Escherichia coli

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 Tritrichomonas suis is described . A T . suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41 kDa antigen in a Triton X-114 membrane-protein fraction . The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants . The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site . Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family . A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli . By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+ . In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite . Our findings with the parabasalid T . suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

J Leukoc Biol, 2001 Feb, 69(2), 289 - 96
Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory properties of secretory IgA; Honorio-Franca AC et al.; Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections . IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied . Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (Fc alphaR, CD89) at levels similar to those of blood neutrophils . Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not . The Fc alphaR on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55-75 kDa) as that identified for blood neutrophils . Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types . In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of gamma chain association with Fc alphaR (gamma-less) exclusively on colostral neutrophils . The functional role of these gamma-less Fc alphaR cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E . coli . No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation . Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells . These results reveal exclusive expression of gamma-less Fc alphaR on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.

J Leukoc Biol, 2001 Feb, 69(2), 253 - 62
CpG-containing oligodeoxynucleotides induce TNF-alpha and IL-6 production but not degranulation from murine bone marrow-derived mast cells; Zhu FG et al.; Mast cells are sentinel cells critical to the initiation of innate immune and inflammatory responses, particularly at mucosal surfaces . To fulfill this function they can be activated by several pathogen-associated stimuli to produce cytokines with or without concurrent degranulation . We examined the ability of immunostimulatory DNA sequences including CpG motifs, which are found in increased quantities in bacterial DNA, to activate mouse bone marrow-derived mast cells (mBMMC) . Mast cells were treated with a range of doses of CpG-containing oligodeoxynucleotides or control oligodeoxynucleotides without CpG within their sequence . There was a dose-dependent increase in the production of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by mast cells treated with the CpG-containing oligodeoxynucleotides . The cytokine levels induced were directly related to the number of CpG within a given length of sequence . Treatment with oligonucleotides containing 3CpG induced an eightfold increase in TNF production over control incubated mast cells . Other cytokines, including granulocyte-macrophage colony-stimulating factor, IL-4, interferon-gamma, and IL-12 were not induced by oligonucleotide treatment . Neither CpG containing oligodeoxynucleotides nor control oligodeoxynucleotides induced degranulation of mast cells . Bacterial DNA from Escherichia coli also induced IL-6 from mBMMC but neither calf thymus DNA nor methylase-treated E . coli DNA had such an effect . Examination of the uptake of Texas red-labeled CpG and non-CpG-containing oligodeoxynucleotides revealed that they were both similarly taken up by the mBMMC . These results have important implications for the mechanism by which mast cells respond to bacteria and for the potential role of mast cells in DNA vaccination.

J Food Prot, 2001 Feb, 64(2), 159 - 63
Efficacy of washing with a commercial flatbed brush washer, using conventional and experimental washing agents, in reducing populations of Escherichia coli on artificially inoculated apples; Annous BA et al.; Conventional and experimental washing formulations were applied with a commercial flatbed brush washer under conditions representative of commercial practice to determine their efficacy in decontaminating apples inoculated with a nonpathogenic Escherichia coli strain . Golden Delicious apples (18 kg) inoculated with E . coli were mixed with approximately 109 kg of uninoculated Fuji apples (distinctly different in appearance) in a wet dump tank containing 1,325 liters of water at 20 degrees C for 15 min . The combined apples were washed in a flatbed brush washer with the following washing solutions: water at 20 degrees C, water at 50 degrees C, 200 ppm of chlorine (pH 6.4) at 20 degrees C, 8% trisodium phosphate at 20 degrees C, 8% trisodium phosphate at 50 degrees C, 5% hydrogen peroxide at 20 degrees C, 5% hydrogen peroxide at 50 degrees C, 1% APL Kleen 245 at 50 degrees C, and two-stage washing treatments using the combination of 1% APL Kleen 245 at 20 or 50 degrees C followed by 5% hydrogen peroxide at 35 or 50 degrees C . None of the washing treatments tested under the conditions of this experiment significantly reduced the E . coli populations on the inoculated apples or in cider made from these apples, probably as a consequence of the inability of this washing system to inactivate or remove the bacterial cells in inaccessible calyx and stem areas of apples . These results are important because they demonstrate the need for new fruit washing technology that can overcome this limitation . Also, there was no significant cross-contamination of the Fuji apples in the dump tank . Significant cross-contamination of cider, made with uninoculated apples, occurred in the hammer mill and/or the press cloth when these units were not sanitized following a trial with inoculated apples.

Electrophoresis, 2000 Nov, 21(17), 3649 - 56
An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis; Sabounchi-Schutt F et al.; In the field of proteomics the need to detect low-abundance cellular components, such as regulatory proteins, is of critical importance . Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most commonly used separation tools for these biological investigations . In this paper we report an alternative micropreparative 2-D PAGE sample application method, called the "paper bridge loading" method . This method makes it possible to apply a larger sample volume to commercially available immobilized pH gradient (IPG) strips . The Vh products required for focusing are only marginally longer than those used in analytical experiments . The method was compared to traditional cup loading and in-gel rehydration . With 18 cm long narrow-range Immobiline DryStrip pH 4.5-5.5, the "paper bridge" method allowed the application of 10 mg human plasma proteins compared to 3 mg with traditional loading methods . The corresponding figures using Escherichia coli sample was found to be 6 mg and less than 2 mg, respectively . The paper bridge method also showed the best results in terms of spot resolution and separation of high molecular weight proteins.

Electrophoresis, 2000 Nov, 21(17), 3639 - 48
A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes; Herbert B et al.; Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis . This was demonstrated with different samples . In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE . Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma . With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.

Arch Microbiol, 2001 Jan, 175(1), 1 - 7
Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca; Schilde C et al.; Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems . Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems . We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP) . gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha . These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid . The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells . gfp expression was monitored by epifluorescence microscopy . The gfp reporter gene plasmids presented here for the model zygomycete A . glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.

Curr Microbiol, 2001 Mar, 42(3), 211 - 6
Purification, refolding of hybrid hIFNgamma-kringle 5 expressed in Escherichia coli; Lu H et al.; The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon gamma and over-produced in the form of inactive inclusion bodies in E . coli . The amount of fusion protein was about 40% of total protein produced . The fusion protein contained in the inclusion bodies was solubilized in 8 M urea and purified by anion-exchange chromatography . We employed the orthogonal experimental design L16(4(5)) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor L-arginine, NaCl, pH, and glycine on the refolding procedure . Our results suggest that the presence of appropriate L-arginine, NaCl, and denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein with biological activity . The refolded fusion protein, gammaIFN/pk5, has in vitro anti-endothelial cell proliferation activity.

Curr Microbiol, 2001 Mar, 42(3), 173 - 7
Enhanced tolerance against salt-stress and freezing-stress of Escherichia coli cells expressing algal bbc1 gene; Tanaka S et al.; The expression of the eukaryotic bbc1 (breast basic conserved) gene (the bbc1 gene of the marine green alga Chlamydomonas sp . W-80 strain) enhanced the tolerance against salt-stress and freezing-stress in E . coli cells . The expression of the BBC1 protein in the E . coli cells carrying the algal bbc1 gene and that in the Chlamydomonas W-80 cells were examined by Western blotting analysis . The result suggests that the eukaryotic BBC1 protein expressed in the E . coli cells has a protective function against the cellular dehydration.

Viral Immunol, 2001, 14(1), 83 - 92
Mapping of T-helper epitopes of Rinderpest virus hemagglutinin protein; Sinnathamby G et al.; Rinderpest virus (RPV) is a highly contagious and often fatal disease of domestic and wild ruminants, caused by rinderpest virus of the genus Morbillivirus under the family Paramyxoviridae . Hemagglutinin (H) and fusion (F) proteins of this enveloped virus confer protective immunity against experimental challenge with virulent rinderpest virus . We have earlier demonstrated that immunization with a single dose of recombinant extracellular baculovirus expressing H protein elicits H-specific humoral and lymphoproliferative responses in cattle . The lymphoproliferative responses are predominantly BoLA class II restricted . In this work, we have analyzed lymphoproliferative responses of peripheral lymphocytes from immunized cattle to truncated H protein fragments expressed in E . coli for locating domains harboring Th epitopes . One region (aa 113-182) recognized by immune T cells is conserved in the H protein of measles virus, which was earlier shown to contain a dominant Th epitope in mouse . Synthetic peptides within this region of measles virus H protein were used to identify a Th epitope conserved in the H protein of RPV virus (aa 123-137) in cattle . A second Th epitope located at the C-terminus of RPV-H was mapped to the region corresponding to aa 512-609 using truncated protein fragments expressed in E . coli . The C-terminal epitope (aa 575-583) was mapped using synthetic peptides corresponding to measles virus H as well as RPV-H protein.

J Dent Res, 2001 Jan, 80(1), 346 - 50
Immunodominant region of Actinobacillus actinomycetemcomitans 40-kilodalton heat shock protein in patients with rheumatoid arthritis; Yoshida A et al.; Bacterial heat shock proteins have been implicated in the pathogenesis of several diseases, and the immunological relationship between rheumatoid arthritis (RA) and Escherichia coli DnaJ has been reported . Since there are similarities in the tissue destruction process of RA and periodontitis, we examined the reactivities of antibodies in sera from RA patients to the DnaJ protein from Actinobacillus actinomycetemcomitans . An enzyme-linked immunosorbent assay showed that IgG titers to the N-terminal conservative region of the DnaJ are significantly higher in RA patients compared with the healthy controls (p < 0.05) . Furthermore, we examined IgG titers of disease controls to determine the specificity of the immune responses to this region in RA patients . The difference between RA and infectious disease patients was also significant (p < 0.05) . These results suggest that the N-terminal region of DnaJ from A . actinomycetemcomitans may contribute to the etiologic analysis of RA.

EMBO Rep, 2000 Oct, 1(4), 323 - 7
New genes with old modus operandi . The connection between supercoiling and partitioning of DNA in Escherichia coli; Dasgupta S et al.; The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis . In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell . As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork . The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity . Thus, genes involved in chromosome topology, once set aside as 'red herrings' in the search for 'true' partition functions, are again recognized as being important for chromosome partitioning in E . coli.

Insect Biochem Mol Biol, 2001 Apr 27, 31(6-7), 521 - 32
Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virus-encoded chitinase gene; Shinoda T et al.; A Chitinase cDNA named Slchi was cloned from the epidermis of the common cutworm, Spodoptera litura, and the enzymatic properties of its recombinant proteins were characterized . The Slchi cDNA encodes 552 amino-acid residues (aa) including a 19 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 60,152 Da . A major transcript of Slchi about 2.8 kb was detected in the epidermis only during molting in the last instar larvae, suggesting its involvement in the digestive system for old cuticle . The E . coli-produced recombinant Slchi exhibited weak chitinolytic activity against 4MU-(GlcNAc)(3)>4MU-(GlcNAc)(2)>4MU-(GlcNAc)(4), in this order, but not against 4MU-(GlcNAc)(1) . A recombinant Slchi with higher specific activity was obtained using recombinant Hyphantria cunea NPV (HycuNPV), which expresses Slchi under polyhedrin promoter . To discriminate chitinase activity of recombinant Slchi from an active chitinase encoded in HycuNPV genome (chiA), we further knocked out the chiA gene from the recombinant virus . The recombinant Slchi expressed in insect cell culture showed a similar substrate specificity against 4MU-(GlcNAc)(n) (n=1-4) to that produced in E . coli, while the viral chitinase showed the highest activity against 4MU-(GlcNAc)(2) . The recombinant Slchi was secreted rapidly into the culture medium from the infected cells, whereas the viral chitinase retained predominantly in the cells.

Eur J Cancer, 2001 Mar, 37(4), 542 - 9
Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma; Roovers RC et al.; Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease . However, only few tumour antigens useful for targeting have been described to date . To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells . Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells . These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues . The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g . Ep-CAM or c-ERB-2) . This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.

FEMS Immunol Med Microbiol, 2001 Mar, 30(2), 87 - 93
Helicobacter pylori mutagenesis by mariner in vitro transposition; Guo BP et al.; We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis . The gene of interest was PCR-amplified and cloned . A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction . After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation . Transformants were selected by plating on kanamycin . Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained . The site of insertion was determined by PCR or sequencing . We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons . Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H . pylori in mixed competition experiments.

