Arch Biochem Biophys, 1998 Aug 1, 356(1), 93 - 8 Human betaine-homocysteine methyltransferase is a zinc metalloenzyme; Millian NS et al.; We have overexpressed recombinant human liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) in Escherichia coli and have purified the enzyme to homogeneity . The Michaelis constants for betaine and l-homocysteine are 2.2 mM and 4 microM, respectively . Analysis of the pure protein for metals by inductively coupled plasma emission spectrometry indicate that the recombinant enzyme contains zinc . Extensive dialysis in buffer containing high levels of EDTA could not strip the protein of zinc . However, dialysis against buffer containing EDTA and methyl methanethiosulfonate, followed by buffer containing EDTA and dithiothreitol, could remove zinc from the enzyme with concomitant loss of activity . Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, completely restored BHMT activity and zinc content . BHMT was also partially purified from human liver . The purest BHMT-containing fractions also contained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme . As with the recombinant enzyme, the partially purified human liver enzyme could be inactivated by treatment with methyl methanethiosulfonate, EDTA, and dithiothreitol . Reconstitution of the zinc-depleted enzyme completely restored activity . We conclude that BHMT is a major zinc metalloenzyme in liver and that cysteineresidues are likely involved in zinc binding . Protein Eng, 1998 May, 11(5), 377 - 81 Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies; Martsev SP et al.; Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7 . At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding . In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition . A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C) . The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy . The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions . The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit. Protein Eng, 1998 May, 11(5), 371 - 6 Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes; Chou WY et al.; Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg- . Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface . In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities . The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6 . Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation . Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme . K3A is also stable towards chemical denaturation . The concentration of urea that causes half unfolding, {urea}0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme . The K3A mutant of malic enzyme might therefore have potential practical applications. Protein Eng, 1998 May, 11(5), 337 - 44 The C-terminal domains of gammaS-crystallin pair about a distorted twofold axis; Basak AK et al.; The 2-domain gammaS-crystallin, a highly conserved early evolutionary off-shoot of the gamma-crystallin family, is located in the water-rich region of eye lenses . The expressed C-terminal domain, gammaS-C, has been crystallized and the 2.56 A X-ray structure determined . There are two domains in the asymmetric unit which pair about a distorted twofold axis . One of the domains has an altered conformation in a highly conserved region of the protein, the tyrosine corner . The distorted gammaS-C dimer of domains is compared with the highly symmetrical, equivalent recombinant dimer of C-terminal domains from gammaB-crystallin . Sequence changes close to the interface, that distinguish gammaS from the other gamma-crystallins, are examined in order to evaluate their role in symmetrical domain pairing. Protein Eng, 1998 May, 11(5), 333 - 5 Remarkable destabilization of recombinant alpha-lactalbumin by an extraneous N-terminal methionyl residue; Ishikawa N et al.; A recombinant bovine alpha-lactalbumin, possessing an additional N-terminal methionyl residue, was expressed in Escherichia coli . In order to address the effects of the N-terminal methionyl residue on conformational stability, the thermal stability of the recombinant alpha-lactalbumin was investigated by measuring temperature-dependence of circular dichroism spectra, and it was compared with that of authentic alpha-lactalbumin from bovine milk . The thermal stability of the recombinant alpha-lactalbumin was significantly lower than that of authentic alpha-lactalbumin . The enthalpy change of unfolding of the recombinant protein was found to be the same as that of the authentic one when compared at the same temperature . Therefore, the N-terminal methionyl residue seems to destabilize the conformation of recombinant alpha-lactalbumin through some entropic effects. Oncogene, 1998 Jun 25, 16(25), 3219 - 25 Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA; Kohno T et al.; The hOGG1 gene encodes a DNA glycosylase that excises 8-hydroxyguanine (oh8Gua) from damaged DNA . Structural analyses of the hOGG1 gene and its transcripts were performed in normal and lung cancer cells . Due to a genetic polymorphism at codon 326, hOGG1-Ser326 and hOGG1-Cys326 proteins were produced in human cells . Activity in the repair of oh8Gua was greater in hOGG1-Ser326 protein than in hOGG1-Cys326 protein in the complementation assay of an E . coli mutant defective in the repair of oh8Gua . Two isoforms of hOGG1 transcripts produced by alternative splicing encoded distinct hOGG1 proteins: one with and the other without a putative nuclear localization signal . Loss of heterozygosity at the hOGG1 locus was frequently (15/ 23, 62.2%) detected in lung cancer cells, and a cell line NCI-H526 had a mutation leading to the formation of the transcripts encoding a truncated hOGG1 protein . However, the oh8Gua levels in nuclear DNA were similar among lung cancer cells and leukocytes irrespective of the type of hOGG1 proteins expressed . These results suggest that the oh8Gua levels are maintained at a steady level, even though multiple hOGG1 proteins are produced due to genetic polymorphisms, mutations and alternative splicing of the hOGG1 gene. Eur Cytokine Netw, 1998 Jun, 9(2), 131 - 8 Interleukin-1-mediated febrile responses in mice and interleukin-1 beta activation of NFkappaB in mouse primary astrocytes, involves the interleukin-1 receptor accessory protein; Zetterstrom M et al.; The endogenous pyrogen interleukin-1 (IL-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation . IL-1 exerts its effects upon binding to the type I IL-1 receptor (IL-1RI) . The IL-1-IL-1RI complex is further thought to associate with the IL-1 receptor accessory protein (IL-1RAcP), which is suggested to be important for most IL-1 signal transduction pathways . With the aim of investigating the importance of the IL-1RAcP in IL-1 signalling, IL-1alpha and IL-1beta induced febrile responses and IL-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using IL-1RAcP-deficient (IL-1RAcP KO) and wild type mice, respectively . It was shown that neither recombinant rat IL-1alpha (rrIL-1alpha, 25 microg/kg), recombinant rat IL-1beta (rrIL-1beta, 40 microg/kg) nor recombinant human IL-1beta (rhIL-1beta, 50 microg/kg) injected i.p . could elicit febrile responses in the IL-1RAcP-deficient mice, while the same doses of rrIL-1alpha/beta or rhIL-1beta injected into wild type mice caused normal fever responses . A febrile response could be induced in the IL-1RAcP-deficient mice by i.p . administration of E . coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice . Furthermore, it was shown that rhIL-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no IL-1beta-induced translocation of NFkappaB could be detected in cultures prepared from IL-1RAcP-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA) . The rhIL-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins. Bioorg Med Chem, 1998 Jun, 6(6), 701 - 6 Chemo-enzymatic synthesis of a new type of enantiomerically pure carbocyclic nucleoside analogues with strong inhibitory effects on terminal deoxynucleotidyl transferase; Theil F et al.; The synthesis of enantiomerically pure carbocyclic adenosine derivatives which have been prepared based on the kinetic resolution of a trans-2-(hydroxymethyl)cyclopentanol derivative is described . Their corresponding triphosphates were evaluated as inhibitors of DNA polymerase beta, terminal deoxynucleotidyl transferase (TdT), telomerase, Escherichia coli DNA polymerase I and reverse transcriptase of human immunodeficiency virus . Surprisingly, the triphosphate of (1S,2R)-1-(6-aminopurin-9-yl)-2-(hydroxymethyl)cyclopentane {(1S,2R)-6} and its enantiomer (1R,2S)-6 emerged as strong inhibitors of TdT (Ki = 0.5 and 1.9 mM, Kmapp dATP = 40 mM), whereas the activities of all other enzymes tested proved to be unaffected. Plant J, 1998 Jun, 14(6), 703 - 13 The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer; St-Pierre B et al.; The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized . The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer . However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme . Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings . The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown . The Mr of approximately 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase . The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes. Plant J, 1998 Mar, 13(6), 743 - 52 Characterization of acyl-ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola; Hawkins DJ et al.; Acyl-acyl-carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils . Acyl-ACP thioesterases with specificity for each of the saturated acyl-ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases . The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight . Acyl-ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1-ACP substrates, but had significant activity with 18:0 relative to that with 16:0-ACP, suggesting a thioesterase might be involved in the production of stearate . Three distinct acyl-ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class . When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1-ACP showed relatively low activity with 16:0-ACP as compared to 18:0-ACP, similar to the substrate preferences shown by the crude seed extract . Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil . These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles. Plant J, 1998 Feb, 13(4), 489 - 505 Two bZIP proteins from Antirrhinum flowers preferentially bind a hybrid C-box/G-box motif and help to define a new sub-family of bZIP transcription factors; Martinez-Garcia JF et al.; Two genes encoding bZIP proteins are expressed in flowers of Antirrhinum majus, predominantly in vascular tissues, carpels and anthers . The sequences of cDNA clones encoding these proteins show them to belong to a distinct sub-family of bZIP proteins which also includes LIP19 from rice and MLIPI5 and OBF1 from maize . The sub-family is characterized by the inclusion of very small proteins consisting of essentially a basic domain and a long leucine zipper . Members also have a conserved upstream open reading frame (uORF) in their 5' leader sequences, implying a common mode of post-transcriptional control . In vitro, the Antirrinum bZIP proteins preferentially bind to a novel hybrid C-box/G-box motif which is found in the promoters of some plant histone genes and of some nuclear-encoded genes with plastidial protein products . Expression of the bZIP proteins in transgenic tobacco under control of the CaMV 35S promoter supports the view that they can regulate expression of genes which contain the preferred target motif within their regulatory sequences, although both enhancement and repression of transcript levels of target genes were observed, indicating that the bZIP proteins probably interact with other factors to modulate transcription in different ways, as has been observed for the small MAF family of bZIP proteins in vertebrates. Plant J, 1998 Jan, 13(2), 249 - 57 Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism; von Schwartzenberg K et al.; Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G . was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain . The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa . Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome . The deduced amino acid sequence had a 52% identity with the human adenosine kinase . The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E . coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue . Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella. Plant J, 1998 Jan, 13(1), 97 - 107 A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L . plants; Rey P et al.; By analysing two-dimensional patterns of chloroplastic proteins from Solanum tuberosum, the authors observed the accumulation of a 32-kDa polypeptide in the stroma of plants subjected to water deficit . N-terminus and internal peptides of the protein, named CDSP 32 for chloroplastic drought-induced stress protein, showed no obvious homology with known sequences . Using a serum raised against the protein N-terminus, a cDNA encoding CDSP 32 was cloned by screening an expression library . The deduced mature CDSP 32 protein is 243 amino acids long and displays typical features of thioredoxins in the C-terminal region (122 residues) . In particular, CDSP 32 contains a CGPC motif corresponding to a thioredoxin active site and a number of amino acids conferring thioredoxin-type structure . The CDSP 32 C-terminal region was expressed as a fusion protein in Escherichia coli and was shown to possess thioredoxin activity based on reduction assay of insulin disulfide bridges . RNA blot analysis showed that CDSP 32 transcript does not accumulate upon mild water deficit conditions corresponding to leaf relative water contents (RWC) around 85%, but high levels of CDSP 32 transcripts were observed for more severe stress conditions (RWC around 70%) . In vivo labelling and immunoprecipitation revealed a substantial increase in CDSP 32 synthesis upon similar stress conditions . Rewatering of wilted plants caused decreases in both transcript and protein abundances . In tomato wild-type plants and ABA-deficient mutants, a similar accumulation of a CDSP 32-related transcript was observed upon water deficit, most likely indicating no requirement for ABA in the regulation of CDSP 32 synthesis . Based on these results, it is proposed that CDSP 32 plays a role in preservation of the thiol: disulfide redox potential of chloroplastic proteins during water deficit. Plant J, 1998 Jan, 13(1), 63 - 70 Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability; Wang SM et al.; Two mutants of Arabidopsis have been isolated that affect ADPG pyrophosphorylase (ADGase) activity . Previously, it has been shown that ADG2 encodes the large subunit of ADGase . This study characterizes the adg1 mutant phenotype and ADG1 gene structure . RNA blot analyses indicate that the adg1-1 mutant accumulates transcripts encoding both the large and small subunits of ADGase, while the adg1-2 mutant accumulates only large subunit transcripts . RFLP analysis and complementation of adg1 mutants with the ADGase small subunit gene demonstrate that ADG1 encodes the small subunit . Sequence analysis indicates that adg1-1 represents a missense mutation within the gene . Western blot analysis confirms that adg1 mutants contain neither the large nor the small subunit proteins, suggesting that the presence of functional small subunits is required for large subunit stability. Plant J, 1998 Jan, 13(1), 17 - 28 A pectate lyase from Zinnia elegans is auxin inducible; Domingo C et al.; The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans-differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate . In a search for genes involved in modifications to cell-wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72-h cDNA library with probes from mRNA at time-points of 24 h and 72 h was done revealing a number of transcripts up-regulated between these times . One of these cDNAs shows homology to pectate lyase, a pectin-degrading enzyme . The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N-terminal signal peptide of about 2 kDa, and one potential N-glycosylation site . Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium . In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia . Recombinant protein made in Escherichia coli possesses calcium-dependent pectate lyase activity . Pectate lyase activity is detected in elongating and differentiating in vitro cell populations . The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed. J Mol Biol, 1998 Aug 7, 281(1), 135 - 47 Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106; Nishida M et al.; Cytosolic glutathione S-transferase is a family of multi-functional enzymes involved in the detoxification of a large variety of xenobiotic and endobiotic compounds through glutathione conjugation . The three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate, N-(N-L-gamma-glutamyl-3-sulfo-L-alanyl)-glycine, has been determined by the multiple isomorphous replacement method and refined to a crystallographic R factor of 0.183 at 2.1 A resolution.The E . coli enzyme is a globular homodimer with dimensions of 58 Ax56 Ax52 A . Each subunit, consisting of a polypeptide of 201 amino acid residues, is divided into a smaller N-terminal domain (residues 1 to 80) and a larger C-terminal one (residues 89 to 201) . The core of the N-terminal domain is constructed by a four-stranded beta-sheet and two alpha-helices, and that of the C-terminal one is constructed by a right-handed bundle of four alpha-helices . Glutathione sulfonate, a competitive inhibitor against glutathione, is bound in a cleft between the N and C-terminal domains . Therefore, the E . coli enzyme conserves overall constructions common to the eukaryotic enzymes, in its polypeptide fold, dimeric assembly, and glutathione-binding site . In the case of the eukaryotic enzymes, tyrosine and serine residues near the N terminus are located in the proximity of the sulfur atom of the bound glutathione, and are proposed to be catalytically essential . In the E . coli enzyme, Tyr5 and Ser11 corresponding to these residues are not involved in the interaction with the inhibitor, although they are located in the vicinity of catalytic site . Instead, Cys10 N and His106 Nepsilon2 atoms are hydrogen-bonded to the sulfonate group of the inhibitor . On the basis of this structural study, Cys10 and His106 are ascribed to the catalytic residues that are distinctive from the family of the eukaryotic enzymes . We propose that glutathione S-transferases have diverged from a common origin and acquired different catalytic apparatuses in the process of evolution . J Mol Biol, 1998 Aug 7, 281(1), 121 - 34 A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure; Williams MK et al.; Kinetic analysis of a mutant version of Escherichia coli aspartate transcarbamoylase in which Thr82 in the regulatory chain (Thr82r) was replaced by Ala results in a shift in the T <==> R equilibrium towards the T-state . In order to understand the structural determinants of this T-state stabilization, the X-ray structure of the unliganded Thr82r-->Ala enzyme was determined at 2 . 6 A resolution and refined to a crystallographic residual of 0.175 . The structure of the mutant r1 regulatory chain is more similar to that of the r6 regulatory chain than observed for the wild-type enzyme, resulting in a more symmetric structure . Furthermore, the structural changes in the mutant enzyme appears to occur only in the r1 chain, while the r6 chain is almost identical in structure to that of the r6 chain of the wild-type enzyme . The structure of the mutant enzyme exhibits alterations in the subunit interfaces between the regulatory and catalytic chains, as well as in the interface between the allosteric and zinc domains within the regulatory chain . Moreover, the regulatory dimers are rotated around their respective 2-fold axes approximately 1 degrees beyond the rotation which occurs in the wild-type T-state enzyme . The structural analysis indicates that the enzyme is an "extreme" T-state, in which a larger rotation of the regulatory dimers is required for the T to R transition compared to the wild-type enzyme . This extreme T-state structure correlates well with the kinetic parameters determined for the mutant enzyme, showing a stabilized T-state . Furthermore, the structural analysis of the mutant enzyme suggests that replacement of Thr82r with Ala alters the local conformation of the nucleotide binding pocket and therefore offers a plausible explanation for the reduced affinity of the enzyme for nucleotides . J Mol Biol, 1998 Aug 7, 281(1), 107 - 19 Hydrophobic forces dominate the thermodynamic characteristics of UvrA-DNA damage interactions; Zou Y et al.; The Escherichia coli DNA repair proteins UvrA, UvrB and UvrC work together to recognize and incise DNA damage during the process of nucleotide excision repair (NER) . To gain an understanding of the damage recognition properties of UvrA, we have used fluorescence spectroscopy to study the thermodynamics of its interaction with a defined DNA substrate containing a benzo{a}pyrene diol epoxide (BPDE) adduct . Oligonucleotides containing a single site-specifically modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, or (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments . All four stereoisomers of DNA-BPDE adducts show an excitation maximum at 350 nm and an emission maximum around 380 to 385 nm . Binding of UvrA to the BPDE-DNA adducts results in a five to sevenfold fluorescence enhancement . Titration of the BPDE-adducted DNA with UvrA was used to generate binding isotherms . The equilibrium dissociation constants for UvrA binding to (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis- BPDE adduct were: 7.4+/-1.9, 15 . 