J Biol Chem, 2001 Apr 20, 276(16), 12614 - 23 Epub 2001 Jan 18. Secoisolariciresinol dehydrogenase purification, cloning, and functional expression . Implications for human health protection; Xia ZQ et al.; Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin . In this study, the approximately 32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity . The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F . intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate . A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of approximately 32 kDa . Expression of this protein in E . coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol via the intermediary lactol . Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting . With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated . Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers. J Biol Chem, 2001 May 11, 276(19), 16501 - 10 Epub 2001 Jan 29. Oxidation of thymine to 5-formyluracil in DNA promotes misincorporation of dGMP and subsequent elongation of a mismatched primer terminus by DNA polymerase; Masaoka A et al.; 5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species . In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential . Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease . Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion . However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently . No incorporation of dCMP and dTMP was observed . When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold . Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold . The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G . The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold) . These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic. J Biol Chem, 2001 May 18, 276(20), 17541 - 9 Epub 2001 Feb 14. Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato; Wang TW et al.; Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv . Match) exposed to environmental stress . DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine . Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A . Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death . Western blot analyses indicated that DHS protein also increases at the onset of senescence . It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division . The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death. J Biol Chem, 2001 Apr 27, 276(17), 14117 - 23 Epub 2001 Jan 30. Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling; Montesano-Roditis L et al.; The Escherichia coli ribosomal protein L7/L12 is central to the translocation step of translation, and it is known to be flexible under some conditions . The assignment of electron density to L7/L12 was not possible in the recent 2.4 A resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J., Moore, P . B., and Steitz, T . A . (2000) Science 289, 905-920) . We have localized the two dimers of L7/L12 within the structure of the 70 S ribosome using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction . First, the structures were determined for ribosomal cores from which protein L7/L12 had been removed by treatment with NH(4)Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles . Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11 . Second, ribosomes were reconstituted using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 A gold derivative . The reconstruction from cryo-electron microscopy images and difference mapping placed the gold at four interfacial positions . The finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of elongation and translocation. J Biol Chem, 2001 May 25, 276(21), 18272 - 81 Epub 2001 Feb 08. Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins; Liendo A et al.; Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion . We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D . L., Hanson, P., Rodriguez, A., and Joiner, K . A . (1998) J . Biol . Chem . 274, 2424-2431) . As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system . To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T . gondii ARF1 (TgARF1) was isolated . Endogenous and transgenic TgARF1 localized to the Golgi of T . gondii, but not to dense granules . An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location . Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker . TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules . To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay . TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay . These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane. J Biol Chem, 2001 Jun 1, 276(22), 19259 - 66 Epub 2001 Mar 02. A mechanistic model for Ncd directionality; Foster KA et al.; Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules . Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism . Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other . Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s(-1), yet rate-limiting ADP release from the second motor domain occurs at 1.4 s(-1) . We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule.Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minus-end of the microtubule . The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head . ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule . The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor. J Biol Chem, 2001 May 4, 276(18), 14710 - 7 Epub 2001 Feb 02. Interactions of the human T-cell leukemia virus type-II integrase with the conserved CA in the retroviral long terminal repeat end; Wang T et al.; Retroviral integrases (INs) interact with termini of retroviral DNA in the conserved 5'-C(A/G)T . For most integrases, modifications of critical moieties in the major and minor grooves of these sequences decrease 3'-processing . However, for human immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the guanine with 6-methylguanine or hypoxanthine not only reduced 3'-processing, but also promoted cleavage at a second site . This novel cleavage activity required an upstream ACA, unique to the HTLV-2 U5 end . 3'-Processing assays with additional isosteric modifications at Gua and filter binding experiments revealed that the mechanism of the second site cleavage differed among the major groove, minor groove, and mismatch modifications . Importantly, the decrease in 3'-processing activity noted with the minor groove and mismatch modifications were attributed to a decrease in binding . Major groove modifications, however, decreased the level of 3'-processing, but did not affect binding . This suggests that integrase binds the viral end through the minor groove, but relies on major groove contacts for 3'-processing . Several modifications were also examined in strand transfer and disintegration substrates . HTLV-2 IN showed reduced activity with strand transfer and disintegration substrates containing major groove, but not minor groove modifications . This suggests major groove interactions at guanine also provide an important role in these reactions. J Biol Chem, 2001 Apr 27, 276(17), 13563 - 72 Epub 2001 Jan 16. Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers; Ortega A et al.; The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy . The calorimetric properties were compared with those of the isolated pore-forming fragment . The unfolding profile of the full-length toxin is consistent with two endothermic transitions . Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain . By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation . The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation . In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%) . Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature . In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure . The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions. J Biol Chem, 2001 Jun 1, 276(22), 18843 - 8 Epub 2001 Mar 13. Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone; Banecki B et al.; The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type . The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone . Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations . Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP . We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein . Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP . ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis . We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid. J Biol Chem, 2001 May 25, 276(21), 18265 - 71 Epub 2001 Jan 16. Cloning and biological activity of epigen, a novel member of the epidermal growth factor superfamily; Strachan L et al.; High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors . We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen . Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily . Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites . Epigen shows 24-37% identity to members of the EGF superfamily including EGF, transforming growth factor alpha, and Epiregulin . Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver . Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor alpha in several assays . In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter . Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1 . Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy. J Biol Chem, 2001 Apr 13, 276(15), 11461 - 4 Epub 2001 Feb 22. An Escherichia coli mutant defective in lipid export; Doerrler WT et al.; Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized . We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA . As shown by (32)P(i) and (14)C-acetate labeling, export of all major lipids to the outer membrane is inhibited by approximately 90% in WD2 after 30 min at 44 degrees C . Transport of newly synthesized proteins is not impaired . Electron microscopy shows reduplicated inner membranes in WD2 at 44 degrees C, consistent with a key role for MsbA in lipid trafficking. Vet Microbiol, 2001 May 3, 80(1), 75 - 83 Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis; Chansiripornchai N et al.; Here we describe the application of a random amplified polymorphic DNA (RAPD) analysis for molecular genetic typing avian pathogenic Escherichia coli (APEC) strains . The RAPD technique was shown to be highly reproducible . Stable banding patterns with a high discriminatory capacity were obtained using two different primers . Overall, 55 E . coli strains were analyzed with a RAPD technique . The RAPD analysis showed that the E . coli strains isolated from poultry in Thailand and Sweden could be grouped into 50 of RAPD types by using these two different primer sets . Most of these different E . coli RAPD types were not geographically restricted . There was, as expected, a tendency of higher genetic relationship among E . coli strains isolated from the same farm . It is suggested that the RAPD technique may provide a rapid, low cost, simple and powerful tool to study the clonal epidemiology of avian E . coli infections. Biochimie, 2001 Feb, 83(2), 251 - 9 Mutagenesis of the downstream region of the Escherichia coli hns promoter; Giangrossi M et al.; The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene . To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence . The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small . Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels. Biochimie, 2001 Feb, 83(2), 231 - 4 Structural basis for preferential binding of H-NS to curved DNA; Dame RT et al.; The Escherichia coli H-NS protein is a nucleoid-associated protein involved in transcription regulation and DNA compaction . H-NS exerts its role in DNA condensation by non-specific interactions with DNA . With respect to transcription regulation preferential binding sites in the promoter regions of different genes have been reported . In this paper we describe the analysis of H-NS-DNA complexes on a preferred H-NS binding site by atomic force microscopy . On the basis of these data we present a model for the specific recognition of DNA by H-NS as a function of DNA curvature. Biochimie, 2001 Feb, 83(2), 213 - 7 DNA supercoiling and transcription in Escherichia coli: The FIS connection; Travers A et al.; The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity . We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase . In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density . On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter . We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density. Biochimie, 2001 Feb, 83(2), 201 - 12 Genome organisation and chromatin structure in Escherichia coli; Ussery D et al.; We have analysed the complete sequence of the Escherichia coli K12 isolate MG1655 genome for chromatin-associated protein binding sites, and compared the predicted location of predicted sites with experimental expression data from 'DNA chip' experiments . Of the dozen proteins associated with chromatin in E . coli, only three have been shown to have significant binding preferences: integration host factor (IHF) has the strongest binding site preference, and FIS sites show a weak consensus, and there is no clear consensus site for binding of the H-NS protein . Using hidden Markov models (HMMs), we predict the location of 608 IHF sites, scattered throughout the genome . A subset of the IHF sites associated with repeats tends to be clustered around the origin of replication . We estimate there could be roughly 6000 FIS sites in E . coli, and the sites tend to be localised in two regions flanking the replication termini . We also show that the regions upstream of genes regulated by H-NS are more curved and have a higher AT content than regions upstream of other genes . These regions in general would also be localised near the replication terminus. Biochimie, 2001 Feb, 83(2), 193 - 200 HU-GFP and DAPI co-localize on the Escherichia coli nucleoid; Wery M et al.; The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria . Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli . By its properties HU shares some traits with histones and HMG proteins . More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor . For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells . To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining . We show that DAPI and HU-GFP colocalize on the E . coli nucleoid . HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell. Biochimie, 2001 Feb, 83(2), 171 - 6 DNA degradation in the terminus region of resolvase mutants of Escherichia coli, and suppression of this degradation and the Dif phenotype by recD; Prikryl J et al.; We recently proposed that guillotining of dimer chromosomes occurs at cell division in resolvase mutants of Escherichia coli . This was based on the abnormal pattern of cell division observed in 10-14% of the cells in microcolonies of xerC, xerD and dif mutants . A prediction of this guillotining is that DNA degradation should occur in the terminus region, in the vicinity of the dif locus . We have tested this by DNA-DNA hybridization and have observed that dif was absent in about 22% of the chromosomes in exponentially growing xerC mutants . A locus 206 kb from dif was not affected by this degradation . We have also observed that degradation did not occur in xerC recD mutants, and that the low efficiency of plating associated with the Dif phenotype was suppressed in this strain . A model is proposed in which rapid degradation of the terminus region does not occur in recD mutants following guillotining, and that this permits the initiation of repair of broken dimer chromosomes prior to completion of cell division. Biochimie, 2001 Feb, 83(2), 161 - 70 Polarization of the Escherichia coli chromosome . A view from the terminus; Capiaux H et al.; The E . coli chromosome replication arms are polarized by motifs such as RRNAGGGS oligomers, found preferentially on leading strands . Their skew increases regularly from the origin to dif (the site in the center of the terminus where chromosome dimer resolution occurs), to reach a value of 90% near dif . Convergent information indicates that polarization in opposite directions from the dif region controls tightly the activity of dif, probably by orienting mobilization of the terminus at cell division . Another example of polarization is the presence, in the region peripheral to the terminus, of small non-divisible zones whose inversion interferes with spatial separation of sister nucleoids . The two phenomena may contribute to the organization of the Ter macrodomain. Biochimie, 2001 Feb, 83(2), 149 - 54 Isolation of the Escherichia coli nucleoid; Cunha S et al.; Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine) . Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate . To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions . Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments . After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations . The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively . RNase treatment caused no dramatic changes in the size of either nucleoid. FEBS Lett, 2001 Mar 23, 493(1), 17 - 20 An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease; Bertani D et al.; The proteolysis of regulatory proteins plays an important role in the control of gene expression . The Escherichia coli heat shock sigma factor RpoH (sigma(32)) is highly unstable . Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH . Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH(1) is highly stable . To determine which regions of E . coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins . Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH . A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36-122) roughly comprising the sigma factor region 2 . This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation. FEBS Lett, 2001 Mar 23, 493(1), 12 - 6 Effect of divalent cations on the ATPase activity of Escherichia coli SecA; Kim J et al.; It was found that Ca(2+) stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome . On the other hand, Mg(2+), the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca(2+) . The enhancement of SecA ATPase activity correlated well with the increase in 8-anilino-1-naphthalene-sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity. Eur J Biochem, 2001 Apr, 268(7), 2179 - 86 Formation and properties of hybrid photosynthetic F1-ATPases . Demonstration of different structural requirements for stimulation and inhibition by tentoxin; Tucker WC et al.; A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure . The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R . rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC) . All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit . In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit . Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes . Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition . However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm . In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid . The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure. Eur J Biochem, 2001 Apr, 268(7), 2141 - 7 Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor; Raza FS et al.; Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation . After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR) . RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals . A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1 . More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism. Eur J Biochem, 2001 Apr, 268(7), 2047 - 54 Exonucleolytic proofreading by p53 protein; Bakhanashvili M; The tumour suppressor p53 protein plays an important role in maintaining genetic integrity . Recently, p53 was shown to have an intrinsic 3'-->5' exonuclease activity . The current study has extended the characterization of purified wild-type recombinant p53-associated 3'-->5' exonuclease function to demonstrate proofreading activity . p53-associated 3'-->5' exonuclease shows clear preference for degradation of ssDNA over dsDNA substrate . On partial duplex structures, this exonucleolytic activity displays a marked preference for excision of a mismatched vs . a correctly paired 3' terminus which enables the p53 protein to act as a proofreader . However, p53 displays variation in excision of mismatched base pairs . The results demonstrate that p53 exhibits mispair excision with a specificity of A:A > A:G > A:C opposite the template adenine residue and with a specificity of G:A > G:G > G:T opposite the template guanine residue . Hence, the observed specificity of mismatch excision shows that p53 exonucleolytic proofreading preferentially repairs transversion mutations . As part of an investigation of the functional interaction between p53 and DNA polymerase, the influence of p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient murine leukemia virus (MLV) reverse transcriptase (RT), representing a relatively low fidelity enzyme . Using an in vitro biochemical assay with 3'-terminal mismatch-containing DNA template primers, it was shown that wild-type recombinant p53 protein enhanced the DNA replication fidelity of MLV RT . A functional interaction between the exonuclease (p53) and polymerase (MLV RT) activities was observed; excision of mispairs by p53 was followed by further elongation onto correctly base-paired 3'-termini by MLV RT . Furthermore, the formation of 3'-mispair and subsequent mispair extension by the enzyme were decreased substantially in the presence of p53 . The fact that the exonuclease-deficient MLV RT is more accurate in the presence of p53, suggests that p53 protein may function as an external proofreading exonuclease for viral enzyme . The observed decrease in initial nucleotide misincorporation and 3'-terminal mispair extension by MLV RT in the presence of p53, indicates the mechanism by which p53 affects the DNA replication fidelity of exonuclease-deficient DNA polymerase. Eur J Biochem, 2001 Apr, 268(7), 2038 - 46 Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity; Manjunatha UH et al.; DNA gyrase is an essential type II topoisomerase found in bacteria . We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis . In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M . smegmatis . Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme . The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria . In contrast, none recognized Escherichia coli GyrA . All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme . MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase . The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner . These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase . In addition, they would be useful for designing peptide inhibitors against DNA gyrase. Eur J Biochem, 2001 Apr, 268(7), 2013 - 9 Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase . A novel bifunctional enzyme in malaria parasites; Clarke JL et al.; Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus . They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria . The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity . This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes . Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned . We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P . falciparum enzyme . Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P . falciparum enzyme . The P . berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P . falciparum enzyme but the insertion in the P . berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P . falciparum insertion . Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag . We show that this enzyme has both Glc6PD and 6PGL activities . Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites. Biotechnol Appl Biochem, 2001 Apr, 33(Pt 2), 117 - 21 Improved preparation and crystallization of 25 kDa human fibroblast growth factor-9; Koyama N et al.; We prepared 25 kDa human fibroblast growth factor-9 (hFGF-9 N33) on a large scale after overproduction in Escherichia coli MM294 (DE3)/pTG931 . The purification was performed by a combination of hydrophobic chromatography and HPLC with an ion exchange column, a heparin affinity column and a gel filtration column . This improved procedure was rapid and simple, and the purified hFGF-9 N33 was found to be homogeneous as judged by various criteria, such as amino acid analysis, N-terminal amino acid sequence, C-terminal amino acid analysis and biological activity . Furthermore, as determined by low endotoxin and DNA content, the protein was of high purity . In addition, the hFGF-9 N33 prepared in the present study was easily crystallized by the vapour diffusion method. Biotechnol Appl Biochem, 2001 Apr, 33(Pt 2), 107 - 15 Biochemical and immunological properties of human cardiac troponin I fragments; Morjana N et al.; Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI) . It is found in human serum within 4-6 h following MI . One of us has shown {Morjana (1998) Biotechnol . Appl . Biochem . 28, 105-111} that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT) . In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties . Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr . The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153) . Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI88, residues 6-96) . TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II TnI Immunoassay . TnI153 exhibits biochemical and immunological properties similar to those of intact TnI . It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT . TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88 . The TnI153-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI88-TnC, and much higher activity than free rTnI, TnI153 and TnI88 . The presence of TnT has no effect on the immunological activity of the TnI153-TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies . Free TnI153 and TnI88 degrade rapidly in human serum . TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88 . The TnI88-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degrees C . However, there was no loss in the immunological activity of the TnI153-TnC complex under the same conditions . A cTnI fragment (TnI80, residues 1-80), expressed in E . coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88. Virology, 2001 Mar 15, 281(2), 294 - 304 An infectious clone of the West Nile flavivirus; Yamshchikov VF et al.; West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent . We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones . This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA . While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication . Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection . In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences . However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired . Fungal Genet Biol, 2001 Feb, 32(1), 21 - 31 Regulation of hisHF transcription of Aspergillus nidulans by adenine and amino acid limitation; Valerius O et al.; The hisHF gene of Aspergillus nidulans encodes imidazole-glycerole-phosphate (IGP) synthase, consisting of a glutamine amidotransferase and a cyclase domain . The enzyme catalyzes the fifth and sixth steps of histidine biosynthesis, which results in an intermediate of the amino acid and an additional intermediate of purine biosynthesis . An A . nidulans hisHF cDNA complemented a Saccharomyces cerevisiae his7Delta strain and Escherichia coli hisH and hisF mutant strains . The genomic DNA encoding the hisHF gene was cloned and its sequence revealed two introns within the 1659-bp-long open reading frame . The transcription of the hisHF gene of A . nidulans is activated upon amino acid starvation, suggesting that hisHF is a target gene of cross pathway control . Adenine but not histidine, both end products of the biosynthetic pathways connected by the IGP synthase, represses hisHF transcription . In contrast to other organisms HISHF overproduction did not result in any developmental phenotype of the fungus in hyphal growth or the asexual life cycle . hisHF overexpression caused a significantly reduced osmotic tolerance and the inability to undergo the sexual life cycle leading to acleistothecial colonies . Nat Struct Biol, 2001 Apr, 8(4), 344 - 8 Simultaneous binding of two proteins to opposite sides of a single transfer RNA; Nomanbhoy T et al.; Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111 . The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules . The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA . Here we present RNA footprints that are consistent with this model . This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA . These opposite locations of binding for these two proteins suggest the possibility of a ternary complex . The formation of a tRNA synthetase--tRNA--Trbp111 ternary complex was detected by two independent methods . The results indicate that the tRNA is sandwiched between the two protein molecules . A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex . These results may have implications for how the translation apparatus is linked to other cellular machinery. Nat Struct Biol, 2001 Apr, 8(4), 334 - 8 Structure of outer membrane protein A transmembrane domain by NMR spectroscopy; Arora A et al.; We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR . The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side . The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction . Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein. Proteins, 2001 May 1, 43(2), 125 - 33 Role of the C-terminal chain in human interferongamma stability: an electrostatic study; Altobelli G et al.; Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model . It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH . The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing . His111 was also found deprotonated at neutral pH . It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions . The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites . This finding suggested that on receptor binding, hIFNgamma adopts energetically more favorable, relaxed, conformation . It was experimentally shown that in contrast to the full-size hIFNgamma, the construct having 21 amino acid residues deleted from the C-terminus is soluble . The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation . On the basis of these results, it was assumed that the electrostatic influence of the C-terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNgamma . Proteins 2001;43:125-133 . Am J Nephrol, 2001 Jan-Feb, 21(1), 28 - 34 Adult-onset minimal change disease among Taiwanese: clinical features, therapeutic response, and prognosis; Huang JJ et al.; There are some racial differences in the prevalence and prognosis of idiopathic nephrotic syndrome; however, reports about minimal change disease (MCD) in Chinese were rare . We retrospectively analyzed 123 Chinese adults with idiopathic nephrotic syndrome, who received percutaneous renal biopsy in our institution within the last 10 years . In total, 46 patients (37.4%) were compatible with the pathological diagnosis of MCD . The male to female ratio was 1.2:1 . The mean age of onset was 30.9 years, and 80% of the patients with MCD were less than 40 years . The mean daily proteinuria was 10.2 g, and serum albumin was 1.8 mg/dl . Azotemia occurred in 16 (35%) of 46 cases; hypertension, 13%; and microscopic hematuria, 13% . High selectivity index for proteinuria (SI <0.1) was noted in 12 (39%) of 31 cases; and high IgE level was found in 83.7% of the study subjects, although only one case had allergic history . Complete remission in 36 MCD patients treated with corticosteroid was achieved by 42% (15/36), 80% (29/36), and 94% (34/36) within 4, 8, and 12 weeks, respectively . The time interval to remission was similar between the younger group (<40 years old, 1.7 months) and older group (>40 years old, 1.6 months) . Nineteen (56%) of 34 cases with steroid response did not relapse, and the other cases (44%) had a mean relapse rate of 1.5 times per patient within a period of 45 months . The age of onset in MCD cases was not significantly correlated with steroid-responsive rate, and the time interval to remission . However, a tendency existed between the onset in the young age and the sequentially relapsing rate (p = 0.06) . Two cases with primary steroid resistance and 5 cases with frequent relapse or steroid dependence responded well to intravenous pulse therapy of cyclophosphamide, except one refractory case . No thrombotic episode was ever noted in our group . Regarding infectious complications, primary peritonitis occurred in one, pneumonia in one, and cellulitis in 6 cases during active nephrotic stage . Two mortality cases, one with E . coli-related necrotizing fasciitis and one from pneumonia, were noted . In brief, compared with children, adult patients with MCD had lesser high selectivity index for proteinuria, the same steroid-responsive rate (94%), but slower response, and significantly lesser relapsing rate . The intravenous pulse therapy of cyclophosphamide may be an alternative regimen for adult patients with steroid resistance or dependency . In addition, the Asian adult-onset MCD had younger age, male predominance, and lesser relapsing rate in comparison to those of the Western population. J Biochem (Tokyo), 2001 Apr, 129(4), 627 - 33 Production of a biologically active epidermal growth factor fusion protein with high collagen affinity; Ishikawa T et al.; Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner . In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain . A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system . This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl . In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin . FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor . In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges . These results suggested that our fusion protein with high collagen affinity would be useful for wound healing. J Biochem (Tokyo), 2001 Apr, 129(4), 569 - 76 Chimeric Na(+)/H(+) antiporters constructed from NhaA of Helicobacter pylori and Escherichia coli: implications for domains of NhaA for pH sensing; Inoue H et al.; In order to delineate regions which play a role in the regulation of Na(+)/H(+) antiporter NhaA activity by pH, we analyzed the antiporter activities of various chimeric mutants constructed from specific portions of NhaA from Escherichia coli and Helicobacter pylori (EC and HP NhaA) . HP NhaA contains 10 residues at the amino-terminus, and 38 residues in a loop region between the eighth and ninth transmembrane spans (loop 8), which are absent in EC NhaA . Deletion from HP NhaA or insertion into EC NhaA of the sequences caused almost no change in pH-dependent antiport activities relative to in the case of the wild-type parent molecules . Chimeras consisting of various combinations of the amino-terminal (amino terminus to sixth or eighth transmembrane span) and carboxy-terminal (seventh or ninth transmembrane span to the carboxy-terminus) regions of EC and HP NhaA showed antiporter activity profiles intermediate between those of the parent molecules . These results show that the two HP-specific sequences are not directly involved in the mechanism of pH sensing by HP NhaA and that the pH sensitivity of NhaA activity is not determined by the amino- or carboxy-terminal regions of NhaA alone, but may be due to interaction between unconserved residues in the two domains . In addition, it was suggested that loop 8 functions primarily as a hinge in both NhaA molecules. J Biochem (Tokyo), 2001 Apr, 129(4), 529 - 35 Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase; Takahashi S et al.; Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney . It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase {Takahashi, S . et al . (1999) J . Biochem . 125, 348-353} and its active site residue was determined to be cysteine 380 by site-directed mutagenesis {Takahashi, S . et al . (1999) J . Biochem . 126, 639-642} . To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells . The expression was detected by Western blotting using anti-rhRnBP antiserum . The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity . Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity. J Biochem (Tokyo), 2001 Apr, 129(4), 513 - 20 Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity; Han CH et al.; A series of truncated forms of gp91phox were expressed in Escherichia coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin . TRX-gp91phox (306-569), which contains the putative FAD and NADPH binding sites, showed weak NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive . Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (306-569), and showed the same K(m) for NADPH as that for superoxide generating activity by the intact enzyme . Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium . In the presence of Rac1, the cytosolic regulatory protein p67phox stimulated the NBT reductase activity, but p47phox had no effect . Truncated p67phox containing the activation domain (residues 199-210) {C.