Mutat Res, 2001 Apr 4, 485(3), 209 - 18
The relative contribution of adduct blockage and DNA repair on template utilization during replication of 1,N2-propanodeoxyguanosine and pyrimido; Fink SP et al.; The role of replication blockage by the exocyclic DNA adducts propanodeoxyguanosine (PdG) and pyrimido{1,2-alpha}purin-10(3H)-one (M1G) was determined through the use of site-specifically adducted M13MB102 genomes containing a C:C-mismatch approximately 3000 base-pairs from the site of adduct incorporation . Genomes containing either dG, PdG, or M1G positioned at site 6256 of the (-)-strand were transformed into repair-proficient and repair-deficient Escherichia coli strains and the percent template utilization was determined by hybridization analysis . Unmodified genomes containing a C:C-mismatch resulted in a percent template utilization of approximately 60 and 40% for the (-)- and (+)-strands, respectively . Transformation of PdG- or M(1)G-adducted genomes resulted in approximately a 60-40% and 50-50% (-)-strand to (+)-strand ratio, respectively, indicating that PdG and M(1)G are negligible blocks to replication in repair-proficient E . coli . This is in contrast to previous studies using (PdG:T)- and (M1G:T)-mismatched M13MB102 genomes, which resulted in a majority of the replication events using the unadducted (+)-strand and suggested that both adducts were significant blocks to replication {J . Biol . Chem . 272 (1997) 11434; Proc . Natl . Acad . Sci . U.S.A . 94 (1997) 8652} . The C:C-mismatch results, though, indicate that the large strand bias detected in the earlier studies is due to repair of the adducts and resynthesis of the (-)-strand using the (+)-strand as a template for repair synthesis . Transformation of adducted C:C-mismatched genomes into E . coli strains deficient in nucleotide excision repair did result in an increased strand bias with only approximately 20 and 34% of the replication events using the (-)-strand for PdG- and M1G-adducted genomes, respectively . The increased strand bias indicates the importance of nucleotide excision repair in the removal of PdG and M1G.

Vet Microbiol, 2001 Apr 19, 79(4), 367 - 74
Dichelobacter nodosus serotype M fimbrial subunit gene: implications for serological classification; Zhou H et al.; Dichelobacter nodosus fimbrial subunit gene (fimA) from a serotype M strain (M-SPAHL) was investigated in this study . A primer set targeting the relatively conserved fimA regions and based on the published sequence from Nepalese serogroup M isolates (Nepalese M), failed to amplify the fimA of M-SPAHL . However, when the downstream primer was substituted with a primer that is specific for other serogroups of D . nodosus, the fimA was successfully amplified . Cloning followed by DNA sequencing, revealed that the M-SPAHL fimA was different to the Nepalese M fimA . The predicted amino acid sequence of the M-SPAHL fimA did not show homology to any known serogroups or serotypes . The most similar sequence was from serotype F1, and not Nepalese M . The consequences of serological relatedness and sequence dissimilarity are discussed.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 239 - 44
Genetic differences between Escherichia coli O26 strains isolated in Brazil and in other countries; Peixoto JC et al.; Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup -- encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains -- was evaluated through random amplified polymorphic DNA (RAPD) analysis . Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters . RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H(-) serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data . The epidemiological significance of these data is discussed.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 223 - 7
Expression and rapid one-step purification of biologically active His-tagged factor C by Ni(2+) affinity column chromatography; Birko Z et al.; Factor C is an unusual extracellular protein capable of inducing cytodifferentiation in certain Streptomyces strains . The protein is produced by Streptomyces griseus 45H at such a low amount that the study of its mode of action was hindered by the shortage of purified protein . We report here the expression of C-terminally hexa-His-tagged factor C in Streptomyces lividans and Escherichia coli . Expression in S . lividans is low while in E . coli it is relatively high, yielding about 5--10 mg of biologically fully active protein per liter culture.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 129 - 33
Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis; Kulik EM et al.; A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease . One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii . These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced . M . oralis was found to be the predominant archaeon in the subgingival dental plaque . Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp . within the Methanobacteriaceae family.

Biochim Biophys Acta, 2001 Mar 19, 1518(1-2), 178 - 82
Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K; Tsuji H et al.; A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated . The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail . The open reading frame was shown to encode a polypeptide composed of 473 amino acids . The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids . The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen . Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein . This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.

Biochem Pharmacol, 2001 Mar 15, 61(6), 651 - 6
Expression of rat liver long-chain acyl-CoA synthetase and characterization of its role in the metabolism of R-ibuprofen and other fatty acid-like xenobiotics; Bruggera R et al.; Our investigations of fatty acid metabolism and epimerization of the 2-arylpropionic acid derivative, R-ibuprofen, resulted in the successful purification of an acyl-CoA synthetase from rat liver microsomes that catalyzes the formation of both palmitoyl-CoA and R-ibuprofenoyl-CoA . To investigate whether R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase (LACS) are identical enzymes, we cloned the cDNA from LACS into the pQE30 expression vector and transformed the construct into Escherichia coli M15{pREP4} . Induction of the bacterial protein synthesis with 0.2 mM isopropyl-beta-D-galactoside resulted in a strong, time-dependent increase in LACS protein as determined by Western blot analysis using a polyclonal rabbit anti-LACS antibody . Incubations of the recombinantly expressed protein with palmitic acid as physiological LACS substrate or R-ibuprofen in the presence of Mg2+, ATP, and CoA resulted in a 5-fold increase in the thioesterification of both substrates . Western blot analysis using tissue homogenates of rat liver, heart, kidney, lung, brain, and ileum showed that LACS was found in every tissue investigated, with the greatest expression in the liver . Similar results were obtained with activity measurements using R-ibuprofen and palmitic acid as substrates . Northern blot analysis revealed a hybridization with a 3.8-kb mRNA transcript in rat liver, heart, and kidney, but no signal was observed in lung, brain and ileum, suggesting the expression of different LACS isoform(s) in these organs . In summary, our results further show that R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase are identical enzymes that are involved in the metabolism of various xenobiotics.

Protein Sci, 2001 Feb, 10(2), 397 - 410
Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike; Haase-Pettingell C et al.; The predominantly beta-sheet phage P22 tailspike adhesin contains eight reduced cysteines per 666 residue chain, which are buried and unreactive in the native trimer . In the pathway to the native trimer, both in vivo and in vitro transient interchain disulfide bonds are formed and reduced . This occurs in the protrimer, an intermediate in the formation of the interdigitated beta-sheets of the trimeric tailspike . Each of the eight cysteines was replaced with serine by site-specific mutagenesis of the cloned P22 tailspike gene and the mutant genes expressed in Escherichia coli . Although the yields of native-like Cys>Ser proteins varied, sufficient soluble trimeric forms of each of the eight mutants accumulated to permit purification . All eight single Cys>Ser mature proteins maintained the high thermostability of the wild type, as well as the wild-type biological activity in forming infectious virions . Thus, these cysteine thiols are not required for the stability or activity of the native state . When their in vivo folding and assembly kinetics were examined, six of the mutant substitutions--C267S, C287S, C458S, C613S, and C635S--were significantly impaired at higher temperatures . Four--C290S, C496, C613S, and C635--showed significantly impaired kinetics even at lower temperatures . The in vivo folding of the C613S/C635S double mutant was severely defective independent of temperature . Since the trimeric states of the single Cys>Ser substituted chains were as stable and active as wild type, the impairment of tailspike maturation presumably reflects problems in the in vivo folding or assembly pathways . The formation or reduction of the transient interchain disulfide bonds in the protrimer may be the locus of these kinetic functions.

Protein Sci, 2001 Feb, 10(2), 384 - 96
Solution nuclear magnetic resonance structure of a protein disulfide oxidoreductase from Methanococcus jannaschii; Cave JW et al.; The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance . The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins . Some aspects of Mj0307's unique behavior can be explained by comparing structure-based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins . It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins . Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.

Protein Sci, 2001 Feb, 10(2), 262 - 76
Plasticity of quaternary structure: twenty-two ways to form a LacI dimer; Swint-Kruse L et al.; The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences . Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure . To elucidate the determinants of assembly, a structure-based alignment has been created for three repressors and four periplasmic binding proteins . Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions . These analyses show few strict requirements for assembly of the core N-subdomain interface . The interfaces of repressor core C-subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282 . However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281 . Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second-site mutation in the core N-subdomain at K84 to L or A . Using the link between LacI assembly and function, we have further identified 22 second-site mutations that compensate the Y282D dimerization defect in vivo . The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism . Thus, the 360-amino acid scaffold of LacI allows plasticity of its quaternary structure . The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.

Protein Sci, 2001 Jan, 10(1), 135 - 48
Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: relative stabilities in experiment and simulations; Sham YY et al.; We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature . The simulations have shown fragments 1--36, 37--88, and 89--159 to be unstable, with a C(alpha)RMSD (C(alpha) root mean squared deviation) >5 A after 3.0 nsec of simulation . The unfolding of fragment 1--36 was immediate, whereas fragments 37--88 and 89--159 gradually unfolded because of the presence of the beta-sheet core structure . In the absence of residues 1--36, the two distinct domains comprising fragment 39--159 associated with each other, resulting in a stable conformation . This conformation retained most of its native structural elements . We have further simulated fragments derived from computational protein cutting . These were also found to be unstable, with the exception of fragment 104--159 . In the absence of alpha(4), the loose loop region of residues 120--127 exhibited a beta-strand-like behavior, associating itself with the beta-sheet core of the protein fragment . The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution . Finally, the critical role of residues 1--36 in allowing the two distinct domains of fragment 104--159 to fold into the final native conformation is discussed.

Protein Sci, 2001 Jan, 10(1), 108 - 15
Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions; Henriksen A et al.; Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties . It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide . Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study . The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS) . This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex . TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance . SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site . A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure . This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.

Protein Sci, 2001 Jan, 10(1), 55 - 62
A model of dynamic side-chain--side-chain interactions in the alpha-lactalbumin molten globule; Bai P et al.; Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology . Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein-folding problem . To explore the nature of side-chain--side-chain interactions in the alpha-lactalbumin (alpha-LA) molten globule, we determined the effective concentration for formation of the 28--111 disulfide bond in 14 double-mutant proteins, each containing two hydrophobic core residues replaced by alanine . We compared our results with those of single-alanine substitutions using the framework of double-mutant cycle analysis and found that, in the majority of cases, the effects of two alanine substitutions are additive . Based on these results, we propose a model of side-chain-side-chain interactions in the alpha-LA molten globule, which takes into consideration the dynamic nature of this partially folded species.

Protein Sci, 2001 Jan, 10(1), 46 - 54
Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: a graph spectral and point mutation study; Kannan N et al.; The formation of alpha(2) dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme . A large number of evidences indicate that the alpha-subunit dimerizes through its N-terminal domain (NTD) . The crystal structures of the alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known . To identify the stabilizing interactions in the dimer interface of the alpha-NTD of E . coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E . coli alpha-NTD . A graph spectral algorithm was used to identify side-chain clusters . This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way . By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core . The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues . Most of the cluster residues in the E . coli enzyme were topologically and sequentially conserved in the T . aquaticus RNAP crystal structure . Residues 35F and 46I were predicted to be important in the stability of the alpha-dimer interface, with 35F forming the center of the cluster . The predictions were tested by isolating single-point mutants alpha-F35A and alpha-I46S on the dimer interface, which were found to disrupt dimerization . Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha-NTD.

Protein Sci, 2001 Jan, 10(1), 12 - 6
Second virial coefficients as a measure of protein--osmolyte interactions; Weatherly GT et al.; The cytoplasm contains high concentrations of cosolutes . These cosolutes include macromolecules and small organic molecules called osmolytes . However, most biophysical studies of proteins are conducted in dilute solutions . Two broad classes of models have been used to describe the interaction between osmolytes and proteins . One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte . To better understand protein--smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein . From these experiments, we determined the second virial coefficients for a series of osmolytes . We have interpreted the second virial coefficient as a measure of both excluded volume and protein--osmolyte binding . We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins.

