phosphate and Ca2+ . Transphosphorylation between these two proteins provides evidence that these M . tuberculosis genes encode functional two-component system regulatory proteins that are members of a signal transduction circuit . Genes Dev, 1999 Mar 15, 13(6), 655 - 65 Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor; Morita MT et al.; Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock sigma-factor sigma32 encoded by the rpoH gene . Increased sigma32 levels result from both enhanced synthesis and stabilization . Previous work indicated that sigma32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5'-coding sequence of rpoH, including the translation initiation region . To understand the mechanism of heat induction of sigma32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock . A clear correlation was found between the stability and expression or the extent of heat induction . Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo . Furthermore, temperature dependence of mRNA-30S ribosome-tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo . Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator. J Food Prot, 1999 Mar, 62(3), 234 - 8 Extent of beef carcass contamination with Escherichia coli and probabilities of passing U.S . regulatory criteria; Sofos JN et al.; In the 1996 U.S . Meat and Poultry Inspection Regulations, Escherichia coli biotype I counts were included as "performance criteria" of the slaughtering process . The criteria were based on a three-class attributes sampling plan applied in a moving window . The values for m and M and c and n were set at 5 and 100 CFU/cm2, and 3 and 13 samples, respectively, for beef carcasses after overnight chilling following slaughter . In this study, beef carcasses were analyzed for counts of E . coli, and the results were expressed according to the above criteria . Furthermore, probabilities of passing E . coli performance criteria were determined . Carcasses were sampled in seven slaughtering plants (four steer and heifer; three cow and bull), during two seasons, and at three plant locations (pre-evisceration, after final carcass washing, and after 24 h of carcass chilling) . Each entire carcass sample (100 cm2 from the brisket, flank, and rump) was analyzed individually for E . coli counts . Compared with the regulation, which set the value of m and the acceptable range based on the 80th percentile of E . coli contamination data from U . S . Food Safety and Inspection Service nationwide baseline studies, our results showed that, on the average and depending on plant and season, 84.2 to 100% of the chilled carcass samples were in the acceptable range . The average percentages of chilled samples in the unacceptable range, set at the 98th percentile, were 0 to 6.7% . Depending on plant and season, the overall probabilities of chilled carcasses passing the regulatory requirement were 0.597 to 1.0 (brisket), 0.471 to 1.0 (flank), and 0.485 to 1.0 (rump) . The results indicated substantial variation among plants and between seasons in ability to meet the E . coli performance criteria. J Trauma, 1999 Mar, 46(3), 374 - 8; discussion 378-9 Synergistic effect of hyperoxia and immunoglobulin A on mucosal barrier defense; Diebel LN et al.; BACKGROUND: Hyperoxia has been reported to be protective against gut-derived sepsis . Although secretory immunoglobulin A (IgA) is primarily responsible for humoral defense of mucosal surfaces, a potential synergistic effect with hyperoxia is unknown . An asanguineous cell monolayer system was used to study these aspects in vitro . METHODS: MDCK cells were grown as polarized monolayers in a two-chamber culture system . Apical chambers were inoculated with 10(8) Escherichia coli M14 with or without polyclonal IgA and incubated in a 21 or 95% O2 environment . Basal medium was sampled at 90 and 180 minutes for bacterial translocation . In a second experiment, MDCK cells were lysed at 90 minutes and intracellular bacteria were quantitated . RESULTS: Bacterial translocation was decreased versus normoxia by the treatment groups IgA without hyperoxia or IgA with hyperoxia at 90 minutes . Bacterial internalization at 90 minutes was reduced to the greatest extent by the combined effects of hyperoxia and IgA . Translocation data at 180 minutes confirmed the additional protective effect of hyperoxia with IgA . CONCLUSION: Hyperoxia exerts a significant protective effect on barrier function independent of enhanced leukocyte function . Hyperoxia has an added effect to the mucosal defense provided by IgA. Res Vet Sci, 1999 Feb, 66(1), 33 - 7 Use of lipopolysaccharide (LPS) as a positive control for the evaluation of immunopotentiating drug candidates in experimental avian colibacillosis models; McGruder ED et al.; The aetiologic agent of avian colibacillosis is Escherichia coli . Colibacillosis is a disease that causes mortality and production performance problems in chickens which results in economic losses for the poultry industry . It will be increasingly important for scientists to identify novel solutions that can be implemented which will provide poultry producers with a tool to manage this economically important disease . The purpose of this investigation was to determine whether lipopolysaccharide (LPS) could be used as a positive control to evaluate novel chemistries for immunopotentiator activity in battery or floor-pen avian colibacillosis models in chickens . In the battery study, subcutaneous administration of LPS to one-day-old broiler