8+/-5.4, 11.3+/-2.7 and 22.4+/-2.0 nM, respectively . There was a large negative change in heat capacity DeltaCpo,obs, (-3.3 kcal mol-1 K-1) accompanied by a relatively unchanged DeltaGoobs with temperature . Furthermore, varying the concentration of KCl showed that the number of ions released upon formation of UvrA-DNA complex is about 3.4, a relatively small value compared to the contact size of UvrA with the substrate . These data suggest that hydrophobic interactions are an important driving force for UvrA binding to BPDE-damaged DNA . J Mol Biol, 1998 Aug 7, 281(1), 49 - 59 Molecular architecture of the c-subunit oligomer in the membrane domain of F-ATPases probed by tryptophan substitution mutagenesis; Groth G et al.; Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction . In order to gain some insight into the architecture of this oligomeric complex, the transmembrane region in the C-terminal membrane-spanning segment was analysed by a site-directed mutagenesis approach . Tryptophan substitution of consecutive residues in positions 61 to 72 of subunit c was used to identify residues oriented towards a helix-helix surface or an accessible phase in the oligomeric complex . Mutants were analysed in functional assays of ATP hydrolysis, ATP synthesis and ATP-dependent proton transport . Function was disrupted according to a pattern that identified inter- and intramolecular contacts in the c-subunit oligomer . Screening experiments on minimal medium support the helix-helix contacts found in the functional assays . The results add strong constraints to the potential orientation of the monomers in the oligomeric complex and are discussed against the background of different structural models that have been proposed for the c-subunit oligomer . J Mol Biol, 1998 Aug 7, 281(1), 31 - 48 Specific correlations between relative synonymous codon usage and protein secondary structure; Oresic M et al.; We found significant species-specific correlations between the use of two synonymous codons and protein secondary structure units by comparing the three-dimensional structures of human and Escherichia coli proteins with their mRNA sequences . The correlations are not explained by codon-context, expression level, GC/AU content, or positional effects . The E . coli correlation is between Asn AAC and the C-terminal regions of beta-sheet segments; it may result from selection for translational accuracy, suggesting the hypothesis that downstream Asn residues are important for beta-sheet formation . The correlation in human proteins is between Asp GAU and the N termini of alpha-helices; it may be important for eukaryote-specific sequential, cotranslational folding . The kingdom-specific correlations may reflect kingdom-specific differences in translational mechanisms . The correlations may help identify residues that are important for secondary structure formation, be useful in secondary structure prediction algorithms, and have implications for recombinant gene expression . J Mol Biol, 1998 Aug 7, 281(1), 17 - 29 Sequence-specificity of Holliday junction resolution: identification of RuvC mutants defective in metal binding and target site recognition; Hagan NF et al.; The RuvC protein of Escherichia coli resolves Holliday intermediates in recombination and DNA repair by a dual strand incision mechanism targeted to specific DNA sequences located symmetrically at the crossover . Two classes of amino acid substitutions are described that provide new insights into the sequence-specificity of the resolution reaction . The first includes D7N and G14S, which modify or eliminate metal binding and prevent catalysis . The second, defined by G114D, G114N, and A116T, interfere with the ability of RuvC to cleave at preferred sequences, but allow resolution at non-consensus target sites . All five mutant proteins bind junction DNA and impose an open conformation . D7N and G14S fail to induce hypersensitivity to hydroxyl radicals, a property of RuvC previously thought to reflect junction opening . A different mechanism is proposed whereby ferrous ions are co-ordinated in the complex to induce a high local concentration of radicals . The open structure imposed by wild-type RuvC in Mg2+ is similar to that observed previously using a junction with a different stacking preference . G114D and A116T impose slightly altered structures . This subtle change may be sufficient to explain the failure of these proteins to cleave the sequences normally preferred . Gly114 and Ala116 residues link two alpha-helices lining the wall of the catalytic cleft in each subunit of RuvC . We suggest that substitutions at these positions realign these helices and interfere with the ability to establish base-specific contacts at resolution hotspots . Mol Microbiol, 1998 Jun, 28(6), 1315 - 22 Multiple cis-acting sites positively regulate Escherichia coli nrd expression; Jacobson BA et al.; Regulation of nrd expression in Escherichia coli by cis-acting elements was found to be more complex than previously reported . At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT-rich region, an inverted repeat and a 10 bp site between the AT-rich region and the inverted repeat . Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression . As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis-inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states . As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle-regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites . Site-directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT-rich region results from deletion of both the Fis binding site and the AT-rich region. Mol Microbiol, 1998 Jun, 28(6), 1307 - 14 A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon; Jacobson BA et al.; Expression of beta-galactosidase from a nrd-lacZ fusion was used to determine the role in nrd regulation of an inverted sequence upstream of the promoter . Removal or replacement of a 45bp inverted repeat with an altered sequence including a 48bp perfect inverted repeat resulted in a mutant phenotype that was low in nrd expression in an exponentially growing culture and that did not increase during DNA synthesis inhibition . Changing the 22 bp in the upstream half of the inverted repeat resulted in the same phenotype, whereas changing the 22 bp in the downstream half of the inverted repeat decreased nrd expression to a lesser extent in an exponentially growing culture and had only a smaller effect on nrd expression during DNA synthesis inhibition . As other mutants with the phenotype of the upstream inverted repeat mutant were found to lack cell cycle regulation, expression of nrd-lac mRNA produced from a plasmid with this mutation in the nrd-lacZ fusion gene was compared with nrd mRNA produced from the chromosomal nrd gene in a synchronized culture . The results indicated that the upstream half of the nrd inverted repeat contains a cis-acting element essential for nrd cell cycle regulation. Mol Microbiol, 1998 Jun, 28(6), 1295 - 306 Characterization and splicing in vivo of a Sinorhizobium meliloti group II intron associated with particular insertion sequences of the IS630-Tc1/IS3 retroposon superfamily; Martinez-Abarca F et al.; By sequence analysis of Sinorhizobium meliloti strain GR4 plasmid pRmeGR4b, we have identified a group II intron named RmInt1 inserted within the insertion sequence ISRm2011-2 of the IS630-Tc1/IS3 retroposon superfamily . Like some other group II introns, RmInt1 possesses, in addition to the structurally conserved ribozyme core, an open reading frame (ORF) with homology to reverse transcriptases . Using a T7 expression system in Escherichia coli, we show that the intron is active in splicing in vivo and that splicing efficiency requires the intron-encoded ORF, which suggests that the putative intron encoded protein has a maturase function . DNA hybridization studies indicate that intron RmInt1 is widespread within S . meliloti native populations and appears to be mostly located within this IS element . Nevertheless, some S . meliloti strains harbour one copy of RmInt1 at a different location . DNA sequence analysis of the 5' exon of one of these heterologous intron insertion sites revealed the presence of a putative IS element closely related to insertion sequence ISRm2011-2 . The intron-binding sites (IBS1 and IBS2 motifs) are conserved, although a transition of a G-->A in the IBS1 has occurred . Our results demonstrate an association of intron RmInt1 with particular insertion sequences of the IS630-Tc1/IS3 retroposon superfamily that may have ensured the spread and maintenance of this group II intron in S . meliloti. Mol Microbiol, 1998 Jun, 28(6), 1199 - 210 The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli; Patzer SI et al.; In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion . Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system . The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+ . In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+ . This high-affinity transport was energized by ATP . The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E . coli . At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain . The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+ . A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN {tetrakis-(2-pyridylmethyl) ethylenediamine} . To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E . coli . A complementing gene, yjbK of the E . coli genome, was identified and named zur (for zinc uptake regulation) . The Zur protein showed 27% sequence identity with the iron regulator Fur . High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain . An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB. Mol Microbiol, 1998 Jun, 28(6), 1121 - 37 Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor; White-Ziegler CA et al.; The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26 degrees C . Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23 degrees C . The histone-like nucleoid structuring protein H-NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not . In vivo analysis of pap DNA methylation patterns indicated that H-NS or a factor regulated by H-NS bound within the pap regulatory region at 23 degrees C but not at 37 degrees C, as evidenced by H-NS-dependent inhibition of methylation of the pap GATC sites designated GATC-I and GATC-II . These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription . Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment containing the GATC-I and GATC-II sites . Moreover, H-NS blocked methylation of these pap GATC sites in vitro: H-NS blocked pap GATC methylation at 1.4 microM but was unable to do so at higher concentrations at which non-specific binding occurred . Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites . These results suggest that the ability of H-NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H-NS with pap regulatory DNA . We hypothesize that a function of H-NS such as oligomerization was altered at 23 degrees C, which enabled H-NS to repress pap gene expression through the formation of a specific nucleoprotein complex. Protein Eng, 1998 Apr, 11(4), 263 - 77 Molecular shape comparisons in searches for active sites and functional similarity; Rosen M et al.; Here we examine the reliability of surface comparisons in searches for active sites in proteins . Detection of a patch of surface on one protein which is similar to an active site in another, may suggest similarities in enzymatic mechanisms, in enzyme functions and implicate a potential target for ligand/inhibitor design . Specifically, we compare the efficacy of molecular surface comparisons with comparisons of surface atoms and of C(alpha) backbone atoms . We further investigate comparisons of specific atoms, belonging to a predefined pattern of catalytic residues versus comparisons of molecular surfaces and, separately, of surface atoms . This aspect is particularly relevant, as catalytic residues may be (partially) buried . We also explore active site comparisons versus comparisons in which the entire molecular surfaces are scanned . While here we focus on the geometrical aspect of the problem, we also investigate the effect of adding residue labels in these comparisons . Our extensive studies cover the serine proteases, containing the highly conserved triad motif, and the chorismate mutases . Since such active site comparisons entail comparisons between unconnected points in 3D space, an order-independent comparison technique is necessary . The geometric hashing algorithm is ideally suited to handling such a task . It can perform both global shape matching for the whole surfaces of large protein molecules and searching for local shape similarities for small surface motifs . Our results show that molecular surface comparisons work best when the similarity is high . As the similarity deteriorates, the number of potential solutions increases rapidly, making their ranking difficult, particularly when scanning entire molecular surfaces . Utilizing atomic coordinates directly appears more adequate under such circumstances. Free Radic Biol Med, 1998 Aug, 25(3), 373 - 7 DNA damage in diabetes: correlation with a clinical marker; Collins AR et al.; Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured . Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics . Strand breaks correlated with body mass index in the diabetic group . A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations . When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal . DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia. J Gen Virol, 1998 Jul, 79 ( Pt 7), 1619 - 29 Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2; Holloway SA et al.; Twenty-two monoclonal antibodies (MAbs) were generated to the gammaherpesvirus equine herpesvirus-2 (EHV-2) . Using Western blot analysis, eight MAbs recognized an Escherichia coli glutathione S-transferase (GST)-glycoprotein B (gB) fusion protein and, using overlapping GST-gB fusion proteins, a neutralization epitope was mapped to amino acids 29-74 . One of the gB-specific MAbs was used to characterize the glycosylation and kinetics of synthesis of EHV-2 gB . EHV-2 gB is synthesized as a 97 kDa polypeptide that is co-translationally modified to a 130 kDa high-mannose precursor that forms a 260 kDa dimer shortly after synthesis . Each 130 kDa precursor is endoproteolytically cleaved to disulphide-linked subunits of 75 and 58 kDa prior to further processing to complex oligosaccharide-containing subunits of 89 and 65/62 kDa . The 89 and 65/62 kDa subunits of EHV-2 gB contain 39 and 17 kDa of N-linked oligosaccharides, respectively, and do not contain any O-linked oligosaccharides . Western blot analysis of purified EHV-2 virions established that gB exists as a 320 kDa dimer in the virion envelope. J Trauma, 1998 Jul, 45(1), 19 - 23; discussion 23-4 Regulation of macrophage eicosanoid generation is dependent on nuclear factor kappaB; Lo CJ et al.; BACKGROUND: Prostaglandin E2 (PGE2) is a major contributor to the production and maintenance of immunosuppression after overwhelming injury, leading to increased infectious morbidity and mortality in trauma patients . Elucidation of the cellular pathways involved in PGE2 production could lead to potential therapeutic interventions . The purpose of this study was to determine the role of cyclooxygenase II (COX-2) in PGE2 production by Mphi and to investigate the cellular mechanism of COX-2 gene activation . METHODS: Mouse macrophages (Mphi), RAW 264.7, were exposed to Escherichia coli lipopolysaccharide (LPS) in the presence of cyclooxygenase inhibitors (ibuprofen or NS398) . COX-1 and COX-2 mRNA expression and PGE2 production were measured by Northern blot assay and enzyme-linked immunosorbent assay, respectively . Nuclear factor kappaB (NFkappaB) activity was measured by electrophoretic mobility shift assay . To elucidate the role of NFkappaB in LPS-induced COX-2 gene activation, Mphi were exposed to LPS in the presence of an NFkappaB inhibitor, TPCK . RESULTS: LPS increased Mphi COX-2 mRNA expression but had no effect on COX-1 mRNA expression . Both ibuprofen and NS398 inhibited COX-2 mRNA as well as PGE2 production by LPS-stimulated Mphi . In addition, LPS-induced NFkappaB activity was attenuated by these agents . Inhibition of NFkappaB with TPCK reduced COX-2 but not COX-1 gene expression and decreased PGE2 production by LPS-stimulated Mphi . CONCLUSION: Our data indicate that COX-2 gene expression by LPS-stimulated Mphi is dependent on NFkappaB . Cyclooxygenase inhibitors reduced PGE2 production by inhibiting both COX-2 mRNA expression and preventing NFkappaB activation. Biochem Mol Biol Int, 1998 Jul, 45(3), 635 - 42 Enhancement by copper, zinc superoxide dismutase of DNA damage and mutagenicity with hydrogen peroxide; Kim RH et al.; Oxidative DNA damage caused by hydrogen peroxide was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks . Hydroxyl radical scavengers such as sodium azide, mannitol and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a metal chelator, diethylenetriamine-pentaacetic acid, and catalase decreased strand breaks and 8-OH-dG formation in DNA . The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with H2O2 . CuZnSOD also caused enhancement of mutation in the pUC18 lacZ' gene in the presence of H2O2 when measured as a loss of alpha-complementation . Based on these results, we interpret the effects of CuZnSOD on hydrogen peroxide induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed predominantly by the reaction of hydrogen peroxide and free Cu2+ released from oxidatively damaged CuZnSOD. Biochem Mol Biol Int, 1998 Jul, 45(3), 567 - 73 Inhibition of mitochondrial gene expression by antisense RNA of mitochondrial transcription factor A (mtTFA); Inagaki H et al.; Mitochondrial transcription factor A (mtTFA) plays an important role in regulating the expression of mitochondrial genes . To gain a better understanding of the relationships between mitochondrial gene expression and mtTFA, the mtTFA gene was inserted into a mammalian expression vector, both in the sense orientation and in the antisense orientation . After construction, these plasmids were transfected into COS-7 cells . In antisense-transformed cells, the expression of the cytochrome c oxidase subunit I and subunit III genes encoded by mitochondrial DNA was inhibited and there was an accompanying reduction of the level of mtTFA protein . These results provide direct evidence that the expression of mitochondrial genes is under the control of mtTFA. Z Naturforsch {C}, 1998 May-Jun, 53(5-6), 416 - 20 Purification and partial characterization of glyceraldehyde-phosphate dehydrogenase from electric organ of Electrophorus electricus (L.); Giovanni-De-Simone S et al.; The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chromatography method on deacetylcolchicine-Sepharose . The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37% . The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa . The purity of the colchicine-Sepharose isolated material was analysed by isoelectrophocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH . Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH. Z Naturforsch {C}, 1998 May-Jun, 53(5-6), 359 - 68 Inhibition of RNA synthesis in vitro by acridines--relation between structure and activity; Piestrzeniewicz MK et al.; The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied . The primary mechanism of action is related to inhibition of the enzyme binding to DNA . The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA . Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives . No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here . The aniline ring seems to decrease the inhibitory potency of a ligand . The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison. J Photochem Photobiol B, 1998 May 15, 43(2), 158 - 63 A fluorescence spectroscopic study on the binding of mRNA 5'-cap-analogs to human translation initiation factor eIF4E: a critical evaluation of the sources of error; Wieczorek Z et al.; Equilibrium constants for the association of human protein translation initiation factor eIF4E with two mRNA 5'-cap analogs, namely 7-methylguanosine 5'-triphosphate and P1-(7-methylguanosine-5') P3-(guanosine-5') triphosphate, and with guanosine 5'-monophosphate have been redetermined by the fluorescence quenching method taking the inner filter effect of the cap-analog into account . It has been shown that neglecting the latter correction may lead to either underestimation or overestimation of the association constant obtained by applying the Eadie-Hofstee plot: the reasonably firm binding of 7-methylated cap-analogs becomes underestimated, while the weak binding of non-methylated nucleotides becomes overestimated. J Photochem Photobiol B, 1998 May 15, 43(2), 101 - 5 Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system; Paretzoglou A et al.; Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers . A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established . DNA seems to be a major target and a variety of types of damage have been observed . In this report we present evidence that histidine can also act as a sensitizer of NUV . Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers . pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan . Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect . These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage . The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites. J Biomol NMR, 1998 Feb, 11(2), 205 - 12 Solvent exchange rates of side-chain amide protons in proteins; Rajagopal P et al.; Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr . The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons . Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8 . An NOE between a side-chain amide proton and a bound water molecule was also observed. Carbohydr Res, 1998 Feb, 306(4), 575 - 8 Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase; Maru I et al.; N-Acetylneuraminate lyase and N-acyl-D-glucosamine 2-epimerase had been cloned and overexpressed in Escherichia coli . Simultaneous use of these two enzymes and feeding of appropriate amounts of pyruvate to the reaction mixture made possible the high conversion of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc) with a 77% conversion rate on a molar basis . As a result, 29 kg of Neu5Ac was obtained from 27 kg of GlcNAc . The product was recovered by direct crystallization, and verified as identical to authentic Neu5Ac. J Cell Biol, 1998 Jul 27, 142(2), 421 - 34 The cytosolic DnaJ-like protein djp1p is involved specifically in peroxisomal protein import; Hettema EH et al.; The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ . DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes . Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome . The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities . Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal . Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant . We conclude that Djp1p is specifically required for peroxisomal protein import. Genes Dev, 1998 Jul 15, 12(14), 2164 - 74 Impaired megakaryopoiesis and behavioral defects in mafG-null mutant mice; Shavit JA et al.; The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo . To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene . mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal . Characterization of the mafG and mafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping . These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation. Gastroenterology, 1998 Aug, 115(2), 281 - 6 Association between intraepithelial Escherichia coli and colorectal cancer; Swidsinski A et al.; BACKGROUND & AIMS: Although multiple studies have focused on Helicobacter pylori, little is known about the mucosa-associated flora of the colon . The aim of this study was to detect bacteria directly in colonic mucosa from patients screened for colorectal cancer . METHODS: Bacteria were quantified with the polymerase chain reaction and identified by comparative sequence analysis in colonoscopic biopsy specimens from 31 asymptomatic and 34 symptomatic controls with normal colonoscopic findings, 29 patients with colonic adenoma, and 31 patients with colorectal carcinoma . In 41 patients, intra- and extracellular location of bacteria was confirmed with the gentamicin protection assay . RESULTS: No bacteria were detected in biopsy specimens from 97% of asymptomatic and 69% of symptomatic controls . In contrast, bacterial concentrations of 10(3)-10(5) colony-forming units per microliter were detected in biopsy specimens from both malignant and macroscopically normal tissue in 90% and 93% of patients with adenoma and carcinoma, respectively . E . coli and coli-like bacteria were shown to colonize the colonic mucosa in 82% of these patients . The gentamicin protection assay indicated that E . coli was partially intracellular in 87% of patients with adenoma and carcinoma and in none of the controls . CONCLUSIONS: The colonic mucosa of patients with colorectal carcinoma but not normal colonic mucosa is colonized by intracellular E . coli. Gene, 1998 Jun 8, 212(2), 295 - 304 Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase; Tillmann H et al.; By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries . From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755 . After overexpression in E . coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate . Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform . This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme. Clin Exp Allergy, 1997 Sep, 27(9), 1086 - 94 Identification of T-cell epitope sequences on an important mite antigen; Fujii S et al.; BACKGROUND: T-cell epitopes on Der 1 and Der 2 groups, the major mite allergens, have been intensively analysed, while those on the other important allergens remain to be elucidated . We have cloned four cDNAs coding for important mite allergens on the basis of frequency and capacity of IgE binding . Stimulatory action of glutathione S-transferase-fused Mag1 on lymphocytes from mite-allergic patients was relatively high among them . OBJECTIVE: To identify T-cell epitopes on Mag1, we studied the stimulating activity of truncated Mag1 proteins and synthetic peptides on proliferative response of lymphocytes from mite antigen-immunized mice and mite-sensitive patients . METHODS: Truncated Mag1 proteins were expressed as a fusion protein with beta-galactosidase in Escherichia coli pop2136 carrying a variety of deleted Mag1 inserts . Murine T-cell epitope regions were estimated by the truncated antigen-induced lymphocyte proliferation assay . Overlapping peptides covering the whole sequence of the presumed T-cell epitope regions were synthesized to identify the epitope core sequences using murine and human Mag1-specific T-cell lines . RESULTS: Amino acid range 56-70 on Mag1 molecule showed remarkable stimulatory action on murine T cells, while amino acid ranges 51-65 and 86-100 had potent stimulatory activity on human T cells . CONCLUSIONS: These results suggest that Mag1 is a valuable antigen suitable for studies on T-cell responses and T-cell epitopes in mice and humans. Plant Mol Biol, 1998 Jul, 37(5), 849 - 57 A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV light and pathogen attack; Christensen AB et al.; A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp . hordei (Bgh) . The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72-79% identity to chalcone synthases (CHS) and 65-68% identity to stilbene synthases . Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS . HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation . The accumulation of HvCHS2 transcripts is also elicited by UV light . To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli . Both HvCHS2 and CHS1 catalyse the formation of chalcones . However, HvCHS2 and CHS1 differ in their substrate requirements . CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme . In contrast, HvCHS2 converts feruloyl-CoA and caffeoyl-CoA at the highest rate whereas cinnamoyl-CoA is a poor substrate . Thus, HvCHS2 is a novel pathogen and UV light induces homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings. Plant Mol Biol, 1998 Jul, 37(5), 839 - 47 Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress; Jang HJ et al.; Plant responses to high salt stress have been studied for several decades . However, the molecular mechanisms underlying these responses still elude us . In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis . Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins . AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin . However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops . We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay . A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift . To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out . The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques . Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment . Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein. Plant Mol Biol, 1998 Jul, 37(5), 773 - 84 Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea; Reddy MK et al.; We have isolated and sequenced the full length cDNA for topoisomerase I . Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template . This fragment was used as a probe to screen a pea cDNA library . Two partial cDNA clones were isolated which were truncated at the 5' end . RACE-PCR was employed to isolate the remaining portion of the gene . The total size of the gene was 3055 bp with an open reading frame of 2676 bp . The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3 . A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related . Pea topoisomerase I has been overexpressed in E . coli system and the recombinant topoisomerase purified to homogeneity . The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+ . In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I . Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei. J Cardiovasc Surg (Torino), 1998 Jun, 39(3), 303 - 5 Intravascular ultrasound diagnosis of aortic graft infection; Duda SH et al.; BACKGROUND: To assess the value of intra-aortic ultrasound (US) for diagnosing intraprosthetic vegetations in atypical aortic graft infection . METHODS: A 66-year-old man presented with fever 12 months after emergency insertion of a straight infrarenal aortic graft because of rupture of an inflammatory abdominal aneurysm . Blood cultures, leukocyte scan, transabdominal US study, and digital angiography were negative . Spiral CT was equivocal . The patient was imaged with a mechanically rotating US transducer at 12.5-MHz from inside the graft . RESULTS: Intravascular catheter ultrasound showed mobile lesions at the graft wall in the absence of periprosthetic fluid . Immediately after the procedure the patient developed several small cutaneous septic infarctions on both feet . At operation the presence of graft infection was confirmed . CONCLUSIONS: This case report suggests that intra-aortic US may constitute a helpful adjunctive modality in suspected atypical infection of prosthetic aortic grafts. Biochem Mol Biol Int, 1998 Jun, 45(2), 289 - 301 Purification of recombinant human pepsinogens and their application as immunoassay standards; Aoki T et al.; Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable . When there were fused to maltose-binding protein (MBP), the fusion proteins (MBP-PGA and MBP-PGC) were expressed as the major products . Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple . MBP-PGA and the PGA segment obtained by factor Xa digestion (designated as r-PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t-PGA) . For PGCs (MBP-PGC, r-PGC and t-PGC) also, the specific activities were almost the same . However, the activities of PGCs were about 3- to 4-hold higher than those of PGAs . In PGA and PGC immunoassay systems, r-PGs (r-PGA and r-PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation . From these results, r-PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems. Biochem Mol Biol Int, 1998 Jun, 45(2), 255 - 60 Hydroxyl group of insulin A19Tyr is essential for receptor binding: studies on (A19Phe)insulin; Du X et al.; One mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe . The mutant proinsulin, (A19Phe)-lysproinsulin (F19KPI) was expressed in E . coli and purified . After trypsin and carboxypeptidase B cleavage and Resource Q separation, (A19Phe)-human insulin (F19HI) was obtained . With native insulin as standard, activity assay and structural analysis were carried out . It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse-phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay . F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity . These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding. Biochem Mol Biol Int, 1998 Jun, 45(2), 215 - 25 Cross-species reactivity of a monoclonal antibody against glutathione S-transferase fusion protein of human beta 2-adrenergic receptor; Shin CY et al.; The purpose of the present study was to produce and characterize a monoclonal antibody against human beta 2-adrenergic receptor . Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human beta 2-adrenergic receptor which was expressed in E.Coli . The immunized splenocytes were fused with myeloma SP2/0-Ag14 cells and the resulting monoclonal antibody was named as mAb beta C02 . The monoclonal antibody beta C02 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography . The results of ELISA, Western blot, and immunocytochemistry showed that mAb beta C02 recognized human beta 2-adrenergic receptor in the beta 2-adrenergic receptor-GST fusion protein and human epidermoid carcinoma cell line A431 with highly specific immunoreactivity . In addition, mAb beta C02 showed cross-species reactivity against beta-adrenergic receptor of hamster lung and rat brain as revealed by Western blot and immunohistochemistry . The monoclonal antibody beta C02 may provide useful tools for the study of the beta-adrenergic receptor of human and other species including rats. J Nat Toxins, 1998 Jun, 7(2), 159 - 72 Purification, cDNA cloning and molecular characteristic of a fibrinolytic enzyme from the venom of Agkistrodon acutus; Du XY et al.; A nonglycoprotein-like fibrinolytic enzyme ((FIB-I) was purified from the crude venom of Agkistrodon acutus by CM-Sepharose CL-6B and DEAE-Sepharose CL-6B ion exchange chromatography and then by FPLC through Superose 12 gel filtration . Its molecular weight is about 23 kDa and isoelectric point is near 6.0 . It not only has fibrinolytic and caseinolytic activity, but also can hydrolyze BAEE . The local hemorrhagic activity was found in mice after the subcutaneous injection of this enzyme . EDTA can inhibit its fibrinolytic activity completely, but PMSF and arrowhead proteinase inhibitor have no such obvious inhibitory effect, thus implying that FIB-I is a metalloproteinase . The N-terminal ten amino acid residues 'STEFQRYMEI' of FIB-I was elucidated . A full-length cDNA gene of this enzyme was cloned by using RT-PCR from the total RNA extracted from the snake venom gland and FIB-I was expressed in E . coli . Having analyzed the sequence, we found that it had a typical zinc-chelating characteristic as 'HEXXHXXGXXHD.' J Food Prot, 1998 Jul, 61(7), 913 - 6 Enumeration of beta-glucuronidase-positive Escherichia coli in foods by using the ISO-GRID method with SD-39 agar; Entis P et al.; A study was undertaken to compare beta-glucuronidase-positive Escherichia coli counts produced by the ISO-GRID hydrophobic grid membrane filter method using SD-39 agar (test method) with those produced by AOAC Official Method 990.11, an existing ISO-GRID method using lactose monensin glucuronate agar and buffered MUG agar (reference method) . The methods were evaluated using 21 food products, with three independent lots of five replicate samples analyzed per product by both methods . The test and reference methods were statistically equivalent for 19 of the 21 products; frozen, raw ground lamb produced significantly higher counts using the reference method, whereas counts obtained from cottage cheese were significantly higher using the SD-39 agar-based method. Mutat Res, 1998 Jul, 408(1), 19 - 25 MNNG-induced {corrected} RecBCD dependent DNA degradation in recA13 mutant cells is not the basis of their hypersensitivity to this agent; Chovanec M et al.; We have examined the hypersensitivity of Escherichia coli recA13 mutant cells to killing by N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and have shown out that despite MNNG-induced adaptation they remained vastly more sensitive to the cytotoxic effect of this agent than wild type cells . Because this might have been a consequence of a different extent of induction of the adaptive response in the recA13 background, we have measured O6-alkylguanine-DNA alkyltransferase (ATase) activity in extracts of adapted and non-adapted recA13 mutant and wild type cells . Adaptation increased ATase levels by 28- and 34-fold in wild type and recA13 mutant cells, respectively . Thus, the adaptive response was no less inducible in recA13 mutant cells than in wild type cells . This indicates that the extreme sensitivity of recA13 cells to MNNG is not caused by an inability to repair the principal toxic lesions induced in DNA . Low doses of MNNG caused substantial degradation of cellular DNA in recA13 mutant cells but not in the wild type cells . This DNA degradation is shown to be the RecBCD-enzyme dependent . Since recA13 recB21 double mutants were even more sensitive to MNNG than recA single mutants, DNA degradation appears not to be the cause of the MNNG-hypersensitivity in recA13 cells. J Biol Chem, 1998 Jul 31, 273(31), 19895 - 901 Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha; Genschel J et al.; A human MSH2-human MSH3 (hMSH2.hMSH3) complex of approximately 1:1 stoichiometry (human MutSbeta (hMutSbeta)) has been demonstrated in several human tumor cell lines and purified to near homogeneity . In vitro, hMutSbeta supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches . Human MutSalpha (hMutSalpha), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSbeta . Thus, the repair specificities of hMutSalpha and hMutSbeta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides . The hMutSalpha level in repair-proficient HeLa cells (1.5 microg/mg nuclear extract) is approximately 10 times that of hMutSbeta . In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSalpha, the hMutSbeta level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line . LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSalpha and hMutSbeta, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells . Coupled with previous findings (J . T . Drummond, J . Genschel, E . Wolf, and P . Modrich (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation. J Biol Chem, 1998 Jul 31, 273(31), 19859 - 65 Crystal structure of recombinant soybean beta-amylase complexed with beta-cyclodextrin; Adachi M et al.; In order to study the interaction of soybean beta-amylase with substrate, we solved the crystal structure of beta-cyclodextrin-enzyme complex and compared it with that of alpha-cyclodextrin-enzyme complex . The enzyme was expressed in Escherichia coli at a high level as a soluble and catalytically active protein . The purified recombinant enzyme had properties nearly identical to those of native soybean beta-amylase and formed the same crystals as the native enzyme . The crystal structure of recombinant enzyme complexed with beta-cyclodextrin was refined at 2 . 07-A resolution with a final crystallographic R value of 15.8% (Rfree = 21.1%) . The root mean square deviation in the position of C-alpha atoms between this recombinant enzyme and the native enzyme was 0.22 A . These results indicate that the expression system established here is suitable for studying structure-function relationships of beta-amylase . The conformation of the bound beta-cyclodextrin takes an ellipsoid shape in contrast to the circular shape of the bound alpha-cyclodextrin . The cyclodextrins shared mainly two glucose binding sites, 3 and 4 . The glucose residue 4 was slightly shifted from the maltose binding site . This suggests that the binding site of the cyclodextrins is important for its holding of a cleaved substrate, which enables the multiple attack mechanism of beta-amylase. J Biol Chem, 1998 Jul 31, 273(31), 19847 - 52 Ribosomal protein L27 participates in both 50 S subunit assembly and the peptidyl transferase reaction; Wower IK et al.; Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations . To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker . The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive . This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene . Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits . Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27 . In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes . Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed . We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center. J Biol Chem, 1998 Jul 31, 273(31), 19729 - 39 Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli; Whitby MC et al.; The RecG protein of Escherichia coli is a junction-specific DNA helicase that drives branch migration of Holliday intermediates in genetic recombination and DNA repair . The reaction was investigated using synthetic X-junctions . RecG dissociates X-junctions to flayed duplex products, although DNA unwinding of the heterologous arms is limited to </=30 base pairs . Junction unwinding requires Mg2+ and the hydrolysis of ATP . X-junction DNA stimulates the ATPase activity of RecG . ATPase activity is also stimulated by linear duplex DNA, although to a lesser extent than by X-DNA, but not by linear single-stranded DNA . In situ 1,10-phenanthroline-copper footprinting shows that RecG binds to the strand cross-over point at the center of the X-junction . Substrate recognition by RecG was investigated using DNAs that represented the various component parts of an X-junction . The minimal DNA structure that RecG forms a stable complex with is a flayed duplex, suggesting that this is the critical feature for junction recognition by RecG . Junction binding and unwinding also depend critically on the concentration of free Mg2+, excess free cation dramatically inhibiting both processes . These inhibitory effects are not mediated specifically by Mg2+; e.g . both Ca2+ and hexamminecobalt(III) chloride also inhibit X-junction binding and unwinding by RecG . The relative abilities of these cations to inhibit RecG-junction binding is correlated with their respective abilities to stack X-junction DNA . From this we conclude that RecG is unable to bind or binds very poorly to fully stacked X-junctions. J Biol Chem, 1998 Jul 31, 273(31), 19532 - 41 Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases; Wells RD et al.; Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG.CAG, CGG.CCG, and GAA.TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli . The presence of G to A polymorphisms in the CTG.CAG sequences served as reporters for the size and location of these instabilities . For the other two repeat sequences, length determinations confirmed the conclusions found for CTG.CAG . These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS . The observation that small and large instabilities for (CTG.CAG)175 fall into distinct size classes (1-8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved . The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells . A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation . This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions . Furthermore, these observations may relate to the high frequency of small deletions versus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients. J Biol Chem, 1998 Jul 31, 273(31), 19391 - 7 Three thiol groups are important for the activity of the liver microsomal glucose-6-phosphatase system . Unusual behavior of one thiol located in the glucose-6-phosphate translocase; Clottes E et al.; Liver microsomal glucose-6-phosphatase (Glc-6-Pase) is a multicomponent system involving both substrate and product carriers and a catalytic subunit . We have investigated the inhibitory effect of N-ethylmaleimide (NEM), a rather specific sulfhydryl reagent, on rat liver Glc-6-Pase activity . Three thiol groups are important for Glc-6-Pase system activity . Two of them are located in the glucose-6-phosphate (Glc-6-P) translocase, and one is located in the catalytic subunit . The other transporters (phosphate and glucose) are not affected by NEM treatment . The NEM alkylation of the catalytic subunit sulfhydryl residue is prevented by preincubating the disrupted microsomes with saturating concentrations of substrate or product . This suggests either that the modified cysteine is located in the protein active site or that substrate binding hides the thiol group via a conformational change in the enzyme structure . Two other thiols important for the Glc-6-Pase system activity are located in the Glc-6-P translocase and are more reactive than the one located in the catalytic subunit . The study of the NEM inhibition of the translocase has provided evidence of the existence of two distinct areas in the protein that can behave independently, with conformational changes occurring during Glc-6-P binding to the transporter . The recent cloning of a human putative Glc-6-P carrier exhibiting homologies with bacterial phosphoester transporters, such as Escherichia coli UhpT (a Glc-6-P translocase), is compatible with the fact that two cysteine residues are important for the bacterial Glc-6-P transport. Biochem J, 1998 Aug 1, 333 ( Pt 3), 765 - 77 Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants; Chang AK et al.; Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides . Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified . Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides . Each enzyme had a pH optimum near 7.5 . The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher . The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined . A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS . Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds . In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold . All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. Biochem J, 1998 Aug 1, 333 ( Pt 3), 671 - 6 Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli; Kim KI et al.; The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity . To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu . All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM . However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP . Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93 . The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve . Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93 . In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present . Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93 . Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could . Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93 . These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures. Biochem J, 1998 Aug 1, 333 ( Pt 3), 527 - 37 Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme; Persson K et al.; All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed . The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine . We have now identified the gene encoding AdoMetDC in T . cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids . The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T . cruzi genomic library . The AdoMetDC gene was located on chromosomes with a size of approx . 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues . The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T . cruzi enzyme . As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs . When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit . The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine . Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T . cruzi enzyme . This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T . cruzi AdoMetDC. J Mol Biol, 1998 Jul 24, 280(4), 721 - 9 Eye lens betaB2-crystallin: circular permutation does not influence the oligomerization state but enhances the conformational stability; Wieligmann K et al.; The related vertebrate eye lens polypeptides, betaB2- and gammaB-crystallin, each fold into two similar beta-sheet domains . The main difference is the state of oligomerization resulting from intermolecular domain interactions in the oligomeric beta-crystallins and intramolecular contacts in the monomeric gamma-crystallins . The question arises whether it is possible to create a monomeric gammaB-like betaB2-molecule by protein engineering methods . We wanted to produce such a molecule by circularly permuting the domains of betaB2-crystallin . The new termini were created from the original connecting peptide, and the new linker from stumps of the original extensions, while the rest of the flexible extensions were deleted . As judged by circular dichroism and fluorescence, the permutation causes little change in the structure of the protein . The circularly permuted protein forms dimers as wild-type betaB2-crystallin . On the other hand, cpbetaB2 shows a slightly enhanced stability against urea with a midpoint of transition of 2.1 M urea versus 1.9 M for the wild-type protein lacking N and C-terminal arms, thus indicating stronger domain interactions . To our knowledge this is the first circularly permuted protein which exhibits a higher stability than the corresponding wild-type protein . J Mol Biol, 1998 Jul 24, 280(4), 687 - 701 The NMR solution structure of human glutaredoxin in the fully reduced form; Sun C et al.; The determination of the nuclear magnetic resonance (NMR) solution structure of fully reduced human glutaredoxin is described . A total of 1159 useful nuclear Overhauser effect (NOE) upper distance constraints and 187 dihedral angle constraints were obtained as the input for the structure calculations for which the torsion angle dynamics program DYANA has been utilized followed by energy minimization in water with the AMBER force field as implemented in the program OPAL . The resulting 20 conformers have an average root-mean-square deviation value relative to the mean coordinates of 0.54 A for all the backbone atoms N, Calpha and C', and of 1.01 A for all heavy atoms . Human glutaredoxin consists of a four-stranded mixed beta-sheet composed of residues 15 to 19, 43 to 47, 72 to 75 and 78 to 81, and five alpha-helices composed of residues 4 to 9, 24 to 34, 54 to 65, 83 to 91, and 94 to 100 . Comparisons with the structures of Escherichia coli glutaredoxin-1, pig liver glutaredoxin and human thioredoxin were made . Electrostatic calculations on the human glutaredoxin structure and that of related proteins provide an understanding of the variation of pKa values for the nucleophilic cysteine in the active site observed among these proteins . In addition, the high-resolution NMR solution structure of human glutaredoxin has been used to model the binding site for glutathione and for ribonucleotide reductase B1 by molecular dynamics simulations . J Mol Biol, 1998 Jul 24, 280(4), 655 - 68 Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex; Knapp JE et al.; The dihydrolipoamide succinyltransferase (E2o) component of the 2-oxoglutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry . The catalytic domain (CD) of the E2o component catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A . The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 A resolution using molecular replacement phases derived from the structure of the catalytic domain from the Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2pCD) . The refined model of the E . coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (Rfree=0.249) for 9696 reflections between 20.0 and 3.0 A resolution . Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in comparison to the less stable A . vinelandii E2pCD 24mer . Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two 3-fold related subunits . Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT . A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from its counterpart in CAT (Asp199) . Binding of the substrates to E2oCD is proposed to induce a change in the conformation of Asp379, allowing this residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT . Computer models of the active site of E2o complexed with dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket . The residues which form this pocket (Ser330, Ser333, and His348) are probably responsible for E2o's substrate specificity . J Mol Biol, 1998 Jul 24, 280(4), 639 - 54 NMR structure determination of the binding site for ribosomal protein S8 from Escherichia coli 16 S rRNA; Kalurachchi K et al.; Many cellular processes involve the preferential interaction of an RNA molecule with a specific protein . A detailed analysis of the individual protein and RNA components of these interactions can provide unique insights into the structural features important for protein-RNA recognition . Ribosomal protein S8 of Escherichia coli plays a key role in 30 S ribosomal subunit assembly through its interaction with 16 S rRNA . The binding site for protein S8 comprises a portion of helix 21, nucleotides G588 to G604 and C634 to C651 . This region forms a base-paired helix that is interrupted by a non-Watson-Crick segment composed of nine phylogenetically conserved nucleotides . We have investigated the detailed structure of the conserved segment and the interaction of this region with metal ions using NMR spectroscopy . Twenty-four of the 40 calculated structures converged to similar conformations and were grouped into two families . The main difference between the families is the orientation of the base of U641 . The rms deviation between the heavy-atoms of the ten lowest-energy structures is 1.24 A . The orientations of the G597.C643 base-pair and A595.(A596.U644) base-triple within the conserved core have been defined and appear to extend the proximal segment of helix 21 into the phylogenetically conserved core . The base of A642 terminates this helix by stacking against C643 and the base of U641 forms hydrogen bonds with core nucleotides . The conserved core also contains a Mg2+-binding site that promotes stabilization of the secondary and tertiary structure elements of the core . A model for the interaction of S8 with its RNA-binding site is proposed . J Mol Biol, 1998 Jul 24, 280(4), 561 - 9 Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo; Sorensen MA et al.; We have deleted the chromosomal rpsA gene, encoding ribosomal protein S1, from an Escherichia coli strain carrying a plasmid where rpsA was controlled by the lac promoter and operator . This exogenous source of protein S1 was essential for growth . Thus we have verified the absolute requirement for protein S1 . To see if translation of individual mRNAs differed in the requirements for protein S1, we removed the inducer and followed the time-course of the synthesis of several individual proteins and of total RNA, DNA and protein . Growth immediately shifted from being exponential to being linear, with a rate of protein synthesis defined by the pre-existing amount of protein S1 . The expression pattern of the individual proteins indicated that the translation of all mRNAs was dependent on protein S1 . Unexpectedly, we found that depletion for protein S1 for extended periods introduced a starvation for amino acids . Such starvation was indicated by an increased synthesis of ppGpp and could be reversed by addition of a mixture of all 20 amino acids . Measurements of the peptide chain elongation rate in vivo showed that ribosomes without protein S1 were unable to interfere with the peptide chain elongation rate of the active ribosomes and that, therefore, protein S1 was unable to diffuse from one ribosome to another during translation . We conclude that protein S1-deficient ribosomes are totally inactive in peptide chain elongation on most, if not all, naturally occurring E . coli mRNAs . Protein Expr Purif . 