Nucleic Acids Res . 2001 Apr 1;29(7):E37.
Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli; Sosio M et al.; We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC) . The procedure makes use of iterative rounds of homologous recombination in Escherichia coli . Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required . They are transferred first into a temperature-sensitive replicon and then into the BAC of choice . We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli-STREPTOMYCES: artificial chromosome (ESAC) . Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1549 - 55
Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease; Farez-Vidal ME et al.; The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease . We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism . The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable . The gene is single copy and is located on a chromosome of approximately 1.9 Mb . A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies . Western blot analysis revealed the existence of a single UDGase species in parasite extracts . Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA . The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity . This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1458 - 63
Site-specifically located 8-amino-2'-deoxyguanosine: thermodynamic stability and mutagenic properties in Escherichia coli; Venkatarangan L et al.; 2-Nitropropane (2-NP), an important industrial solvent and a component of cigarette smoke, is mutagenic in bacteria and carcinogenic in rats . 8-Amino-2'-deoxyguanosine (8-amino-dG) is one of the types of DNA damage found in liver, the target organ in 2-NP-treated rats . To investigate the thermodynamic properties of 8-amino-dG opposite each of the four DNA bases, we have synthesized an 11mer, d(CCATCG*CTACC), in which G* represents the modified base . By annealing a complementary DNA strand to this modified 11mer, four sets of duplexes were generated each containing one of the four DNA bases opposite the lesion . Circular dichroism studies indicated that 8-amino-dG did not alter the global helical properties of natural right-handed B-DNA . The thermal stability of each duplex was examined by UV melting measurements and compared with its unmodified counterpart . For the unmodified 11mer, the relative stability of the complementary DNA bases opposite G was in the order C > T > G > A, as determined from their -DeltaG degrees values . The free energy change of each modified duplex was lower than its unmodified counterpart, except for the G*:G pair that exhibited a higher melting transition and a larger -DeltaG degrees than the G:G duplex . Nevertheless, the stability of the modified 11mer duplex also followed the order C > T > G > A when placed opposite 8-amino-dG . To explore if 8-amino-dG opposite another 8-amino-dG has any advantage in base pairing, a G*:G* duplex was evaluated, which showed that the stability of this duplex was similar to the G*:G duplex . Mutagenesis of 8-amino-dG in this sequence context was studied in Escherichia coli, which showed that the lesion is weakly mutagenic (mutation frequency approximately 10(-3)) but still can induce a variety of targeted and semi-targeted mutations.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1426 - 32
Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions; Brannvall M et al.; Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA . In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions . Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example . We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine . Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe) . We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites . Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.

J Cell Biol, 2001 Jan 22, 152(2), 411 - 7
Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import; Ben-Efraim I et al.; Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins . The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system . We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality . To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively . These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import . Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153 . Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import . These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Arch Virol, 2001, 146(1), 59 - 69
Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli; Lopez Vazquez AL et al.; All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conservation . Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus . Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates . In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate specificity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates . Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a "copy-back" mechanism that initiated at the template 3'-terminal OH.

Arch Virol, 2001, 146(1), 15 - 25
Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus; Olsen BS et al.; The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA . The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail . Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides . The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537 . Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses . Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator . Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA . Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions . Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield . Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus . Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus . Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

Mol Cells, 2001 Feb 28, 11(1), 48 - 54
Molecular cloning and cultivar specific expression of MAP kinases from Capsicum annuum; Shin HJ et al.; Two MAP kinases, MK1 and MK2, were cloned from Capsicum annuum (pepper) cv . Subicho using a parsley MAP kinase gene as a heterologous probe . MK1 and MK2 encode stress-inducible protein kinases that can contribute to the response to wounding, UV-C, and cold . MK1 has a 92% amino acid identity with WIPK of tobacco . It was transcriptionally induced in response to wounding . In contrast, no detectable MK1 transcript was found in unwounded leaves of pepper . MK2 has a high level of amino acid homology to MAP kinases, such as NTF4 and SIPK and was constitutively expressed in all tissues . Both MK transcripts were downregulated by UV-C treatment . Each MK protein activation was independently wound-inducible in a cultivar dependent manner . MK1 is phosphorylated in cv . Pungchon but not cv . Subicho; whereas, the MK2 protein activation by wounding is restricted to cv . Subicho . In addition, de novo synthesis of the MK1 protein and tyrosine phosphorylation was rapidly and transiently induced in cv . Pungchon by wounding . In contrast, it is highly unlikely that the MK1 protein is produced in cv . Subicho, even though there is an abundant expression of MK1 mRNA after wounding in this cultivar . In Escherichia coli, which overexpresses MK1, autophosphorylation is observed at conserved threonine and tyrosine phosphorylation sites.

EMBO Rep, 2000 Aug, 1(2), 183 - 9
The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer; Braun T et al.; GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily . To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity . Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length . Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF . Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution . Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers . In projection, GlpF is similar to AQP1, the erythrocyte water channel . However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

Free Radic Res, 2001 Dec, 33(6), 757 - 770
Cross-Talk between No and Oxyradicals, a Supersystem that Regulates Energy Metabolism and Survival of Animals; Inoue M et al.; Mammalian tissues have large amounts of available ATP which are generated by oxidative phosphorylation in mitochondria . For the maintenance of the human body, a large amount of oxygen is required to regenerate these ATP molecules . A small fraction of the inspired oxygen is converted to superoxide radical and related metabolites even under physiological conditions . Most reactive oxygen species react rapidly with a variety of molecules thereby interfering with cellular functions and induce various diseases . Nitric oxide (NO) is an unstable gaseous radical with high affinity for various molecules, such as hemeproteins, thiols, and related radicals . NO easily penetrates through cell membrane/lipid bilayers, forms dissociable complexes with these molecules and modulates cellular metabolism and functions . Because NO has an extremely high affinity for the superoxide radical, the occurrence of the latter might decrease the biological function of NO . Thus, superoxide radicals in and around vascular endothelial cells play critical roles in the pathogenesis of hypertension and vasogenic tissue injury . Because NO also reacts with molecular oxygen, it rapidly loses its biological activity, particularly under ambient atmospheric conditions where the oxygen tension is unphysiologically high . Thus, biological functions of NO are determined by the local concentrations of molecular oxygen and superoxide radicals . NO also inhibits electron transfer reaction and ATP synthesis in mitochondria and aerobic bacteria, such as E . coli; the inhibitory effects are also enhanced by hypoxia . Thus, the cross-talk between NO, molecular oxygen and oxyradicals play critical roles in the regulation of energy metabolism, fates and the survival of aerobic organisms . The present work describes the pathophysiological significance of the supersystem driven by the cross-talk between NO and oxyradicals.

Eur J Clin Invest, 2001 Mar, 31(3), 264 - 71
Closed-circuit organ perfusion technique for gene transfer into the lungs . An experimental trial on farm pigs; Parpala-Sparman T et al.; In an attempt to develop gene therapy for lung diseases, we have explored a closed-circuit surgical perfusion method for gene transfer into the lung . For gene transfer we used a replication defective type 5 adenovirus carrying the E . coli beta-galactosidase gene as a reporter gene . The middle lobe of the right lung of eight young farm pigs was perfused in vivo via thoracotomy for up to 60 min with the viral solution . The gene transfer was performed using a closed-circuit organ perfusion method in vivo . The efficiency of gene transfer was assessed visually by analysis of histologic sections after X-gal, PAS and immunohistochemical stainings . The lung perfusion resulted in transgene expression in the alveolar epithelial cells, capillary endothelial cells, airway epithelial cells and alveolar macrophages of the lung examined seven days after perfusion . The present results suggest that operatively performed closed-circuit warm lung perfusion method may be used for gene transfer in treatment of diseases that have pulmonary manifestations.

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 614 - 5
Purification, crystallization and preliminary X-ray studies of thermostable alkaline phosphatase from Thermus sp . 3041; Ji CN et al.; Thermostable alkaline phosphatase from Thermus sp . 3041 has been expressed in Escherichia coli, purified and crystallized . The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A . Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 592 - 5
Human phosphoglucose isomerase: expression, purification, crystallization and preliminary crystallographic analysis; Cordeiro AT et al.; Phosphoglucose isomerase (PGI) is the second enzyme in the glycolytic pathway and catalyzes an aldose-ketose isomerization . Outside the cell, PGI has been found to function as both a cytokine and as a growth factor . The human pgi gene was cloned and the expressed enzyme was purified to homogeneity . Isomorphous crystals were obtained under two conditions and belong to the P2(1)2(1)2(1) space group, with unit-cell parameters a = 80.37, b = 107.54, c = 270.33 A . A 94.7% complete data set was obtained and processed to a limiting resolution of 2.6 A . The asymmetric unit contains two hPGI dimers according to density calculations, a self-rotation function map and molecular-replacement solution.

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 506 - 15
The structure of human mitochondrial branched-chain aminotransferase; Yennawar N et al.; X-ray crystal structures of three forms of human mitochondrial branched-chain aminotransferase (BCAT) were solved by molecular-replacement methods, using Escherichia coli BCAT as the search model . The enzyme is a homodimer and the polypeptide chain of each monomer has two domains . The small domain is composed of residues 1--175 and the large domain is composed of residues 176--365 . The active site is close to the dimer interface . The 4'-aldehyde of the PLP cofactor is covalently linked to the epsilon-amino group of the active-site lysine, Lys202, via a Schiff-base linkage in two of the structures . In the third structure, the enzyme is irreversibly inactivated by Tris . The overall fold of the dimer in human mitochondrial BCAT is similar to the structure of two bacterial enzymes, E . coli BCAT and D-amino acid aminotransferase (D-AAT) . The residues lining the putative substrate-binding pocket of human BCAT and D-AAT are completely rearranged to allow catalysis with substrates of opposite stereochemistry . In the case of human mitochondrial branched-chain aminotransferase, a hydrogen-bond interaction between the guanidinium group of Arg143 in the first monomer with the side-chain hydroxyl of Tyr70 in the second monomer is important in the formation of the substrate-binding pocket.

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 498 - 505
Three-dimensional structure of human RNase 1 delta N7 at 1.9 A resolution; Pous J et al.; Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A . Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI) . This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized . Its crystal structure has been determined and refined using data to 1.9 A resolution . The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily . The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite . These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

Mol Biol Evol, 2001 Apr, 18(4), 530 - 41
Giardia lamblia expresses a proteobacterial-like DnaK homolog; Morrison HG et al.; We identified a novel gene encoding molecular chaperone HSP70 in the amitochondriate parasite Giardia lamblia . The predicted protein is similar to bacterial DnaK and mitochondrial HSP70s . The gene is transcribed and translated at a constant level during trophozoite growth and encystation . Alignment of the sequence with a data set of cytosolic, endoplasmic reticulum (ER), mitochondrial, and DnaK HSP70 homologs indicated that the sequence was extremely divergent and contained insertions unique to giardial HSP70s . Phylogenetic analyses demonstrated that this sequence was distinct from the cytosolic and ER forms and was most similar to proteobacterial and mitochondrial DnaKs . However, a specific relationship with the alpha proteobacterial and mitochondrial sequences was not strongly supported by phylogenetic analyses of this data set, in contrast to similar analyses of cpn60 . These data neither confirm nor reject the possibility that this gene is a relic of secondary mitochondrial loss; they leave open the possibility that it was acquired in a separate endosymbiotic event.

J Biol Chem, 2001 Jun 8, 276(23), 20387 - 96 Epub 2001 Mar 22.
Imidazole glycerol phosphate synthase from Thermotoga maritima . Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex; Beismann-Driemeyer S et al.; Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family . In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF . Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide . In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized . Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound . Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics . Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate . The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 219 - 27
Toward the three-dimensional structure of the Escherichia coli DNA-binding protein H-NS: A CD and fluorescence study; Schroder O et al.; The DNA-binding protein H-NS compacts DNA and acts as a specific transcription factor regulating the expression of various bacterial genes . The small abundant protein binds to curved DNA without apparent sequence specificity and the exact nature of its DNA interaction is still unknown . H-NS lacks any common DNA-binding or oligomerization motif and except for a C-terminal fragment of the protein no high resolution structural information is available today . Since the complete structure of H-NS is of considerable interest for understanding its versatile regulatory features, and in lack of high-resolution data for the complete molecule, we have combined circular dichroism (CD) and fluorescence measurements to collect secondary- and higher-order structural information on H-NS . Comparison of CD analyses of wild type H-NS and functional defective mutants allowed assigning secondary structure elements to the N-terminal oligomerization domain of the protein . Moreover, according to fluorescence energy-transfer data we calculate a 45 A distance between the DNA-binding and the oligomerization domain of H-NS .

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 131 - 41
Identification and structure of the nerve growth factor binding site on TrkA; Robertson AG et al.; Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease . NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains . We have expressed the second Ig-like domain as a recombinant protein in E . coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor . This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation . We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution .

Acc Chem Res, 2001 Feb, 34(2), 145 - 57
The crotonase superfamily: divergently related enzymes that catalyze different reactions involving acyl coenzyme a thioesters; Holden HM et al.; Synergistic investigations of the reactions catalyzed by several members of an enzyme superfamily provide a more complete understanding of the relationships between structure and function than is possible from focused studies of a single enzyme alone . The crotonase (or enoyl-CoA hydratase) superfamily is such an example whereby members catalyze a wide range of metabolic reactions but share a common structural solution to a mechanistic problem . Some enzymes in the superfamily have been shown to display dehalogenase, hydratase, and isomerase activities . Others have been implicated in carbon-carbon bond formation and cleavage as well as the hydrolysis of thioesters . While seemingly unrelated mechanistically, the common theme in this superfamily is the need to stabilize an enolate anion intermediate derived from an acyl-CoA substrate . This apparently is accomplished by two structurally conserved peptidic NH groups that provide hydrogen bonds to the carbonyl moieties of the acyl-CoA substrates and form an "oxyanion hole".

Arthritis Rheum, 2001 Mar, 44(3), 712 - 22
Activation of a fibroblast-specific enhancer of the proalpha2(I) collagen gene in tight-skin mice; Denton CP et al.; OBJECTIVE: Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis . METHODS: Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding . Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates . Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells . RESULTS: There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age . This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001) . Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture . CONCLUSION: These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene . Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.

Life Sci, 2001 Feb 23, 68(14), 1677 - 85
Metabolic activation of mitomycin C by NADPH-ferredoxin reductase in vitro; Jiang HB et al.; Mammalian NADPH-ferredoxin reductase (EC 1.18.1.2) functions in the mitochondrial electron transport chain for cytochrome P-450-dependent steroid hydroxylation . Significant homology of three-dimensional structure exists in the surroundings of FAD between NADPH-ferredoxin reductase and NADH-cytochrome b5 reductase . The latter is involved in the bioreduction of mitomycin C (MC), a prototype antitumor agent . In this study, we assessed the capacity of NADPH-ferredoxin reductase to activate MC . Mitomycin C increased the NADPH oxidase activity of NADPH-ferredoxin reductase . In the absence of ferredoxin, the Km value of NADPH-ferredoxin reductase for MC was 73.5 +/- 2.3 microM . While in the presence of 500 nM ferredoxin, a Lineweaver-Burk plot exhibited a biphasic curve . NADPH-ferredoxin reductase-mediated reduction of MC resulted in the formation of an alkylated complex of 4-(p-nitrobenzyl) pyridine and an increase in plasmide DNA single-strand breaks under hypoxic conditions . With the addition of 500 nM ferredoxin, the amount of the alkylated complex of 4-(p-nitrobenzyl) pyridine and the plasmide DNA single-strand breaks increased by 40% and 37%, respectively . However, neither alkylated complex of 4-(p-nitrobenzyl) pyridine nor DNA strand breaks was observed in the presence of SOD and catalase under aerobic conditions . These findings demonstrate that NADPH-ferredoxin reductase is capable of catalyzing the bioactivation of mitomycin C under hypoxic conditions in vitro.

Transpl Int, 2001, 14(1), 44 - 7
Hemorrhagic colitis due to a novel Escherichia coli serotype (O121:H19) in a transplant patient; Stock KJ et al.; Infection due to enterohemorrhagic Escherichia coli (EHEC) has not been described in immunosuppressed patients . We recently saw a case of EHEC infection caused by a novel Shiga toxin II-producing Escherichia coli serotype (O121:H19) that caused hemorrhagic colitis in a patient with renal and cardiac transplants . The patient's signs, symptoms, and colon pathology were similar to reports of EHEC infection in immunocompetent patients . This case suggests that the immunosuppressed state may not alter the clinical presentation or histopathologic findings of this disorder . Assays for EHEC are not routinely done at most hospitals . Therefore, clinicians caring for transplant patients should be aware of the typical clinical presentation of EHEC infection, so that they can initiate appropriate laboratory investigation in suspected cases.

EMBO Rep, 2000 Dec, 1(6), 494 - 9
Escherichia coli cell cycle control genes affect chromosome superhelicity; Weitao T et al.; We have used ethidium bromide titration for direct measurement of the changes in the negative supercoiling of Escherichia coli chromosome caused by mutations inactivating the cell cycle functions mukB and seqA . The amounts of the intercalative agent required to relax the supercoiled chromosome in mukB and seqA mutants were lower and higher, respectively, than for the wild-type parent, confirming that these cell cycle genes modulate the topology of the E . coli chromosome . Plasmid superhelicity measured in these mutant strains showed similar effects albeit of reduced magnitude . As the effects of mukB and seqA mutations were not restricted to the chromosome alone, MukB and SeqA proteins possibly interact with factors involved in the maintenance of intracellular DNA topology . To our knowledge, this is the first direct demonstration of the influence of mukB and seqA genes on the superhelicity of the E . coli chromosome.

EMBO Rep, 2000 Dec, 1(6), 484 - 8
The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity; Wagner J et al.; The recent discovery of a new family of ubiquitous DNA polymerases involved in translesion synthesis has shed new light onto the biochemical basis of mutagenesis . Among these polymerases, the dinB gene product (Pol IV) is involved in mutagenesis in Escherichia coli . We show here that the activity of native Pol IV is drastically modified upon interaction with the beta subunit, the processivity factor of DNA Pol III . In the absence of the beta subunit Pol IV is strictly distributive and no stable complex between Pol IV and DNA could be detected . In contrast, the beta clamp allows Pol IV to form a stable initiation complex (t 1/2 approximately 2.3 min), which leads to a dramatic increase in the processivity of PoI IV reaching an average of 300-400 nucleotides . In vivo, the beta processivity subunit may target DNA Pol IV to its substrate, generating synthesis tracks much longer than previously thought.

EMBO Rep, 2000 Dec, 1(6), 479 - 83
Limiting DNA replication to once and only once; Boye E et al.; In Escherichia coli cells, the origin of chromosomal replication is temporarily inactivated after initiation has occurred . Origin sequestration is the first line of defence against over-initiation, providing a time window during which the initiation potential can be reduced by: (i) titration of DnaA proteins to newly replicated chromosomal elements; (ii) regulation of the activity of the DnaA initiator protein; and (iii) sequestration of the dnaA gene promoter . This review represents the first attempt to consider together older and more recent data on such inactivation mechanisms in order to analyze their contributions to the overall tight replication control observed in vivo . All cells have developed mechanisms for origin inactivation, but those of other bacteria and eukaryotic cells are clearly distinct from those of E . coli . Possible differences and similarities are discussed.

Pac Symp Biocomput . 2001;:532-43.
Simulating the growth of viruses; You L et al.; To explore how the genome of an organism defines its growth, we have developed a computer simulation for the intracellular growth of phage T7 on its E . coli host . Our simulation, which incorporates 30 years of genetic, biochemical, physiological, and biophysical data, is used here to study how the intracellular resources of the host, determined by the specific growth rate of the host, contribute toward phage development . It is also used to probe how changes in the linear organization of genetic elements on the T7 genome can affect T7 development . Further, we show how time-series trajectories of T7 mRNA and protein levels generated by the simulation may be used as raw data to test data-mining strategies, specifically, to identify partners in protein-protein interactions . Finally, we suggest how generalization of this work can lead to a knowledge-driven simulation for the growth of any virus.

Pac Symp Biocomput . 2001;:139-50.
A structure-based approach for prediction of protein binding sites in gene upstream regions; Mandel-Gutfreund Y et al.; The challenge of identifying DNA regulatory sequences based on sequence information only has been emphasized in view of the fast accumulation of new genes in the databases . While most predictive algorithms are based on multiple alignments of already known binding sites, here we examine the usefulness of a novel approach that is based on structural information of the protein-DNA complex . It has already been shown that specific recognition between a protein and its DNA target is achieved by stereo-chemical complementarity between the protein amino acids and the DNA bases . The proposed computational scheme uses crystallographic information to define the set of amino acid-base contacts between the proteins of a given DNA-binding protein family and their DNA targets . The compatibility of a given protein to bind to putative regulatory DNA sequences is then evaluated by knowledge-based parameters for amino acid-base interactions . By this procedure gene upstream regions may be screened for potential binding sites for regulatory proteins . Predictions are demonstrated for the E . coli cyclic AMP receptor protein (CRP) which recognizes the DNA via the helix-turn-helix motif, and for various Zif268-like proteins which belong to the Cys2His2 zinc finger family . The advantages and limitations of this approach are discussed.

Pac Symp Biocomput . 2001;:103-14.
Stress-induced DNA duplex destabilization in transcriptional initiation; Benham CJ; Stress-induced destabilization of the DNA double helix (SIDD) is involved in several mechanisms by which transcription is regulated . This paper describes a computational method for predicting the locations and extents of destabilization as functions of DNA sequence and imposed superhelical stress . This method is used to investigate several transcriptional regulatory events . These include IHF-mediated activation of gene expression in E . coli, the bimodal control of the initiation of transcription from the human c-myc gene, and the determination of the minimal requirements for transcriptional activity in yeast . Collaborations with experimental groups have established the central role of SIDD in each of these processes.

J Biol Chem, 2001 Jun 15, 276(24), 21571 - 7 Epub 2001 Mar 21.
An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria; Ohtsuki T et al.; We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans . Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu . The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu . Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature . The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so . A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs . This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

Anal Biochem, 2001 Apr 1, 291(1), 109 - 17
Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins; McEwen DP et al.; Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically . Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively . Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline . Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM) . The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS . Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins . Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base . Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching . The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding .

Anal Biochem, 2001 Apr 1, 291(1), 89 - 95
Optical determination of glutamine using a genetically engineered protein; Dattelbaum JD et al.; We have developed a reagentless optical biosensor for glutamine based on the Escherichia coli glutamine binding protein (GlnBP) . Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes . The fluorescence emission of acrylodan and 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35%, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 microM; K(Dapp) 160 nM) . No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine, and arginine . Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay . Anisotropy decay measurements for GlnBPS179C-acrylodan showed a 13-ns rotational correlation time in the ligand-free state, whereas multiple correlation times were assigned in the glutamine-bound conformation . The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine . The engineered GlnBP is a first step toward the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures .

Anal Biochem, 2001 Apr 1, 291(1), 74 - 83
Adenosine triphosphate-dependent degradation of a fluorescent lambda N substrate mimic by Lon protease; Lee I et al.; Escherichia coli Lon exhibits a varying degree of energy requirement toward hydrolysis of different substrates . Efficient degradation of protein substrates requires the binding and hydrolysis of ATP such that the intrinsic ATPase of Lon is enhanced during protein degradation . Degradation of synthetic tetrapeptides, by contrast, is achieved solely by ATP binding with concomitant inhibition of the ATPase activity . In this study, a synthetic peptide (FRETN 89-98), containing residues 89-98 of lambda N protein and a fluorescence donor (anthranilamide) and quencher (3-nitrotyrosine), has been examined for ATP-dependent degradation by E . coli and human Lon proteases . The cleavage profile of FRETN 89-98 by E . coli Lon resembles that of lambda N degradation . Both the peptide and protein substrates are specifically cleaved between Cys93 and Ser94 with concomitant stimulation of Lon's ATPase activity . Furthermore, the degradation of FRETN 89-98 is supported by ATP and AMPPNP but not ATPgammaS nor AMPPCP . FRETN 89-98 hydrolysis is eight times more efficient in the presence of 0.5 mM ATP compared to 0.5 mM AMPPNP at 86 microM peptide . The ATP-dependent hydrolysis of FRETN 89-98 displays sigmodial kinetics . The k(cat), {S}(0.5), and the Hill coefficient of FRETN 89-98 degradation are 3.2 +/- 0.3 s(-1), 106 +/- 21 microM, and 1.6 respectively .