cockerels caused a significant reduction (P < 0.003) in all parameters of colibacillosis evaluated, i.e . mean air sac lesion scores, per cent air sac lesions, E . coli re-isolation and per cent mortality . However, in the floor-pen study, subcutaneous administration to one-day-old broiler chicks resulted in a numerical, but not statistically significant reduction (P < 0.1) in mortality associated with colibacillosis . These data indicate that LPS can be used as a positive control to evaluate the efficacy of immunopotentiator drug candidates in avian colibacillosis models. Mol Membr Biol, 1998 Oct-Dec, 15(4), 203 - 11 Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine; Ritzel MW et al.; Two Na(+)-dependent nucleoside transporters implicated in adenosine and uridine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: CNT1 is selective for pyrimidine nucleosides but also transports adenosine; CNT2 (also termed SPNT) is selective for purine nucleosides but also transports uridine . Both proteins belong to a gene family that includes the NupC proton/nucleoside symporter of E . coli . cDNAs encoding members of the CNT family have been isolated from rat tissues (jejunum, brain, liver; rCNT1 and rCNT2/SPNT) and, most recently, human kidney (hCNT1 and hSPNT1) . Here, the molecular cloning and functional characterization of a CNT2/SPNT-type transporter from human small intestine are described . The encoded 658-residue protein (hCNT2 in the nomenclature) had the same predicted amino acid sequence as human kidney hSPNT1, except for a polymorphism at residue 75 (Arg substituted by Ser), and was 83 and 72% identical to rCNT2 and hCNT1, respectively . Sequence differences between hCNT2 and rCNT2 were greatest at the N-terminus . In Xenopus oocytes, recombinant hCNT2 exhibited the functional characteristics of a Na(+)-dependent nucleoside transporter with selectivity for adenosine, other purine nucleosides and uridine (adenosine and uridine K(m) app values 8 and 40 microM, respectively) . hCNT2 transcripts were found in kidney and small intestine but, unlike rCNT2, were not detected in liver . Deoxyadenosine, which undergoes net renal secretion in humans, was less readily transported than adenosine . hCNT2 also mediated small, but significant, fluxes of the antiviral purine nucleoside analogue 2',3'-dideoxyinosine . hCNT2 is, therefore potentially involved in both the intestinal absorption and renal handling of purine nucleosides (including adenosine), uridine and purine nucleoside drugs . The gene encoding hCNT2 was mapped to chromosome 15q15. Int J Oncol, 1999 Apr, 14(4), 681 - 5 Analysis of antibodies to p16INK4A tumor suppressor gene products in lung cancer patients; Namikawa O et al.; We examined by immunoblotting antibodies that bind to p16INK4A tumor suppressor gene products in the sera of patients with lung cancer . Sera from 15 to 102 patients, including 6 with adenocarcinoma, 3 with squamous cell carcinoma, 2 with large cell carcinoma and 4 with small cell carcinoma, reacted with a glutathione-S-transferase (GST) fusion protein containing whole human anti-p16INK4A, produced by Escherichia coli . However, sera from 30 normal volunteers did not react with the GST-p16INK4A fusion protein . The background such as age, gender, performance status, histology, stage, smoking history, and prior treatment was not significantly different between the patients with and without anti-GST-p16INK4A antibodies . Circulating p16INK4A tumor suppressor products were not detected in any individuals with lung cancer or in the normal controls. Plasmid, 1999 Mar, 41(2), 135 - 40 Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors; Chawla M et al.; Escherichia coli-mycobacteria shuttle vectors, derived from pAL5000 (a mycobacterial plasmid) and pUC19, were frequently found to undergo structural alterations due to transposition of IS1096, a Mycobacterium smegmatis transposable element, at a cluster of sites located within a small region of 60 bp, immediately upstream of a kanamycin resistance gene present in these vectors . The structural alterations led to deletion of large regions of the vector which, in several cases, were found to extend into the ORF2 (RepB) coding sequences of the pAL5000 replication region without affecting its replication capability . This suggests that the entire ORF2 coding sequences of the pAL5000 replication region may not be essential for replication of pAL5000-derived vectors . The deletion derivatives, which contain the minimal sequences required for replication and selection in mycobacteria, were found to be structurally stable and therefore these could be potentially used as stable vector systems for the transformation of mycobacteria . Plasmid, 1999 Mar, 41(2), 110 - 9 Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12; Atlung T et al.; The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time . In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives . However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold . The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min . Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid . To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184 . In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid . The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth . We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells . J Periodontal Res, 1999 Jan, 34(1), 34 - 40 Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates production of interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and interleukin-1 receptor antagonist in human whole blood; Schytte Blix IJ et al.; Actinobacillus actinomycetemcomitans (A . actinomycetemcomitans) is supposed to be an important etiological agent in localized juvenile periodontitis (LJP) . We have studied the effect of lipopolysaccharide (LPS) extracted from these periodontopathogenic bacteria on synthesis of the proinflammatory cytokines, interleukin-1beta(IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) in human whole blood . LPS from A . actinomycetemcomitans in concentrations > or =1 ng/ml induced a significant production of all these proinflammatory cytokines, whereas LPS from Escherichia coli (E . coli), strain 026:B6 had to be added in concentrations > or =1 microg/ml to obtain a similar effect . Similarly, LPS from A . actinomycetemcomitans > or =0.1 ng/ml resulted in production of IL-1ra, while LPS from E . coli 026:B6 had to be added at > or =10 ng/ml to obtain similar effects . It has been suggested that the ratio between production of proinflammatory and anti-inflammatory cytokines may influence the outcome of periodontal diseases . Other in vitro and in vivo studies have, however, indicated that very large excesses (100-1000 times) of IL-1ra compared to IL-1beta are required to shift the IL-1ra:IL-1beta ratio in favor of an inhibition of IL-1 bioactivity . In our ex vivo system, we found that stimulation with extremely low doses of A . actinomycetemcomitans LPS (0.1-1 ng/ml) resulted in IL-1ra production solely, without concomitant production of IL-1beta, the excess of IL-1ra over IL-1beta peaking at 1 ng/ml, which accordingly should suggest that LPS from A . actinomycetemcomitans primarily has proinflammatory effects. Eur J Med Res, 1999 Mar 26, 4(3), 85 - 94 Interleukin-10: effects on phagocytosis and adhesion molecule expression of granulocytes and monocytes in a comparison with prednisolone; Buchwald UK et al.; Interleukin-10 (IL-10) has potent anti-inflammatory and immunosuppressive properties . The potential therapeutic benefit may be compromised by the down-regulation of the non-specific immune system and an increased risk of infection . We studied the effects of IL-10 on important functions of native and granulocyte-macrophage colony-stimulating factor (GM-CSF) activated neutrophils and monocytes, namely phagocytosis and membrane expression of the beta superset2-integrins and of the intercellular adhesion molecule-1 (ICAM-1) . In order to simulate the in vivo situation closely, we used whole blood flowcytometric assays . The effects of IL-10 (0.05, 1, 10, 100 ng/ml) were compared to those of prednisolone (10 superset-8-10 superset-5 Mol/l), an approved immunosuppressive drug which is known to impair phagocyte function . - Incubation with IL-10 for three hours significantly attenuated the ability of neutrophils to phagocytose E.coli, particularly in lower concentrations . On the other hand, high IL-10 concentrations (10, 100 ng/ml) slightly augmented monocyte phagocytosis . Similarly, expression of the beta subset2-integrins and of ICAM-1 on monocytes was markedly enhanced with IL-10 concentrations in the range from 1 to 100 ng/ml and IL-10 showed strong synergistic effects with GM-CSF in the enhancement of monocyte receptor expression . Neutrophil adhesion molecule expression was not affected . Prednisolone suppressed the phagocytosis of both cell types in a dose-dependent fashion but hardly altered the receptor numbers . Our study indicates that IL-10 can behave as a de-activator as well as an activator on the non-specific immune system, depending on the cell type and the concentration. J Cell Sci, 1999 Apr, 112 ( Pt 8), 1257 - 71 The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle; Gachet Y et al.; The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts . P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast . Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated . Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner . (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies . (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle . At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition . (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro . The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B . (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology . Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability. J Cell Sci, 1999 Apr, 112 ( Pt 8), 1191 - 201 Myosin IB from Entamoeba histolytica is involved in phagocytosis of human erythrocytes; Voigt H et al.; Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery in humans . The disease is prevalent worldwide . Infection with E . histolytica results in invasion of the intestine by the parasite, followed by tissue damage and inflammation . During this invasive process, parasites kill and phagocytose human epithelial cells, immune cells and erythrocytes . Expression of amoebic pathogenicity requires a dynamic cytoskeleton that allows movement, tissue penetration and changes in parasite morphology . Myosin IB is a member of the myosin I family of motor proteins . Studies conducted both with Dictyostelium discoideum, a non-pathogenic amoeba, and with the yeast Saccharomyces cerevisiae indicate the involvement of myosin IB in cellular processes including movement, phagocytosis and endocytosis . Recently, we isolated the gene encoding myosin IB from E . histolytica . Thus, we decided to analyze the role of myosin IB in