-H . Han, J.R . Freeman, T . Lee, S.A . Motalebi, and J.D . Lambeth (1998) J . Biol . Chem . 273, 16663-16668} stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity . Our data indicate that: (i) TRX-gp91phox (306-569) contains binding sites for both pyridine and flavin nucleotides; (ii) this flavoprotein domain shows weak diaphorase activity; and (iii) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox. J Biochem (Tokyo), 2001 Apr, 129(4), 501 - 7 Functions of RecQ family helicases: possible involvement of Bloom's and Werner's syndrome gene products in guarding genome integrity during DNA replication; Enomoto T; Escherichia coli RecQ helicase is a component of the RecF pathway of recombination whose components are required to reassemble a replisome complex at the site of the replication fork after the removal of a lesion . There are at least five RecQ homologues in human cells, including BLM and WRN . The genes encoding BLM and WRN are mutated in the cancer-prone disorder Bloom's syndrome (BS) and the plogeroid disorder Werner's syndrome (WS), respectively . These syndromes are characterized by a high degree of genomic instability, including chromosomal breaks, multiple large deletions, and translocations, and cells derived from BS and WS patients show defects in DNA replication . Recently, it has become clear that a Holliday junction-like structure is formed at stalled replication forks to result in the formation of double-stranded breaks, and recombination plays an important role in the repair of stalled or broken replication forks, leading to the reinitiation of replication . Defects in the processing of stalled replication forks could lead to aberrant recombination events resulting in genetic instability . Recent studies on BLM, WRN, and the RecQ homologue of Saccharomyces cerevisiae, Sgs1, indicate that these RecQ homologues interact with proteins involved in DNA replication, and function in a pathway from the DNA replication check point to homologous recombination. Free Radic Biol Med, 2001 Apr 1, 30(7), 709 - 14 Modulation of catalase peroxidatic and catalatic activity by nitric oxide; Brunelli L et al.; Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch . Biochem . Biophys . 316:327-334, 1995) . Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase . We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide . Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex . Catalase consumed more nitric oxide upon the addition of hydrogen peroxide . Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide . Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis . Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide . The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide. Pediatr Neurol, 2001 Feb, 24(2), 149 - 51 Multilocular hydrocephalus: ultrasound studies of origin and development; Prats JM et al.; Multilocular hydrocephalus is a complication of neonatal hydrocephalus . Its main feature is the presence of multiple cysts inside the ventricles, which requires a specific therapeutic approach . The case of a preterm infant with intracranial hemorrhage grade II-III and central nervous system infection is reported . The cysts developed at the subependymal layer in the posterior area of the patient's thalamus . Their growth and development were charted by ultrasound imaging for several weeks . These types of cysts may grow to occupy the totality of the lateral ventricles, isolating the temporal horns . Of all the reviewed pathogenic mechanisms, we support the hypothesis of an inflammatory vasculitis at the subependymal level, with the subsequent infarct giving rise to the cysts . The osmotic pressure within the cavities, rather than intraventricular fluid, would account for the enlargement of the cysts. Trends Genet, 2001 Apr, 17(4), 175 - 7 Transcription unit conservation in the three domains of life: a perspective from Escherichia coli; Moreno-Hagelsieb G et al.; Here we address the question of the degree to which genes within experimentally characterized operons in one organism (Escherichia coli) are conserved in other genomes . We found that two genes adjacent within an operon are more likely both to have an ortholog in other organisms, regardless of relative position, than genes adjacent on the same strand but in two different transcription units . They are also more likely to occur next to, or fused to, one another in other genomes . Genes frequently conserved adjacent to each other, especially among evolutionarily distant species, must be part of the same transcription unit in most of them. Biochem Pharmacol, 2001 Apr 1, 61(7), 915 - 20 Proteolytic activation of membrane-bound guanylate cyclase; Chen ZJ et al.; Membrane-bound guanylate cyclase-A (GC-A), the receptor for atrial natriuretic factor (ANF), has been shown to be regulated by its kinase-like domain . To resolve the nature of this regulation, we measured the effects of various proteases on the activity of guanylate cyclase in rat lung membranes, and on the activity of the bacterial-expressed catalytic domain (GC-c) and on a recombinant peptide composed of both the kinase-like and catalytic domain (GC-kc) of guanylate cyclase . Pronase increased rat guanylate cyclase activity in a biphasic manner with a maximal effect at about 10-20 microg per assay tube . Thermolysin had effects similar to those of pronase on the activity of guanylate cyclase in rat lung membranes . In the case of bacterial-expressed proteins, pronase increased the activity of GC-kc, but not GC-c . These results indicate that GC-A contains an autoinhibitory site on its kinase-like domain, and that removal of the autoinhibitory site by limited proteolysis leads to enzyme activation . GC-A was poorly activated by ANF and ATP after the rat lung membrane was pretreated with pronase, suggesting that ANF/ATP and pronase activate guanylate cyclase through the same mechanism . It is suggested that the binding of ANF and ATP to GC-A may induce a conformational change of the receptor that releases the inhibitory constraint on enzyme activity leading to enzyme activation. Biochem Pharmacol, 2001 Apr 1, 61(7), 803 - 9 Modulation of inositol 1,4,5-trisphosphate binding to the various inositol 1,4,5-trisphosphate receptor isoforms by thimerosal and cyclic ADP-ribose; Vanlingen S et al.; Three different genes encode the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an intracellular Ca2+ channel involved in cellular Ca2+ signaling . The IP3-binding characteristics of the various IP3R isoforms differ, but until now no specific activators or inhibitors of IP3 binding have been described . We compared the effects of oxidizing reagents, in particular thimerosal, and of cyclic ADP-ribose (cADPR) on IP3 binding to the various IP3R isoforms . We therefore expressed the N-terminal 581 amino acids of the three IP(3)R isoforms as recombinant proteins in the soluble fraction of Escherichia coli (ligand-binding sites {lbs} 1, 2, and 3) as well as the full-length IP3R1 and IP3R3 in Spodoptera frugiperda (Sf9) insect cells . Thimerosal (100 microM) stimulated IP3 binding to lbs-1 (1.4-fold) and lbs-3 (2.5-fold), but had no effect on lbs-2 . Thimerosal acted on lbs-1 and lbs-3 by decreasing the Kd for IP3 binding (from 46 +/- 4 nM to 20 +/- 2 nM and from 54 +/- 21 nM to 19 +/- 7 nM for lbs-1 and -3, respectively) without modifying the Bmax . Similarly, IP3 binding to microsomes of Sf9 insect cells overexpressing the full-length IP3R1 was 1.2-fold stimulated by thimerosal . Thimerosal, however, did not affect IP3 binding to Sf9-IP3R3 microsomes, suggesting that in situ thimerosal will only directly affect ligand binding to the type 1 isoform . cADPR (50 microM) stimulated IP3 binding to Sf9-IP3R1 microsomes (1.5-fold), but not to Sf9-IP3R3 microsomes . In addition, cADPR inhibited IP3 binding to lbs-1 and lbs-2 by decreasing the affinity for IP3 1.8- and 2.8-fold, respectively, while IP3 binding to lbs-3 was not affected . These results suggest that a regulatory site for cADPR is present in the ligand-binding domain of IP3R1 and 2, but not of IP3R3. Protein Sci, 2001 Apr, 10(4), 879 - 86 Reduction of the amyloidogenicity of a protein by specific binding of ligands to the native conformation; Chiti F et al.; It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases . A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation . Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated . We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP . By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost . These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo. Protein Sci, 2001 Apr, 10(4), 845 - 53 Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study; Griko Y et al.; Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme . T7RNAP is a large monomeric enzyme (99 kD) . Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains . Interactions between these structural domains and their stability strongly depend on solvent conditions . The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations . Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state . Urea-induced unfolding at 25 degrees C reveals a two-step process . The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea . The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C . The second transition leads to a mixture of poly(Pro)II and unordered conformations . As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation . Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture . Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding. Protein Sci, 2001 Apr, 10(4), 779 - 87 Prediction of the transmembrane regions of beta-barrel membrane proteins with a neural network-based predictor; Jacoboni I et al.; A method based on neural networks is trained and tested on a nonredundant set of beta-barrel membrane proteins known at atomic resolution with a jackknife procedure . The method predicts the topography of transmembrane beta strands with residue accuracy as high as 78% when evolutionary information is used as input to the network . Of the transmembrane beta-strands included in the training set, 93% are correctly assigned . The predictor includes an algorithm of model optimization, based on dynamic programming, that correctly models eight out of the 11 proteins present in the training/testing set . In addition, protein topology is assigned on the basis of the location of the longest loops in the models . We propose this as a general method to fill the gap of the prediction of beta-barrel membrane proteins. Protein Sci, 2001 Apr, 10(4), 771 - 8 Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves; Baladi S et al.; Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT . To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP) . The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein . N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C . C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP) . The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+ . Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain . Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT . Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction. Protein Sci, 2001 Apr, 10(4), 715 - 24 Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability; Krowarsch D et al.; It is widely accepted that solvent-exposed sites in proteins play only a negligible role in determining protein energetics . In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T(den) difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2 degrees C at pH 2.0 . The DeltaH(den) versus T(den) plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal* mole(-1) * K(-1); 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal*mole(-1) . Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) =7.1 degrees C) . Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences . A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position . The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation . The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities . In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution. Protein Sci, 2001 Apr, 10(4), 707 - 14 Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses; Muroya A et al.; The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses . The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H . Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family . Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme . Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid. Protein Sci, 2001 Apr, 10(4), 697 - 706 Three-dimensional structures of the three human class I alcohol dehydrogenases; Niederhut MS et al.; In contrast with other animal species, humans possess three distinct genes for class I alcohol dehydrogenase and show polymorphic variation in the ADH1B and ADH1C genes . The three class I alcohol dehydrogenase isoenzymes share approximately 93% sequence identity but differ in their substrate specificity and their developmental expression . We report here the first three-dimensional structures for the ADH1A and ADH1C*2 gene products at 2.5 and 2.0 A, respectively, and the structure of the ADH1B*1 gene product in a binary complex with cofactor at 2.2 A . Not surprisingly, the overall structure of each isoenzyme is highly similar to the others . However, the substitution of Gly for Arg at position 47 in the ADH1A isoenzyme promotes a greater extent of domain closure in the ADH1A isoenzyme, whereas substitution at position 271 may account for the lower turnover rate for the ADH1C*2 isoenzyme relative to its polymorphic variant, ADH1C*1 . The substrate-binding pockets of each isoenzyme possess a unique topology that dictates each isoenzyme's distinct but overlapping substrate preferences . ADH1*B1 has the most restrictive substrate-binding site near the catalytic zinc atom, whereas both ADH1A and ADH1C*2 possess amino acid substitutions that correlate with their better efficiency for the oxidation of secondary alcohols . These structures describe the nature of their individual substrate-binding pockets and will improve our understanding of how the metabolism of beverage ethanol affects the normal metabolic processes performed by these isoenzymes. Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3976 - 81 Epub 2001 Mar 13. L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis; Qian F et al.; L-selectin mediates homing of lymphocytes to lymph nodes (LN) . Transgenic mice that express rat insulin promoter regulated simian virus 40 Tag (RIP-Tag) develop large, local cancers that metastasize to liver but not LN . To test whether this lack of LN metastases reflects their absence from the circulation, transgenic mice were produced that express Tag (T), L-selectin (L), and Escherichia coli LacZ (Z), in pancreatic beta cells . LTZ mice developed insulinomas that specifically had LN metastases; metastasis was blocked by an anti L-selectin mAb . LacZ(+) tumor cells from these LN homed to secondary LN upon transfer . These results suggest that the highly vascularized islet carcinomas are shedding tumor cells into the bloodstream, which is a necessary but insufficient condition for metastasis to occur; L-selectin can facilitate homing of such tumor cells to LN, resulting in metastasis. Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3762 - 7 RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin; Gautschi M et al.; The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family . Our current understanding of which homologs functionally interact is incomplete . Zuotin is a DnaJ homolog bound to the yeast ribosome . We have now identified the DnaK homolog Ssz1p/Pdr13p as zuotin's partner chaperone . Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit . RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP . In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperone-like effect on nascent chains . In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo . However, overexpression of zuotin partly rescued the growth defect of the Delta ssz1 strain, whereas overexpression of Ssz1p did not affect the Delta zuo1 strain, suggesting a pivotal function for the DnaJ homolog. Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3679 - 84 Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-A resolution; Allard J et al.; 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate . The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate . A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6 . Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues . Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps . Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group . In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group . Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133) . Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues. Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4486 - 91 Epub 2001 Mar 27. Multicopy plasmids are clustered and localized in Escherichia coli; Pogliano J et al.; We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters . In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions . The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle . Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics . A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI . Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions . Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations. Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4776 - 81 Epub 2001 Mar 27. Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation; Bian S et al.; Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening . However the structural basis of Ca(2+) binding to BK channels is unknown . To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli . We purified Dslo-C280 in soluble form and used a (45)Ca(2+)-overlay protein blot assay to detect Ca(2+) binding . Dslo-C280 exhibits specific binding of (45)Ca(2+) in comparison with various control proteins and known EF-hand Ca(2+)-binding proteins . A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces (45)Ca(2+)-binding activity by 56% . By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca(2+) sensitivity for activation and a shift of approximately +80 mV in the midpoint voltage for activation . This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca(2+) and a reduction in apparent Ca(2+) affinity, suggesting the loss of one Ca(2+)-binding site per monomer . These results demonstrate a functional correlation between Ca(2+) binding to a specific region of the BK protein and Ca(2+)-dependent activation, thus providing a biochemical approach to study this process. Physiol Rev, 2001 Apr, 81(2), 685 - 740 Molecular basis of mechanotransduction in living cells; Hamill OP et al.; The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes . When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer . Localized changes in bilayer thickness and curvature may compensate for this mismatch . The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers . Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects . As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations . Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension . However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer . Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli . This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension . Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity . In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer. J Am Soc Nephrol, 2001 Apr, 12(4), 800 - 6 Detection of verocytotoxin bound to circulating polymorphonuclear leukocytes of patients with hemolytic uremic syndrome; Te Loo DM et al.; The epidemic form of hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children and is characterized by a prodromal phase of sometimes bloody diarrhea . The role of verocytotoxin (VT)-producing Escherichia coli has been strongly implicated . Although antibodies against VT have been detected in the serum of patients with HUS, VT itself has never been detected in circulating blood . In this study, VT-2 was detected in the systemic circulation in 9 of 10 patients with the epidemic form of HUS . In those cases, VT-2 was bound exclusively to polymorphonuclear leukocytes (PMN) . The detection of VT-2 bound to PMN was associated with the presence of diarrhea at the time the blood samples were obtained . The one patient for whom VT was not detected presented with atypical HUS . For 5 of the 10 patients with HUS who were studied, the time course of VT binding was analyzed; binding decreased in four patients . The finding of VT bound to PMN in the systemic circulation of patients with HUS is important for a clearer understanding of the pathogenesis of HUS and suggests new approaches for treatment in the future. J Biol Chem, 2001 Jun 15, 276(24), 21594 - 600 Epub 2001 Mar 26. Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins; DeMarco SJ et al.; Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission . Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains . The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen . The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b . Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs . Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells . In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95 . These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs . PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation. J Biol Chem, 2001 Jun 15, 276(24), 20866 - 75 Epub 2001 Mar 23. Characterization of the Escherichia coli sigma E regulon; Dartigalongue C et al.; Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes . Efinal sigma(E) RNA polymerase controls one of the two heat shock regulons of E . coli . This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E . coli . final sigma(E) (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors . Here we asked how many genes are activated by Efinal sigma(E) RNA polymerase and what is the identity of these genes . Using two independent genetic approaches, 20 E . coli promoters were identified which activate reporter gene transcription in a final sigma(E)-dependent manner . In all cases examined, a canonical final sigma(E) binding site could be revealed upon mapping transcriptional start sites . 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E . coli envelope proteins (dsbC, fkpA, skp, and surA) . The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA-ecfM) . Two of these ecf genes were found to be essential for E . coli growth. J Bacteriol, 2001 Apr, 183(8), 2700 - 3 Purification of the RelB and RelE proteins of Escherichia coli: RelE binds to RelB and to ribosomes; Galvani C et al.; The direct interaction of the Escherichia coli cytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the relB and relE genes . In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation. J Bacteriol, 2001 Apr, 183(8), 2691 - 5 Rapid dephosphorylation of the TorR response regulator by the TorS unorthodox sensor in Escherichia coli; Ansaldi M et al.; Induction of the torCAD operon, encoding the trimethylamine N-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability . TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443-->Asp723-->His850-->Asp(TorR) . In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed . Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)-->His850-->Asp723, since His850 and Asp723 are both essential in this process . By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of tor operon transcription in less than 2 min . Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators. J Bacteriol, 2001 Apr, 183(8), 2677 - 81 Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses; Ekaza E et al.; Pathogens often encounter stressful conditions inside their hosts . In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins . Under high-temperature stress conditions, ClpB of B . suis was induced, and an isogenic B . suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH . The effects were reversible by complementation . Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress . In B . suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C . We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins. J Bacteriol, 2001 Apr, 183(8), 2646 - 53 Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element; Kobayashi K et al.; The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli . E . coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30 . Suppressor mutants showing high solvent resistance were isolated from OST5500 . These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins . The suppressor mutants lost solvent resistance on inactivation of the acrEF operon . The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS . It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation . The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac . The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF . Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump. J Bacteriol, 2001 Apr, 183(8), 2614 - 23 Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus; Cann IK et al.; Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya . The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates . RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis . The P . furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL) . Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) and P . furiosus PCNA (PfuPCNA) . The ATPase activity of PfuRFC itself was as strong as that of RFCS . However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions . RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA . The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II from P . furiosus . We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved in Archaea and Eucarya. J Bacteriol, 2001 Apr, 183(8), 2535 - 42 Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1; Collis CM et al.; The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites, attI sites, found in integrons, and members of the 59-be family, found in gene cassettes . The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between attI1 and a 59-be, or between two attI1 sites, were compared . Recombination events involving two attI1 sites were significantly less efficient than the reactions in which a 59-be participated, and the attI1 x 59-be reaction was generally preferred over the 59-be x 59-be reaction . Recombination of attI1 with secondary sites was less efficient than the 59-be x secondary site reaction. J Bacteriol, 2001 Apr, 183(8), 2476 - 84 Involvement of H-NS in transpositional recombination mediated by IS1; Shiga Y et al.; IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition . Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements . Host factors involved in the IS1 transposition reaction, however, are not known . We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA . Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant . The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene . These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo . Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence . The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not . The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain . We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase. J Bacteriol, 2001 Apr, 183(8), 2454 - 62 Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection; Endley S et al.; Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations . Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells . Transposon (Tn5) mutagenesis of B . abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival . One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration . The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage . B . abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection . Virulence was restored by an intact copy of cydAB or by B . abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase . These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction . Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain. J Bacteriol, 2001 Apr, 183(8), 2445 - 53 Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins; Otto K et al.; Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood . By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins . A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E . coli strains, which was at least partly caused by proteolysis . Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains . Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells . While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain . Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC . While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced . Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins . This change alters the surface characteristics of the cell envelope and may thus influence adhesion. J Bacteriol, 2001 Apr, 183(8), 2399 - 404 Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli; Vazquez-Laslop N et al.; Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium . This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises approximately 10% of the total bacterial protein content . As we demonstrate here, the same proteins are released from electroporated E . coli cells pretreated with EDTA . Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation . Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E . coli homogenate . This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope . As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells. Genes Dev, 2001 Mar 15, 15(6), 737 - 47 Tn7 recognizes transposition target structures associated with DNA replication using the DNA-binding protein TnsE; Peters JE et al.; We report that the bacterial transposon Tn7 selects targets by recognizing features associated with DNA replication using the transposon-encoded DNA-binding protein TnsE . We show that Tn7 transposition directed by TnsE occurs in one orientation with respect to chromosomal DNA replication, indicating that a structure or complex involved in DNA replication is likely to be a critical determinant of TnsE insertion . We find that mutant TnsE proteins that allow higher levels of transposition also bind DNA better than the wild-type protein . The increased binding affinity displayed by the TnsE high-activity mutants indicates that DNA binding is relevant to transposition activity and suggests that TnsE interacts directly with target DNAs . In vitro, TnsE interacts preferentially with certain DNA structures, indicating a mechanism for the TnsE-mediated orientation and insertion preference . The pattern of TnsE-mediated insertion events around the Escherichia coli chromosome provides insight into how DNA replication forks proceed in vivo. J Mol Biol, 2001 Mar 30, 307(3), 785 - 98 Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy; Sorensen MA; Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation . One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type . Previous work had shown that the charging levels of tRNAs were the same in Rel(+) and Rel(-) strains and reduced, at most, two- to fivefold in both strains during starvation . The present reinvestigation of the charging levels of tRNA(2)(Arg), tRNA(1)(Thr), tRNA(1)(Leu) and tRNA(His) during starvation of isogenic Rel(+) and Rel(-) strains showed that starvation reduced charging levels tenfold to 40-fold . This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier . The determination of the charging levels of tRNA(2)(Arg) and tRNA(1)(Thr) during starvation were accurate enough to demonstrate that charging levels were at least fivefold lower in the Rel(-) strain compared to the Rel(+) strain . Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome . J Mol Biol, 2001 Mar 30, 307(3), 755 - 69 Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli; Magliery TJ et al.; Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis . In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops . Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG . The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon . Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli . We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo . In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors . Biosci Biotechnol Biochem, 2001 Jan, 65(1), 79 - 85 Direct expression of the extracellular portion of human FcepsilonRIalpha chain as inclusion bodies in Escherichia coli; Takai T et al.; The extracellular portion of the alpha chain of the human high-affinity IgE receptor (FcepsilonRIalpha) was expressed as inclusion bodies in Escherichia coli . In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcepsilonRIalpha monoclonal antibodies . N-terminal sequencing showed that the two bands were equivalent to the soluble FcepsilonRIalpha with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-terminal 22 residues of the soluble FcepsilonRIalpha have been removed, possibly by degradation in E . coli cells . IgE-binding to CHO cells expressing FcepsilonRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies . The system for the expression of soluble human FcepsilonRIalpha in E . coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcepsilonRIalpha interaction. Biosci Biotechnol Biochem, 2001 Jan, 65(1), 185 - 9 A simple, rapid, and highly efficient gene expression system for multiheme cytochromes c; Ozawa K et al.; The genes of tetraheme cytochrome c3 and hexadecaheme high-molecular-weight cytochrome c from Desulfovibrio vulgaris could be overexpressed as holoproteins in Shewanella oneidensis TSP-C using pUC-type vectors of E . coli . Surprisingly, S . oneidensis was transformed directly by pUC-type vectors through electroporation . The yields of the recombinant proteins in this expression system were much higher than the previously reported ones. Photochem Photobiol, 2001 Feb, 73(2), 128 - 34 Alternative forms of formamidopyrimidine-DNA glycosylase from Arabidopsis thaliana; Gao MJ et al.; Formamidopyrimidine-DNA glycosylase (FPG) catalyzes the initial steps in the repair of DNA containing oxidized purines . Two complementary DNA clones encoding homologs of bacterial FPG, designated Atfpg-1 and Atfpg-2, have been isolated from Arabidopsis thaliana . They are products of alternative splicing of the transcript of a single gene . Proteins encoded by both clones, AtFPG-1 and AtFPG-2, engineered to contain oligohistidine sequences on their C-terminal ends, were expressed in Escherichia coli and purified, and their activities were assayed . Both proteins cleaved DNA that contained apurinic sites, indicating that they have abasic lyase activity . AtFPG-1, but not AtFPG-2, showed significant cleavage of a double-stranded oligonucleotide that contained 8-oxo-guanine, indicating that the structural differences between the two proteins influence their enzymatic activities . However, both proteins were able to cleave the same sites in DNA that was treated with visible light in the presence of methylene blue. J Comput Aided Mol Des, 2001 Feb, 15(2), 117 - 28 Simulation of carbohydrate-protein interactions: computer-aided design of a second generation GM1 mimic; Bernardi A et al.; The oligosaccharide of ganglioside GM1 {Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer} is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E . coli (LT) . We recently reported that the pseudosaccharide 2 {Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)DCCHD} is a high-affinity ligand for CT . and thus a functional mimic of GM1 (Bernardi, A., Checchia, A., Brocca, P., Sonnino, S . and Zuccotto, F., J . Am . Chem . Soc., 121 (1999) 2032-2036) . In this paper we describe the design of a second-generation mimic, formally obtained from 2 by inverting the configuration of a single stereocenter, thus transforming a N-acetyl galactosamine into a N-acetyl glucosamine . The design process involved modeling of the free ligand and its LT complex, followed by qualitative and quantitative comparison with the corresponding structures of 2 . The protocol employed relied on both conformational search and molecular dynamics methodologies to account for the flexibility of both the ligand and the protein receptor . The conformational search of the LT:inhibitor complex showed that, compared to 2, the new compound can insert one more hydroxy group within the protein binding site . Molecular dynamics simulations showed that, in turn, this may trigger a series of rearrangements and reorientations of side chains and crystallographic water molecules in the toxin, leading to new H-bond contacts which may result in enhanced affinity of the new inhibitor . FEP calculations were performed by mutating the structure of 2 in solution and in the protein complex, and the prediction was made that the second-generation mimic should be a stronger binder than its parent compound. Parasitology, 2001 Feb, 122(Pt 2), 125 - 32 A novel EF-hand calcium-binding protein in the flagellum of the protozoan Tritrichomonas suis; Felleisen RS et al.; The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tri