Biosens Bioelectron, 2001 Jan, 16(1-2), 1 - 8
Genomic DNA hybridizes with the same rate constant on the QCM biosensor as in homogeneous solution; Towery RB et al.; Hybridization rates of sheared, genomic E . coli DNA in 0.14 M, pH 6.7 phosphate buffer at 65 degrees C were determined by: (1) observing the rate of absorbance decrease at 260 nm due to self-hybridization in solution; and (2) measurement of the rate of mass increase caused by hybridization between DNA in solution and DNA photografted to polystyrene . The latter measurement was done using a quartz crystal microbalance (QCM) . In both the spectrophotometric and QCM experiments the probe was identical to the target, as both were taken from the same sample of sheared E . coli DNA . In the QCM measurements, viscoelastic effects were made negligible by drying the biopolymer layer on the QCM's surface before taking the frequency readings . Our purpose was to explore the effect of immobilizing DNA on its hybridization rate constant . A second-order constant of 2.32 +/- 0.09 x 10(-6) ml microg(-1) s(-1), n = 14, for hybridization in solution was obtained spectrophotometrically, while the QCM experiment gave a constant of 2.2 +/- 0.3 x 10(-6) ml microg(-1) s(-1), n = 6 . These values are not statistically different . The reaction half-lives for the spectrophotometric and QCM experiments were 6.5 h and 13 min, respectively . The shorter half-life on the QCM can be explained solely by the much greater reactant concentration in the QCM experiment . About 25% of the DNA was inactivated by the attachment reaction . After correcting for this, the surface-attached DNA hybridized with the same rate constant as DNA free in solution . Therefore, it is concluded that, in these specific experiments with genomic DNA, the immobilized regions must have been short compared to the length of the molecules . The data demonstrate the high hybridization rate obtainable when nucleic acids are hybridized in a thin-film, micro-volume reaction on a non-porous surface.

Mol Microbiol, 2001 Mar, 39(6), 1572 - 84
CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli; Yamanaka K et al.; CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli . Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication . CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity . CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro . Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA . We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.

Mol Microbiol, 2001 Mar, 39(6), 1550 - 61
RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II; Cairrao F et al.; In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment . Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability . RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E . coli extracts . We have identified and characterized a new E . coli gene, which was named gmr (gene modulating RNase II) . The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity . Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain . Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability . RNase II protein turnover is slower in the gmr deletion strain . We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr . The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.

Mol Microbiol, 2001 Mar, 39(6), 1504 - 22
The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215; Weyand NJ et al.; Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli . Both in vitro and in vivo analyses indicate that binding of cAMP-CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription . CAP-dependent activation of papBA requires binding of the leucine-responsive regulatory protein (Lrp) at binding sites that extend from -180 to -149 relative to the start site of papBA . Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites . Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP-dependent promoters . Also, like class I promoters, the C-terminal domain of the alpha-subunit of RNA polymerase appears to play a role in transcription activation . Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site . Using an 'oriented heterodimer' approach with wild-type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter-distal subunit is not . Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H-NS . The results presented here show that AR1 of the promoter-proximal CAP subunit was required for papBA transcription even in the absence of the histone-like protein H-NS . These results show that the promoter-proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C-terminal domain of the alpha-subunit of RNA polymerase.

Mol Microbiol, 2001 Mar, 39(6), 1427 - 33
Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids; Matsumoto K; The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane . However, we successfully constructed a null pgsA mutant of E . coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable . In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor . This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E . coli . The anionic phospholipids may play dual roles in E . coli as (i) substrates for head group-specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group-specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity . These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.

Genes Cells, 2001 Feb, 6(2), 121 - 9
Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs; Martin GW 3rd et al.; BACKGROUND: Selenocysteine incorporation has been reported to be inefficient in all systems studied, including Escherichia coli, baculovirus-insect cell systems, rabbit reticulocyte in vitro translation systems, transiently transfected mammalian cells, and intact animals . Nonetheless, full-length selenoproteins containing up to 17 selenocysteine residues are produced in animals, indicating that the efficiency observed in manipulated systems might not accurately reflect the true efficiency of this process in nature . RESULTS: To begin to address this apparent discrepancy, we have examined the polysome profiles of endogenously expressed selenoprotein mRNAs in a mammalian cell line, and compared them with nonselenoprotein mRNAs . We report that three selenoprotein mRNAs, type 1 deiodinase, glutathione peroxidase and selenoprotein P, are under-loaded with ribosomes, based on their predicted open reading frame sizes . The average numbers of ribosomes per mRNA correspond to the sizes predicted by termination at the UGA selenocysteine codons . Appropriate loading on the type 1 deiodinase mRNA is seen following substitution of a cysteine codon for the selenocysteine codon, indicating that the UGA codon confers a translational penalty on the mRNA . Surprisingly, ribosomal loading is also increased by the expression of eukaryotic release factors eRF1 and eRF3 . CONCLUSIONS: These results suggest that the presence of a selenocysteine codon confers a translational penalty on selenoprotein mRNAs, and that increased levels of release factors may alter the kinetics of termination.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3685 - 9 Epub 2001 Mar 20.
The Sm domain is an ancient RNA-binding motif with oligo(U) specificity; Achsel T et al.; Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms . These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches . Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins . Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli . This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer . It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer . Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer . These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3720 - 5 Epub 2001 Mar 20.
Solution structure of the A loop of 23S ribosomal RNA; Blanchard SC et al.; The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA . The A loop is highly conserved and contains a ubiquitous 2'-O-methyl ribose modification at position U2552 . Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification . Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552 . NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry . Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555 . We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure {Ban, N., Nissen, P., Hansen, J., Moore, P . B . & Steitz, T . A . (2000) Science 289, 905--920; Nissen, P., Hansen, J., Ban, N., Moore, P . B . & Steitz, T . A . (2000) Science 289, 920--930} . The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center . Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2'-O-methylation in the mechanism of translation, are discussed.

Mol Pharmacol, 2001 Apr, 59(4), 692 - 8
Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle; Li M et al.; The motility of gastrointestinal tract is regulated by classical neurotransmitters, neuropeptides, and humoral agents . Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein, Bv8, and a nontoxic protein of mamba snake venom . These human cDNAs encode two secreted proteins of 86 and 81 amino acids . Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach . Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation . Mass spectrometry has confirmed the formation of five pairs of disulfide bonds . The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range . The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle . To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins . Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle . The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors . Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle . In summary, we have identified two novel endogenous regulators of gastrointestinal motility . The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.

J Neurochem, 2001 Mar, 76(6), 1887 - 94
Direct binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor; Cen B et al.; beta-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia and tolerance/dependence . This study was carried out to measure the direct interaction between beta-arrestins and opioid receptor . Immunoprecipitation experiments demonstrated that beta-arrestin 1 physically interacts with delta opioid receptor (DOR) co-expressed in human embryonic kidney 293 cells in an agonist-enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction . In vitro data from glutathione-S-transferase pull-down assay showed that the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR are both capable of and either domain is sufficient for binding to beta-arrestin 1 and 2 . Surface plasmon resonance determination further revealed that binding of CT and I3L of DOR to beta-arrestin is additive, suggesting these two domains bind at distinctly different sites on beta-arrestin without considerable spatial hindrance . This study demonstrated for the first time the direct binding of beta-arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.

J Biol Chem, 2001 Jun 15, 276(24), 21821 - 7 Epub 2001 Mar 20.
The pyrimidine ring-opened derivative of 1,N6-ethenoadenine is excised from DNA by the Escherichia coli Fpg and Nth proteins; Speina E et al.; It was previously shown that 1,N(6)-ethenoadenine (epsilonA) in DNA rearranges into a pyrimidine ring-opened derivative of 20-fold higher mutagenic potency in Escherichia coli (AB1157 lacDeltaU169) than the parental epsilonA (Basu, A . K., Wood, M . L., Niedernhofer, L . J., Ramos, L . A., and Essigmann, J . M . (1993) Biochemistry 32, 12793-12801) . We have found that at pH 7.0, the stability of the N-glycosidic bond in epsilondA is 20-fold lower than in dA . In alkaline conditions, but also at neutrality, epsilondA depurinates or converts into products: epsilondA --> B --> C --> D . Compound B is a product of water molecule addition to the C(2)-N(3) bond, which is in equilibrium with a product of N(1)-C(2) bond rupture in epsilondA . Compound C is a deformylated derivative of ring-opened compound B, which further depurinates yielding compound D . Ethenoadenine degradation products are not recognized by human N-alkylpurine-DNA glycosylase, which repairs epsilonA . Product B is excised from oligodeoxynucleotides by E . coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Nth) . Repair by the Fpg protein is as efficient as that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA . Ethenoadenine rearrangement products are formed in oligodeoxynucleotides also at neutral pH at the rate of about 2-3% per week at 37 degrees C, and therefore they may contribute to epsilonA mutations.

J Biol Chem, 2001 Jun 8, 276(23), 20641 - 7 Epub 2001 Mar 20.
Oxyanion binding alters conformation and quaternary structure of the c-terminal domain of the transcriptional regulator mode . Implications for molybdate-dependent regulation, signaling, storage, and transport; Gourley DG et al.; The molybdate-dependent transcriptional regulator ModE of Escherichia coli functions as a sensor of intracellular molybdate concentration and a regulator for the transcription of several operons that control the uptake and utilization of molybdenum . We present two high-resolution crystal structures of the C-terminal oxyanion-binding domain in complex with molybdate and tungstate . The ligands bind between subunits at the dimerization interface, and analysis reveals that oxyanion selectivity is determined primarily by size . The relevance of the structures is indicated by fluorescence measurements, which show that the oxyanion binding properties of the C-terminal domain of ModE are similar to those of the full-length protein . Comparisons with the apoprotein structure have identified structural rearrangements that occur on binding oxyanion . This molybdate-dependent conformational switch promotes a change in shape and alterations to the surface of the protein and may provide the signal for recruitment of other proteins to construct the machinery for transcription . Sequence and structure-based comparisons lead to a classification of molybdate-binding proteins.

J Biol Chem, 2001 Jun 15, 276(24), 21601 - 7 Epub 2001 Mar 20.
Rad23 provides a link between the Png1 deglycosylating enzyme and the 26 S proteasome in yeast; Suzuki T et al.; In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome . We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p . Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses . The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p . We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities.

J Biol Chem, 2001 Jun 8, 276(23), 19937 - 44 Epub 2001 Mar 20.
Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework . Analysis of global gene expression in Escherichia coli K12; Long AD et al.; We describe statistical methods based on the t test that can be conveniently used on high density array data to test for statistically significant differences between treatments . These t tests employ either the observed variance among replicates within treatments or a Bayesian estimate of the variance among replicates within treatments based on a prior estimate obtained from a local estimate of the standard deviation . The Bayesian prior allows statistical inference to be made from microarray data even when experiments are only replicated at nominal levels . We apply these new statistical tests to a data set that examined differential gene expression patterns in IHF(+) and IHF(-) Escherichia coli cells (Arfin, S . M., Long, A . D., Ito, E . T., Tolleri, L., Riehle, M . M., Paegle, E . S., and Hatfield, G . W . (2000) J . Biol . Chem . 275, 29672-29684) . These analyses identify a more biologically reasonable set of candidate genes than those identified using statistical tests not incorporating a Bayesian prior . We also show that statistical tests based on analysis of variance and a Bayesian prior identify genes that are up- or down-regulated following an experimental manipulation more reliably than approaches based only on a t test or fold change . All the described tests are implemented in a simple-to-use web interface called Cyber-T that is located on the University of California at Irvine genomics web site.

J Biol Chem, 2001 Jun 1, 276(22), 18905 - 7 Epub 2001 Mar 19.
The internal repeats in the Na+/Ca2+ exchanger-related Escherichia coli protein YrbG have opposite membrane topologies; Saaf A et al.; We have determined the topology of the Escherichia coli inner membrane protein YrbG, a putative Na(+)/Ca(2+) exchanger with homology to a family of eukaryotic ion exchangers . Our results show that the two homologous halves of YrbG both have five transmembrane segments but opposite membrane orientations . This has implications for our understanding of the function of Na(+)/Ca(2+) exchangers and provides an example of "divergent" evolution of membrane protein topology.

J Biol Chem, 2001 Jun 1, 276(22), 18968 - 76 Epub 2001 Mar 19.
Retention of heme in axial ligand mutants of succinate-ubiquinone xxidoreductase (complex II) from Escherichia coli; Maklashina E et al.; Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ubiquinone reduction by succinate . In the E . coli enzyme, heme b(556) is ligated between SdhC His(84) and SdhD His(71) . Contrary to a previous report (Vibat, C . R . T., Cecchini, G., Nakamura, K., Kita, K., and Gennis, R . B . (1998) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in both SdhC H84L and SdhD H71Q mutants of SdhCDAB . EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L (g(z) = 2.92) mutant and high spin heme in the SdhD H71Q mutant (g = 6.0) . The presence of low spin heme in the SdhC H84L mutant suggests that the heme b(556) is able to pick up another ligand from the protein . CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin heme of the SdhC H84L mutant . The g = 2.92 signal of the SdhC H84L mutant titrates with a redox potential at pH 7.0 (E(m)(,7)) of approximately +15 mV, whereas the g = 6.0 signal of the SdhD H71Q mutant titrates with an E(m)(,7) of approximately -100 mV . The quinone site inhibitor pentachlorophenol perturbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant . This finding suggests that the latter residue also plays an important role in defining the quinone binding site of the enzyme . The SdhC H84L mutation also results in a significant increase in the K(m) and a decrease in the k(cat) for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters . Overall, these data indicate that SdhC His(84) has an important role in defining the interaction of SdhCDAB with both quinones and heme b(556).