pathogenesis of amoeba . Using a specific anti-myosin IB antibody, this protein was localized in cell regions including the pseudopod, vesicles and underneath the plasma membrane . When E . histolytica was activated for erythrophagocytosis, myosin IB was markedly recruited to both the phagocytic cup and around internalized phagosomes . To analyze the role of myosin IB in phagocytosis, a strain overexpressing the myosin IB gene was constructed . This strain synthesizes threefold more myosin IB than the wild-type strain . Challenge of the transfected cell line with erythrocytes showed that these amoebae were deficient in erythrophagocytosis mainly in the uptake step, suggesting a role for myosin IB in the pathogenic activity of a human parasite. J Cell Sci, 1999 Apr, 112 ( Pt 8), 1181 - 90 Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation? Perennes C, Glab N, Guglieni B, Doutriaux MP, Phan TH, Planchais S, Bergounioux C. Progression of BY-2 tobacco cells through the cell cycle was followed after treatments with ultra violet (UV) and salicylic acid (SA) used as a potent inhibitor of the octadecanoid pathway which can mediate response to UV irradiation . Cells in S phase were more sensitive than G0/G1 or G2 cells to UV irradiation . Although SA efficiently blocked cells in G0/G1 or G2, it did not block S phase synchronized cells . UV and SA applied simultaneously to cells in G0/G1 delayed the cell cycle progression more than each one separately . Therefore UV irradiation and SA act as agonists to arrest BY-2 cells at cell cycle entry . To further investigate the signalling pathway mediating UV response, we complemented a UV-sensitive Escherichia coli strain with a Nicotiana xanthi cDNA expression library . A cDNA (arcA3) whose coding sequence is identical to the 2,4-D induced arcA cDNA cloned by Ishida et al . (1993) was isolated . We show that arcA3 transcription is induced at cell cycle entry but not directly by the 2,4-D treatment . Moreover, arcA3 transcription is induced prior to the restriction point as shown with the CDK inhibitor roscovitine . The arcA3 transcription level is increased by UV irradiation but prevented by SA . Indeed, addition of SA prior to UV irradiation blocks the induction of arcA3 transcription . This suggests that arcA3 gene is modulated in both UV and SA responses, the SA effect preceding the UV step . Since arcA3 is 67% similar to RACK1 (functional homology), a rat intracellular receptor for protein kinase C, and possesses identical PKC fixation motifs, it is hypothesised that the arcA3 gene is involved in UV and SA cell cycle arrest. Biochem J, 1999 Apr 1, 339 ( Pt 1), 201 - 7 Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes; Pirard M et al.; Human tissues contain two types of phosphomannomutase, PMM1 and PMM2 . Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome {Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat . Genet . 19, 88-92} . The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1 . PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates . The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1 . Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate . PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively . Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate . Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively. J Biol Chem, 1999 Mar 26, 274(13), 8993 - 7 SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle; Qi HY et al.; Recent work has demonstrated that the signal recognition particle (SRP) is required for the efficient insertion of many proteins into the Escherichia coli inner membrane (IM) . Based on an analogy to eukaryotic SRP, it is likely that bacterial SRP binds to inner membrane proteins (IMPs) co-translationally and then targets them to protein transport channels ("translocons") . Here we present evidence that SecA, which has previously been shown to facilitate the export of proteins targeted in a post-translational fashion, is also required for the membrane insertion of proteins targeted by SRP . The introduction of SecA mutations into strains that have modest SRP deficiencies produced a synthetic lethal effect, suggesting that SecA and SRP might function in the same biochemical pathway . Consistent with this explanation, depletion of SecA by inactivating a temperature-sensitive amber suppressor in a secAam strain completely blocked the membrane insertion of AcrB, a protein that is targeted by SRP . In the absence of substantial SecA, pulse-labeled AcrB was retained in the cytoplasm even after a prolonged chase period and was eventually degraded . Although protein export was also severely impaired by SecA depletion, the observation that more than 20% of the OmpA molecules were translocated properly showed that translocons were still active . Taken together, these results imply that SecA plays a much broader role in the transport of proteins across the E . coli IM than has been previously recognized. J Biol Chem, 1999 Mar 26, 274(13), 8806 - 12 Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy; Yanaga F et al.; Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique . The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations . An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity . A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation . Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity . Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity . These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