1998 Jul;13(2):IV. Papers to appear in forthcoming issues Overproduction and one-step purification of the N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri. Max-Planck-Institut fur terrestrische Mikrobiologie, Fachbereich Biologie, Philipps-Universitat, Marburg, GermanyN5,N10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO2 and H2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98 degrees C . The gene mch from M . kandleri was cloned, sequenced, and expressed in Escherichia coli . The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate . From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained . The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M . kandleri cells . The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85 degrees C), from Methanobacterium thermoautotrophicum (65 degrees C), and from Methanosarcina barkeri (37 degrees C) . Of the four enzymes, that from M . kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition . Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine . Some of these properties are unusual for enzymes from hyperthermophilic organisms . They may reflect the observation that the cyclohydrolase from M . kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism. Anim Biotechnol, 1998, 9(1), 1 - 14 Partial cloning and expression of the bovine leptin gene; Ji S et al.; The product of the leptin (i.e., obese) gene may be an important regulator of energy metabolism, adiposity, and reproduction, and is perhaps linked to meat quality determinants such as marbling . Molecular probes were developed using polymerase chain reaction (PCR) technology to evaluate leptin expression in adipose depots and to evaluate the tissue-dependent nature of expression reported in other species . A 438 bp fragment representing the coding region of the bovine leptin gene excluding the N-terminal secretory signal was amplified, cloned into a plasmid vector (pASK75), and expressed in E . coli . Sequence analysis of the cDNA and the corresponding polypeptide indicate that, overall, both share approximately 87% homology with the mouse and human leptin genes and polypeptides . Amino terminal sequencing (30 amino acid residues) of the recombinant bovine leptin (rBL) protein revealed 100% homology with mouse and human leptin . The bovine leptin gene is expressed as a 3,090 nt mRNA which is detected in adipose tissue, but is not found in brain (despite the appreciable fat content and lipid metabolism) or other tissues . Leptin gene expression in several adipose depots (subcutaneous, renal, and omental) was similar (P = .73) in finished cattle. Plant J, 1998 Jun, 14(5), 613 - 22 Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli; Knight ME et al.; A full length cDNA clone encoding a starch synthase (zSS) from maize endosperm (inbred line W64) was isolated and characterized . The cDNA clone (Ss1) is 2907 bp in length and contains an open reading frame of 1866 bp corresponding to a polypeptide of 622 amino acid residues including a transit peptide of 39 amino acids . The Ss1 cDNA clone was identified as zSSI by its direct alignment with sequences to: (i) the N-terminus obtained from the granule-associated form of the zSSI polypeptide, (ii) four internal peptide fragments obtained from the granule-associated form of the zSSI protein, and (iii) one internal fragment from the soluble form of the zSSI protein . The deduced amino acid sequence of Ss1 shares 75.7% sequence identity with rice soluble Ss and contains the highly conserved KSGGLGDV putative ADP-Glc binding site . Moreover, Ss1 exhibited significant activity when expressed in E . coli and the expressed protein is recognized by the antibody raised against the granule associated zSSI protein . Ss1 transcripts were detected in endosperm beginning at 15 days after pollination, but were not found in embryo, leaf or root . Maize contains a single copy of the Ss1 gene, which maps close to the Waxy locus of chromosome 9. FEMS Microbiol Lett, 1998 Jul 1, 164(1), 125 - 31 Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein; Pereira CM et al.; The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats . Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen . Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera . The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera . This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8. Trends Microbiol, 1998 Jun, 6(6), 228 - 33 Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk; Tesh VL; Many bacterial exotoxins, originally defined by cytopathic effects, may also possess additional biological activities . The capacity of exotoxins to elicit the synthesis and secretion of pro- or anti-inflammatory cytokines may be as important as their direct toxic effects in pathogenesis . One example of such 'molecular crosstalk' occurs between Shiga toxins and the cytokines made in response to these toxins during the development of disease. Carbohydr Res, 1998 Feb, 307(3-4), 375 - 9 Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from {14C} maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase; Pajatsch M et al.; Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism . 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues . The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase . The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K . oxytoca M5a1. Carbohydr Res, 1998 Feb, 307(3-4), 233 - 42 Synthesis of some amino and carboxy analogs of galabiose; evaluation as inhibitors of the pilus protein PapGJ96 from Escherichia coli; Hansen HC et al.; The 2'-amino-2'-deoxy, 6-amino-6-deoxy, and 6-carboxy analogs of the reference inhibitors 2-(trimethylsilyl)ethyl (alpha-D-galactopyranosyl)-(1-->4)-ss-D-galactopyranoside were synthesized and evaluated as inhibitors of the binding of the Escherichia coli-derived pilus protein PapGj96, using an ELISA assay . The inhibitory efficiencies (Krel; relative to the reference inhibitor) were: 157,13, and < 8, respectively . The results support the previously proposed combining site model, where the protein carries a negatively charged amino acid residue near HO-2' and HO-6 of the galabio-side. Biochim Biophys Acta, 1998 Jul 17, 1372(2), 311 - 22 Effect of cysteine replacements on the properties of the turgor sensor KdpD of Escherichia coli; Jung K et al.; Escherichia coli responds rapidly to K+-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase . This process is controlled by the membrane-bound histidine kinase KdpD and the response regulator KdpE . Here, it is demonstrated that replacements of the native Cys residues at positions 409, 852, and 874 influence distinct activities of KdpD, whereas replacements of Cys residues at positions 32, 256, and 402 have no effect . Replacements of Cys409 in KdpD reveal that transmembrane domain I is important for perception and/or propagation of the stimulus . When Cys409 is replaced with Ala, kdpFABC expression becomes constitutive regardless of the external stimuli . In contrast, when Cys409 is replaced with Val or Tyr, induction of kdpFABC expression in response to different stimuli is drastically reduced . KdpD with Ser at position 409 supports levels of kdpFABC expression comparable to those seen in wild-type . Since neither the kinase nor phosphatase activity of these proteins is affected, it is proposed that different amino acid side-chains at position 409 alter the switch between the inactive and active forms of the kinase . When Cys852 or Cys874 is replaced with Ala or Ser, kinase activity is reduced to 10% of the wild-type level . However, kinetic studies reveal that the apparent ATP binding affinity is not affected . Surprisingly, introduction of Cys852 and Cys874 into a KdpD protein devoid of Cys residues leads to full recovery of the kinase activity . Labeling studies support the idea that a disulfide bridge forms between these two residues. Biochim Biophys Acta, 1998 Jul 28, 1386(1), 179 - 88 Purification and characterisation of the phosphoglycerate kinase isoenzymes of Trypanosoma brucei expressed in Escherichia coli; Zomer AW et al.; The Trypanosoma brucei phosphoglycerate kinase (PGK) glycosomal and cytosolic isoenzymes have been overexpressed in Escherichia coli and purified to near-homogeneity . Both enzymes were similar to the corresponding natural proteins with respect to their physicochemical and kinetic properties . In addition, a mutant of the glycosomal PGK lacking the 20 amino acid long C-terminal extension was overexpressed and purified . Various properties of this truncated glycosomal PGK were examined and it was found that in some aspects the protein behaved quite differently when compared with its natural counterpart . This was notably the case for the apparent Km for 3-phosphoglyceric acid, its sensitivity to inhibitors and its response to salts and guanidine HCl . However, its Vmax was found to be similar to that of the natural glycosomal PGK . These results suggest that the changes in the C-terminus caused a conformational change effecting the 3-phosphoglyceric acid binding site located at the N-terminal domain of the protein. Biochim Biophys Acta, 1998 Jul 28, 1386(1), 121 - 31 Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity; Vorum H et al.; By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No . IEF SSP 9302 as the human homologue of calumenin . The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin . The deduced protein contains a 19 amino acid N-terminal signal sequence, 7 EF-hand domains and, at the C-terminus, a HDEF sequence which has been reported to function as retrieval signal to the ER . The calumenin transcript is ubiquitously expressed in human tissue, at high levels in heart, placenta and skeletal muscle, at lower levels in lung, kidney and pancreas and at very low levels in brain and liver . Calumenin belongs to a family of multiple EF-hand proteins that include the ER localized proteins reticulocalbin and ERC-55 and the Golgi localized Cab45 . Since its Ca2+ binding may be important for the function of the protein we have used microdialysis experiments in order to analyse for the affinity and the capacity of recombinant human (rh) calumenin . All 7 EF-hands of the protein are functional and bind Ca2+, each with an affinity of 1.6x103 M-1 . The relatively low affinity for the EF-hands may suggest a role for the protein in Ca2+-dependent processes in the ER. Mutat Res, 1998 Jun 18, 402(1-2), 93 - 102 Antimutagenic role of base-excision repair enzymes upon free radical-induced DNA damage; Laval J et al.; As a consequence of oxidative stress, reactive oxygen species are generated in the cells . They interact with DNA and induce various modifications . Among them, oxidised purines (such as C8-oxoguanine and purines whose imidazole ring is opened), oxidised pyrimidines (such as thymine and cytosine glycols, ring saturated and fragmented pyrimidines), ethenobases and hypoxanthine . These various lesions have either miscoding properties or are blocks for DNA and RNA polymerases during replication and transcription, respectively . Most of these lesions are repaired by the base excision pathway in which the first step