Biophys J, 2001 Apr, 80(4), 1837 - 50
Structural studies of the HIV-1 accessory protein Vpu in langmuir monolayers: synchrotron X-ray reflectivity; Zheng S et al.; Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain . It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4 . Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLgPC) at the water-air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC . Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles . The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer . At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure.

Biol Reprod, 2001 Apr, 64(4), 1147 - 52
Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa; Govind CK et al.; Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N:-terminal signal sequence and the C:-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein . A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies . Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12) . The solubilized protein was refolded in the presence of oxidized and reduced glutathione . The circular dichroism spectra revealed the presence of both alpha helical and beta sheet components in the secondary structure of the refolded r-bmZPB . The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB . The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa . In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region . Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB . These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization.

Virology, 2001 Mar 30, 282(1), 206 - 13
The basic loop of the RNase H domain of MLV RT is important both for RNase H and for polymerase activity; Boyer PL et al.; Escherichia coli RNase H has a basic extension that is involved in binding nucleic acid substrates . This basic extension is present in the RNase H of Moloney murine leukemia virus reverse transcriptase (MLV RT), but has been deleted from the RNase H of HIV-1 RT . Previous work showed that removing the basic loop from MLV RT (the mutant is called DeltaC) blocked viral replication; however, DeltaC MLV RT retained RNase H activity in an in situ gel assay . We prepared recombinant DeltaC MLV RT and compared its activity to wild-type MLV RT . The DeltaC mutant is impaired in both polymerase and RNase H activity; the pattern of defects suggests that the basic loop is involved in the binding of MLV RT to a heteropolymeric template-primer.

Virology, 2001 Mar 30, 282(1), 87 - 101
The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp); Agrawal S et al.; Hepatitis E virus (HEV) is the major cause of acute epidemic and sporadic hepatitis in the developing world . It is a positive-strand RNA virus with a genome length of about 7.2 kb . The replication mechanism of this virus is virtually unexplored . Identification of the regulatory elements involved in initiation of replication may help in designing specific inhibitors for therapy . In the positive-stranded RNA viruses the initiation of replication requires interaction of the 3' end of genome with its RNA-dependent RNA polymerase (RdRp) and possibly host-derived cofactors for synthesis of the minus-strand replicative intermediate . Secondary structure prediction of the conserved 3' end of the infectious HEV genome was carried out to identify possible stem-loop structures necessary for RNA-protein interaction and the model was confirmed by structure probing experiments . Electrophoretic mobility-shift assays showed specific binding of purified and refolded recombinant HEV RdRp protein to the 3' end of its RNA genome containing the poly(A) stretch . Mutations at the 3' end, in which the stem-loop structures were partially or completely destroyed or recreated revealed that the two stem-loop structures SL1 and SL2 at the 3' end and the poly(A) stretch are necessary for this binding . The interacting nucleotides in such an interaction were further identified by generating footprints of the complex by Pb(II)-induced hydrolysis . This specific binding of viral RdRp to the 3' end of HEV RNA directs the synthesis of complementary-strand RNA and thus such a binding domain might assume the role of a possible cis-acting element as a potential site for the initiation of replication .

Inorg Chem, 2001 Feb 26, 40(5), 919 - 27
Methylation of (2-methylethanethiol-bis-3,5-dimethylpyrazolyl)methane zinc complexes and coordination of the resulting thioether: relevance to zinc-containing alkyl transfer enzymes; Hammes BS et al.; A series of zinc complexes using a new tripodal, N(2)S, heteroscorpionate ligand (L3SH) that is isostructural and isoelectronic with the well-known N(3) trispyrazolylborates have been methylated in solution and the coordination properties of the resulting thioether examined . This system models the reactivity of zinc-containing enzymes involved in alkyl group transfers such as the DNA repair protein Ada from E . coli, or farnasyl transferase where it has been shown that the thioether resulting from alkyl group transfer remains in the coordination sphere of the zinc . The following complexes have been structurally characterized: {(L3S)ZnI} (1), {(L3SCH(3))ZnI(2)} (2), {(L3SCH(3))ZnI}BF(4) (3), {(L3SCH(3))Zn-mu-bis-acetato-mu-hydroxo-Zn(L3SCH(3))}BF(4) (5), {(L3SCH(3))ZnSPh(F5)}ClO(4) (7), and {(L3SCH(3))(2)Zn}(BF(4))(2) (8) . Complexes 3, 4, 5, 7, and 8 all display thioether coordination . Thus in the absence of superior anionic ligands, thioethers are reasonably good donors to zinc in either a tetrahedral or octahedral geometry . The methylation of the complex {(L3S)ZnSPh(F5)}, which contains two different thiols, produces a single product, 7, where only the aliphatic thiol has been alkylated . This observation validates the suggestion that reactivity in enzymes with multiple zinc-bound thiols could be controlled by differences in thiol pK(a) (Hammes, B . S.; Warthen, C . R.; Crans, D.; Carrano, C . J . J . Biol . Inorg . Chem . 2000, 6, 82 . Compound 7 is also of interest in that it resembles the metal ion-binding site of the blue copper protein, azurin.

Chem Res Toxicol, 2001 Feb, 14(2), 222 - 7
The lethal interaction and formation of a lipophilic ternary complex between 2,4,5-trichlorophenol and the Cu(II)-bis(1,10-phenanthroline) complex; Zhu BZ; When nonlethal levels of 2,4,5-trichlorophenol (0.2 mM) and the Cu(II)-bis(1,10-phenanthroline) complex {Cu(II)(OP)2} (0.1 microM) were combined, a remarkable synergistic cytotoxicity was observed as measured by the extent of bacterial inactivation . In contrast, no such synergism was observed for the combination of 2,4,5-trichlorophenol with the Cu(II)-bis(bathophenanthroline disulfonate) complex {Cu(II)(BPS)2} which has a chemical structure similar to Cu(II)(OP)2, except for the net charge . The synergism observed for 2,4,5-trichlorophenol and Cu(II)(OP)2 was found to be due to the neutralization of their opposite charge and formation of a lipophilic ternary complex which facilitated copper transport into the bacterial cells.

Biochemistry, 2001 Mar 20, 40(11), 3403 - 12
Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences; Gerchman Y et al.; Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained {Williams, K . A., Kaufer, U . G., Padan, E., Schuldiner, S . and Kuhlbrandt, W . (1999) EMBO J., 18, 3558-3563} . In these crystals NhaA exists as a dimer . Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer . Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters . Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column . The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments . Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place . Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE . The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers . Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA.

Biochemistry, 2001 Mar 20, 40(11), 3370 - 7
Solution structure of the DNA-binding domain of TraM; Stockner T et al.; The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented . The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy . It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical . The very first amino acids of the N-terminus are unstructured.

Biochemistry, 2001 Mar 20, 40(11), 3308 - 15
RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli; Wettich A et al.; An RNA that replicates with core RNA polymerase from E . coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI . Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP . Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons . The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates . Both RNA were single-stranded with much internal base-pairing but low melting points . Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters . There was evidence for 3'-terminal elongation on an intramolecular template . No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process . The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.

Biochemistry, 2001 Mar 20, 40(11), 3257 - 63
Identification of a stability determinant on the edge of the Tet repressor four-helix bundle dimerization motif; Schubert P et al.; Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues . Their active state is a homodimer formed by a four-alpha-helix bundle as the main interaction motif . We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle . The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible denaturation followed by fluorescence and circular dichroism . Both methods yield identical results . The deltaG(o)U (H2O) values are 60 and 75 kJ x mol(-1) . We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ x mol(-1) stability difference . Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability . The P184Q exchange leads to a more stable protein, whereas the G181D exchange located at the solvent's exposed edge of the four-helix bundle is solely responsible for the reduced stability . Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring residues . Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles . We assume that the larger conformational flexibility of Gly and the reduction of the negative surface charge could favor formation of the turn on the edge of the four-helix bundle.

Biochemistry, 2001 Mar 13, 40(10), 3215 - 21
Significance of nucleobase shape complementarity and hydrogen bonding in the formation and stability of the closed polymerase-DNA complex; Dzantiev L et al.; DNA polymerases insert a dNTP by a multistep mechanism that involves a conformational rearrangement from an open to a closed ternary complex, a process that positions the incoming dNTP in the proper orientation for phosphodiester bond formation . In this work, the importance and relative contribution of hydrogen-bonding interactions and the geometric shape of the base pair that forms during this process were studied using Escherichia coli DNA polymerase I (Klenow fragment, 3'-exonuclease deficient) and natural dNTPs or non-hydrogen-bonding dNTP analogues . Both the geometric fit of the incoming nucleotide and its ability to form Watson-Crick hydrogen bonds with the template were found to contribute to the stability of the closed ternary complex . Although the formation of a closed complex in the presence of a non-hydrogen-bonding nucleotide analogue could be detected by limited proteolysis analysis, a comparison of the stabilities of the ternary complexes indicated that hydrogen-bonding interactions between the incoming dNTP and the template increase the stability of the complex by 6-20-fold . Any deviation from the Watson-Crick base pair geometry was shown to have a destabilizing effect on the closed complex . This degree of destabilization varied from 3- to 730-fold and was found to be correlated with the size of the mismatched base pair . Finally, a stable closed complex is not formed in the presence of a ddNTP or rNTP . These results are discussed in relation to the steric exclusion model for the nucleotide insertion.

Biochemistry, 2001 Mar 13, 40(10), 3184 - 8
Helix packing in the lactose permease of Escherichia coli: distances between site-directed nitroxides and a lanthanide; Voss J et al.; By exploiting substrate protection of Cys148 in lactose permease, a methanethiosulfonate nitroxide spin-label was directed specifically to one of two Cys residues in a double-Cys mutant, followed by labeling of Cys148 with a thiol-reactive chelator that binds Gd(III) quantitatively . Distances between bound Gd(III) and the nitroxide spin-label were then studied by electron paramagnetic resonance . The results demonstrate that the Gd(III)-induced relaxation effects on nitroxides at positions 228, 226 (helix VII), and 275 (helix VIII) agree qualitatively with results obtained by studying spin-spin interactions {Wu, J., Voss, J., et al . (1996) Proc . Natl . Acad . Sci . U.S.A . 93, 10123-10127} . Thus, a nitroxide attached to position 228 (helix VII) is closest to the lanthanide at position 148 (helix V), a nitroxide at position 275 (helix VIII) is further away, and the distance between positions 226 (helix VII) and 148 is too long to measure . However, the Gd(III)-spin-label distances are significantly longer than those estimated from nitroxide-nitroxide interactions between the same pairs due to the nature of the chelator . Although the results provide strong confirmation for the contention that helix V lies close to both helices VII and VIII in the tertiary structure of lactose permease, other methods for binding rare earth metals are discussed which do not involve the use of bulky chelators with long linkers.

Biochemistry, 2001 Mar 13, 40(10), 3101 - 8
Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model; Pyle LE et al.; The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were investigated in an in vitro model . ProapoA-I, mutants and wild type, were expressed in the PGEX/E . coli expression system as fusion proteins with glutathione S-transferase (GST) . Use of GST-proapoA-I and truncated forms of proapoA-I enabled quantitation of the amount of GST and apoA-I formed as a result of cleavage following incubation with human serum . Deletion of the pro-peptide (GST-apoA-I) resulted in complete inhibition of the reaction . Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction . Substitution of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide . The reaction was inhibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta-mercaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane . Calcium was essential for the activation of the cleavage enzyme, but it had a biphasic effect on the cleavage, activating it at concentrations below 1.5 mM and inhibiting at concentrations above 1.75 mM . Manganese alone was not essential for activation of the enzyme nor did it modify the effect of low concentration of calcium . However, a high concentration of manganese partially reverted the inhibitory effect of a high calcium concentration . Thus, residues within -2 to +4 are involved in forming the cleavage site for the maturation enzyme . The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.

Biochemistry, 2001 Mar 6, 40(9), 2923 - 31
Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair; Zou Y et al.; DNA damage recognition plays a central role in nucleotide excision repair (NER) . Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands . DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold . However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6) . These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA . Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5 . These data suggest that E . coli NER features a dynamic two-stage recognition mechanism.

Biochemistry, 2001 Mar 6, 40(9), 2816 - 25
Semisynthetic derivatives of concanamycin A and C, as inhibitors of V- and P-type ATPases: structure-activity investigations and developments of photoaffinity probes; Drose S et al.; V-type ATPases are inhibited by the plecomacrolides bafilomycin and concanamycin, which exert their inhibitory potential at nanomolar concentrations . In addition, some P-type ATPases are inhibited at micromolar concentrations . We initiated intensive structure-activity investigations with semisynthetic concanamycin derivatives to approach the following two questions: (i) What is the pharmacophor, the structural key element, of the plecomacrolides that leads to their inhibitory potential against V- and P-type ATPases? (ii) Where is the binding site within these two different types of ATPases? In a first step, we examined where chemical modifications (O-acylations, substitutions, eliminations) could be placed without seriously affecting the inhibitory potential of the macrolides . In a second step, we used the knowledge of these structure-activity investigations to introduce traceable elements (fluorescent or radioactive) or nitrene-generating azido or carbene-generating diazirine-groups able to bind the inhibitors to their target covalently . These studies led finally to the synthesis of two photoaffinity probes that were used in labeling experiments with the purified plasma membrane V-type ATPase of Manduca sexta (described in a following paper, Huss, M., Gassel, M., Ingenhorst, G., Drose, S., Zeeck, A., Altendorf, K., Wieczorek, H., manuscript submitted).

Biochemistry, 2001 Mar 6, 40(9), 2754 - 65
The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography; Canyuk B et al.; The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi . Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis . Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph . Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate . However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant . Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands . Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure . The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer . The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis . Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

Biochemistry, 2001 Mar 6, 40(9), 2647 - 52
Identification of hydrogen bonds between Escherichia coli DNA polymerase I (Klenow fragment) and the minor groove of DNA by amino acid substitution of the polymerase and atomic substitution of the DNA; Spratt TE; DNA polymerases replicate DNA with high fidelity despite the small differences in energy between correct and incorrect base pairs . X-ray crystallographic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fidelity . The current hypothesis is that polymerases check the geometry of the base pairs through hydrogen bonds and steric interactions with the minor groove of the DNA . The mechanisms by which these interactions are related to catalysis and fidelity are not known . In this manuscript, we have studied these interactions using a combination of site-specific mutagenesis of Escherichia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA . Crystal structures have predicted hydrogen bonds from Arg668 to the terminal base on the primer (P1) and Gln849 to its base pair partner (T1) . Kinetic studies, however, have implicated the minor groove of the primer terminus but not its base pair partner as being important to catalysis and fidelity . Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A . Hydrogen bonds from the DNA were probed with three oligodeoxynucleotides which have a guanine or 3-deazaguanine (3DG) at P1, T1, or T2 . We found that the pre-steady-state parameter k(pol) was decreased with R668A (40-fold) and Q849A (150-fold) or with 3DG at P1 (300-fold) or T2 (25-fold) . When R668A was combined with 3DG at P1 the decrease in rate was only 80-fold, consistent with a hydrogen bond between Arg668 and P1 . In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold . Similar reactions between R668A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.

Int J Radiat Biol, 2001 Mar, 77(3), 281 - 93
Redox reactivity of guanyl radicals in plasmid DNA; Milligan JR et al.; PURPOSE: It has been previously argued that gamma-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E . coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG), and there that derive from an intermediate guanyl radical species . The wish was to characterize the reactivity of this intermediate with reducing agents . MATERIALS AND METHODS: Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10(-3) to 10(-1) mol dm(-3)) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10(-7) to 10(-3) mol dm(-3)) were subjected to 137Cs gamma-irradiation (662 keV) . After irradiation, the plasmid was incubated with FPG . Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions . RESULTS: The very high yields of FPG-sensitive sites in the presence of SCN- or Br- decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least . CONCLUSIONS: From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5 x 10(5), 2 x 10(5), 10(7) and 10(7) dm3 mol(-1) s(-1) for ferrocyanide, nitrite, promethazine and tryptophan respectively).

EMBO Rep, 2001 Feb, 2(2), 108 - 12
Chromosomal DNA demethylation specified by protein binding; Lin IG et al.; In the present study, we utilize the well-characterized Escherichia coli lac repressor/operator system to demonstrate that protein binding can lead to demethylation at the binding sites in the chromosome . Similar to the findings using the episome, we found that the presence of LacI in the cells can lead to demethylation of methylated lacO in the chromosome and the LacI inhibitor, isopropyl-beta-D-thiogalactopyranoside (IPTG), can prevent demethylation of the methylated lacO . The lacO sites become progressively more demethylated over time with the presence of LacI, supporting the role of protein occupancy in demethylation targeting . These results validate our earlier conclusions using a stable episomal system, and establish for the first time that protein binding can specify sites of demethylation in the chromosome.

Antisense Nucleic Acid Drug Dev, 2001 Feb, 11(1), 29 - 40
Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation; Engdahl HM et al.; This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system . This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA . One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA . As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA . One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability . To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends . The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo . As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments) . In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.

EMBO Rep, 2000 Nov, 1(5), 441 - 6
The class 2 selenophosphate synthetase gene of Drosophila contains a functional mammalian-type SECIS; Hirosawa-Takamori M et al.; Synthesis of monoselenophosphate, the selenium donor required for the synthesis of selenocysteine (Sec) is catalyzed by the enzyme selenophosphate synthetase (SPS), first described in Escherichia coli . SPS homologs were identified in archaea, mammals and Drosophila . In the latter, however, an amino acid replacement is present within the catalytic domain and lacks selenide-dependent SPS activity . We describe the identification of a novel Drosophila homolog, Dsps2 . The open reading frame of Dsps2 mRNA is interrupted by an UGA stop codon . The 3'UTR contains a mammalian-like Sec insertion sequence which causes translational readthrough in both transfected Drosophila cells and transgenic embryos . Thus, like vertebrates, Drosophila contains two SPS enzymes one with and one without Sec in its catalytic domain . Our data indicate further that the selenoprotein biosynthesis machinery is conserved between mammals and fly, promoting the use of Drosophila as a genetic tool to identify components and mechanistic features of the synthesis pathway.

EMBO Rep, 2000 Nov, 1(5), 435 - 40
One RNA polymerase serving two genomes; Hedtke B et al.; The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3) . The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively . Here we show that the transit peptide of RpoT;2 possesses dual targeting properties . Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles . Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes . RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.

Arzneimittelforschung, 2001 Feb, 51(2), 180 - 7
Influence of a co-stimulation of human leucocytes with an Escherichia coli preparation and fixed immunoglobulins on cytokine release in the presence of hydrocortisone; Gebauer F et al.; Pharmaceuticals of biological origin consisting of bacterial culture suspensions (BCS) as active ingredients have long been used for the treatment of hemorrhoidal diseases and chronic anal pruritogenic eczemas . However, some of these pharmaceuticals often contain glucocorticoids such as hydrocortisone as an anti-inflammatory supplement . Therefore, the question arises whether the claimed immunostimulatory capacity of the bacterial culture suspension might be altered by the steroid . Up to now, numerous reports support the evidence that the stimulation of the different Fc-receptor subtypes on leucocytes result in profound immunoregulatory activities influencing phagocytosis and antigen processing, antibody-dependent cytotoxicity or secretory functions thereby enhancing the overall activities of the immune system towards foreign antigens/pathogens . With these findings in mind it was investigated whether the immunomodulatory capacity(s) of the BCS in the presence of hydrocortisone will be modified by solid-phase bound immunoglobulins (Igs) . For this purpose freshly prepared human peripheral blood leucocytes (PBLs) were incubated with different concentrations of the BCS (0.1, 1, 10 micrograms/ml), either with or without fixed human immunoglobulins in the presence of increasing concentrations of hydrocortisone . As a parameter of PBL activation the secretion of different cytokines was measured, e.g . tumor necrosis factor alpha (TNF-alpha), interleukin-10 (IL-10) and granulocyte-macrophage colony stimulating factor (GM-CSF) . Cytokines were determined with specific sandwich ELISAs . With this modified cell culture system it was demonstrated that the immunosuppressive activities, normally caused by hydrocortisone, were partially antagonized by the combination of BCS plus fixed Igs . TNF-alpha and GM-CSF were significantly more produced, even in the presence of hydrocortisone, whereas the synthesis of IL-10 was diminished by fixed Igs . However, this effect could be reversed with increasing concentrations of hydrocortisone . These results raise the possibility that in the natural environment, e.g . the rectal mucosa, antigens derived from the BCS are bound by specific Igs, thereby modifying secretory and effector functions of locally present leucocytes in another way as free antigens . The biological relevance of these in vitro data with respect to the therapeutic benefit of the BCS preparations with hydrocortisone will be discussed considering recent findings in the literature.

Appl Biochem Biotechnol, 2001 Jan, 90(1), 1 - 10
Inhibition of platelet aggregation of a mutant proinsulin molecule engineered by introduction of a native Arg-Gly-Asp sequence; Yang ZH et al.; A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques . The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli . The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations . The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses . This RGD-proinsulin showed an inhibitory activity of adenosine 5'-diphosphate-induced human platelet aggregation with an IC50 value of 200 nM . Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.

FEMS Microbiol Lett, 2001 Mar 1, 196(1), 77 - 83
Synthesis of the Streptomyces lividans maltodextrin ABC transporter depends on the presence of the regulator MalR; Schlosser A et al.; During growth with maltotriose or amylose, Streptomyces lividans and Streptomyces coelicolor A3(2) synthesize a maltodextrin uptake system with highest specificity for maltotriose . The transport activity is absent in mutants of S . coelicolor A3(2) lacking a functional MalE binding protein . Cloning and sequencing data suggest that the mal operon of S . coelicolor A3(2) corresponds to the one of S . lividans and that the deduced S . lividans Reg1 amino acid sequence is identical to that of MalR from S . coelicolor A3(2) . It can be concluded that both strains have the same ABC transport system for maltodextrins . The S . lividans malR was cloned in Escherichia coli in frame with six histidine-encoding codons . The resulting, purified 6HisMalR(SI) was shown to bind to two motifs within the S . lividans malR-malE intergenic region and to dissociate in the presence of maltopentaose.

FEMS Microbiol Lett, 2001 Mar 1, 196(1), 19 - 23
The immunological dissection of the Escherichia coli molecular chaperone DnaJ; Al-Herran S et al.; In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes . DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions . Truncated DnaJ polypeptides (DnaJ 1-108, DnaJ Delta1-108, DnaJ Delta1-199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies . Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains . These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation . In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation . The C-terminal region had only partial involvement in luciferase protection.

Biochim Biophys Acta, 2001 Mar 9, 1546(1), 216 - 25
Activation of phosphatidylinositol transfer protein alpha and beta isoforms from inclusion bodies; Bouma B et al.; Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies . Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles . Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e . long-chain PtdCho and phosphatidylinositol, did not stimulate activation . Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e . up to 500 microg ml(-1)) . The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E . coli culture . Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e . 10 microg ml(-1)) were required . In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days . This inactivation could be prevented by addition of beta-alanine . In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.

Biochim Biophys Acta, 2001 Mar 9, 1546(1), 196 - 204
Importance of the N-terminal sequence of the extrinsic 23 kDa polypeptide in Photosystem II in ion retention in oxygen evolution; Ifuku K et al.; The function of the extrinsic 23 kDa polypeptide (OEC23) in Photosystem II (PS II) is to retain Ca(2+) and Cl(-) during the S-state turnover of the Mn cluster in photosynthetic oxygen evolution . Recombinant OEC23s from several plant species were produced in Escherichia coli to characterize the molecular mechanism of the OEC23 function then used in reconstitution experiments . One tobacco isoform, OEC23 (2AF), had much less oxygen-evolving activity than the spinach and cucumber OEC23s when PS II activities were reconstituted in salt-washed spinach PS II particles . The fact that the OEC23s had similar binding affinities for PS II particles suggests different ion-retention capacities for the individual OEC23s: The chimeric OEC23s produced between spinach and 2AF and those produced between cucumber and 2AF show that 58 N-terminal amino acid residues are important for PS II activity . Further dissection of the sequence and site-directed mutagenesis indicated the importance of 20 N-terminal amino acid residues for activity, in particular the asparagine at the 15th position . In spinach the N15D mutation decreased PS II activity, whereas in 2AF the D15N mutation increased it . This shows the importance of the N-terminal sequence of OEC23 in ion retention during the water-splitting process.

FEBS Lett, 2001 Mar 16, 492(3), 228 - 32
The C-terminus of dUTPase: observation on flexibility using NMR; Nord J et al.; The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1H-(15)N nuclear magnetic resonance spectroscopy . The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity . In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range . No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility.

FEBS Lett, 2001 Mar 16, 492(3), 193 - 8
The TIM-barrel fold: a versatile framework for efficient enzymes; Wierenga RK; Recent studies on triosephosphate isomerase (TIM)-barrel enzymes highlight the remarkable versatility of the TIM-barrel scaffold . At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C-terminal end of the eight parallel beta-strands of the barrel . Sequence and structure comparisons now suggest that many of the TIM-barrel enzymes are evolutionarily related . Common structural properties of TIM-barrel enzymes are discussed.

FEBS Lett, 2001 Mar 16, 492(3), 177 - 82
The molecular peculiarities of catalase-peroxidases; Zamocky M et al.; In developing ideas of how protein structure modifies haem reactivity, the activity of Class I of the plant peroxidase superfamily (including cytochrome c peroxidase, ascorbate peroxidase and catalase-peroxidases (KatGs)) is an exciting field of research . Despite striking sequence homologies, there are dramatic differences in catalytic activity and substrate specificity with KatGs being the only member with substantial catalase activity . Based on multiple sequence alignment performed for Class I peroxidases, we present a hypothesis for the pronounced catalase activity of KatGs . In their catalytic domains KatGs are shown to possess three large insertions, two of them are typical for KatGs showing highly conserved sequence patterns . Besides an extra C-terminal copy of the ancestral hydroperoxidase gene resulting from gene duplication, these two large loops are likely to control the orientation of both the haem group and of essential residues in the active site . They seem to modulate the access of substrates to the prosthetic group at the distal side as well as the flexibility and character of the bond between the proximal histidine and the ferric iron . The hypothesis presented opens new possibilities in the rational engineering of peroxidases.

Vaccine, 2001 Mar 21, 19(17-19), 2742 - 8
Plant-based vaccines: unique advantages; Streatfield SJ et al.; Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants . The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy . Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination . We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Vaccine, 2001 Mar 21, 19(17-19), 2701 - 7
Transcutaneous immunization: T cell responses and boosting of existing immunity; Hammond SA et al.; Transcutaneous immunization (TCI) is a novel immunization strategy by which antigen and adjuvant are applied topically to intact, hydrated skin to induce potent antibody and cell-mediated immune responses specific for both the antigen and the adjuvant . Using tetanus toxoid as a model antigen, we examined the T cell response to tetanus toxoid after topical immunization with a variety of adjuvants . TCI readily induced systemic antigen specific T cell responses with a mixed Th1/Th2 phenotype but with a Th2 bias . We also investigated whether priming by the intramuscular route, which is known to induce T cell memory, could be followed by a boosting immunization on the skin to induce secondary responses . TCI could augment existing immunity, but interestingly, this strategy induced potent responses only if the antibody titer was low at the time of TCI boosting . These and previous observations suggest that TCI follows known immunological principles that govern other routes of vaccine delivery . Furthermore, booster immunization using tetanus toxoid may provide a useful model for further development of important patch and formulation concepts for TCI, and act as an early candidate for validating product feasibility of TCI in humans.

Vaccine, 2001 Mar 21, 19(17-19), 2534 - 41
Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants; Pizza M et al.; Most vaccines are still delivered by injection . Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes . However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems . Cholera toxin (CT) and Escherichia coli enterotoxin (LT) are powerful mucosal adjuvants when co-administered with soluble antigens . However, their use in humans is hampered by their extremely high toxicity . During the past few years, site-directed mutagenesis has permitted the generation of LT and CT mutants fully non toxic or with dramatically reduced toxicity, which still retain their strong adjuvanticity at the mucosal level . Among these mutants, are LTK63 (serine-to-lysine substitution at position 63 in the A subunit) and LTR72 (alanine-to-arginine substitution at position 72 in the A subunit) . The first is fully non toxic, whereas the latter retains some residual enzymatic activity . Both of them are extremely active as mucosal adjuvants, being able to induce very high titers of antibodies specific for the antigen with which they are co-administered . Both mutants have now been tested as mucosal adjuvants in different animal species using a wide variety of antigens . Interestingly, mucosal delivery (nasal or oral) of antigens together with LTK63 or LTR72 mutants also conferred protection against challenge in appropriate animal models (e.g . tetanus, Helicobacter pylori, pertussis, pneumococci, influenza, etc) . In conclusion, these LTK63 and LTR72 mutants are safe adjuvants to enhance the immunogenicity of vaccines at the mucosal level, and will be tested soon in humans.

J Gen Virol, 2001 Apr, 82(Pt 4), 909 - 18
Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4; Johne R et al.; Agnoproteins, encoded by the 5'-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions . In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed . Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication . Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles . Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells . With proteins expressed in E . coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind . A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding . Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.

Biochem J, 2001 Apr 1, 355(Pt 1), 51 - 8
The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly; Reid E et al.; In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor . A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm . Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH . We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid . An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly . The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants . The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH . The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.

Bioorg Khim, 2001 Jan-Feb, 27(1), 40 - 4
{Methods of preparation of recombinant proteins-cytokines . IV . Renaturation of recombinant human interleukin-3}; Tikhonov RV et al.; Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized . Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions . An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy . It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one . The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.

Bioorg Khim, 2001 Jan-Feb, 27(1), 32 - 9
{Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate}; Grigor'eva OV et al.; The interaction of inorganic pyrophosphatase from E . coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate . The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate . The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex . The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium . The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.

J Cell Biochem, 2001, 81(3), 535 - 46
Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity; Swamy N et al.; Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification . DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone . Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass . Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf . The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment . This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities . Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides . Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts . The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied . The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function . Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity . The activated form of a full length recombinant DBP, expressed in E . coli showed no activity in the in vitro assay . Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity . The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein . Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E . coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated . These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf .

Gene, 2001 Mar 7, 265(1-2), 205 - 12
Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit; Deshpande KL et al.; Queuosine (Q) is a 7-deazaguanosine found in the first position of the anticodon of tRNAs that recognize NAU and NAC codons (Tyr, Asn, Asp and His) . Eukaryotes synthesize Q by the base-for-base exchange of queuine (Q base) for guanine in the unmodified tRNA, a reaction catalyzed by TGT . A search of the human EST database for sequences with significant homology to the well studied TGT from Escherichia coli identified several candidates for full-length (1.3-1.4 kb) cDNA clones . Three candidate cDNA clones, available from IMAGE Consortium, LLNL, (Lennon et al., 1996, Genomics 33, 151-152) were obtained: IMAGE Clone Id Nos . 611146, 1422928, and 72154 . Here we report the complete sequences of these clones . IMAGE:72154 contains an ORF encoding a 44 kDa polypeptide with high homology to bacterial TGTs and was subcloned into the mammalian expression vector pMAMneo-Cat . When this construct was transfected into the TGT-negative cell line, GC(3)/c1 (Gunduz et al., 1992, Biochim . Biophys . Acta 1139, 229-238), it restored the ability of the cells to form Q-containing tRNA . This TGT cDNA sequence is encoded in human chromosome 19 clone CTC-539A10 (GenBank accession no . AC011475), enabling determination of the exon-intron boundaries for the TGT gene . The sequence of IMAGE:611146 is 5'-truncated by 76 bp compared to that from IMAGE:72154 and, except for two differences in the 3'-non-coding region, the remainder of the sequence is identical to that of IMAGE:72154 . IMAGE:1422928 is a 1390 bp chimera: the 5'-portion, bp 1-708, is identical to a genomic DNA sequence from chromosome 15 (GenBank accession no . AC067805, bp 148976-149683); the 3'-end, bp 726-1390, is identical to the 3'-end of the TGT cDNA sequence from IMAGE:611146.

Biochimie, 2001 Jan, 83(1), 125 - 9
FtsZ rings in mukB mutants with or without the Min system; Yu XC et al.; The site of cell division in Escherichia coli is defined by formation of the Z ring between the two segregated daughter nucleoids . Positioning of the Z ring, composed of the highly conserved and tubulin-like FtsZ protein, appears to be negatively regulated by both the nucleoid and the oscillating MinCD inhibitor proteins . MukB protein is probably involved in nucleoid condensation, and in the absence of MukB, the negative effect of the nucleoid on Z rings appears to be partially suppressed . In this study, we examined the localization of Z rings in cells lacking both the Min system and MukB . In the Deltamin DeltamukB double null mutant, essentially all nucleoid-free zones, either at the cell poles or at non-polar sites between nucleoids, contained Z rings . However, a significant proportion of Z rings also formed on top of nucleoids . Interestingly, Z ring clusters often formed at gaps between nucleoids, and some of the rings within the clusters were clearly positioned on top of nucleoids . These results provide further evidence that the negative topological effect of nucleoids in cells lacking MukB is partially but not totally suppressed, and that the absence of the Min system allows more promiscuous Z ring formation.

Biochimie, 2001 Jan, 83(1), 121 - 4
Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells; Pas E et al.; Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA) . Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface . These observations are consistent with the view (Woldringh et al., J . Bacteriol . 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them.

Biochimie, 2001 Jan, 83(1), 83 - 9
Cell-cycle research with synchronous cultures: an evaluation; Helmstetter CE et al.; The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth . In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates . Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony . We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state . The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.

Biochimie, 2001 Jan, 83(1), 67 - 74
Experiments on movement of DNA regions in Escherichia coli evaluated by computer simulation; Roos M et al.; During the cell cycle of Escherichia coli DNA is replicated and segregated over two prospective daughter cells . Nucleoids as a whole separate gradually in line with cell elongation, but sub-nucleoid DNA regions may behave differently, separating non-gradually . We tested the ability of three models to predict the outcome of a fluorescent in situ hybridisation (FISH) experiment . We did this by comparing computer-simulated data with experimental data . The first model predicts gradual separation in line with cell elongation . The second model predicts that origins stick together for some time after duplication before one copy jumps to the other side of the cell (non-gradual separation) . The simulated data of these models are very similar, indicating that FISH is not a suitable method to distinguish between these two models . The third model predicts that origins may be anywhere within the nucleoid(s) . We found that simulated data using the third model resemble the experimental data most . However, DNA regions are not randomly localised in the cell, although their localisation is fuzzy . We propose that movement of DNA regions is the result of a combination of factors . Nucleoid segregation (or the forces behind it) dictates the overall direction of movement . Other factors, of which we show that diffusion could be an important one, move DNA in other directions giving rise to non-gradual movement in individual cells and contributing to variation in intracellular position per cell length in a population of cells.

Biochimie, 2001 Jan, 83(1), 49 - 51
The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology; Skarstad K et al.; The SeqA protein was identified as a factor that prevents reinitiation of newly replicated, hemimethylated origins . SeqA also seems to inhibit initiation of fully methylated origins, thus contributing to the regulation of chromosomal replication . The SeqA protein was found to bind to two sites in the left part of the origin, near the AT-rich region where strand separation takes place during initiation of replication . The same binding sites seemed to be preferred irrespective of whether the origin was in the newly replicated (hemimethylated) state or not . In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU . The restraint of supercoils by SeqA was, in contrast to that of HU, cooperative.

Biochimie, 2001 Jan, 83(1), 41 - 8
Partitioning of the Escherichia coli chromosome: superhelicity and condensation; Nordstrom K et al.; Segregation in Escherichia coli, the process of separating the replicated chromosomes into daughter progeny cells, seems to start long before the duplication of the genome reaches completion . Soon after initiation in mid-cell region, the daughter oriCs rapidly move apart to fixed positions inside the cell (quarter length positions from each pole) and are anchored there by yet unknown mechanism(s) . As replication proceeds, the rest of the chromosome is sequentially unwound and then refolded . At termination, the two sister chromosomes are unlinked by decatenation and separated by supercoiling and/or condensation . Muk and Seq proteins are involved in different stages of this replication-cum-partition process and thus can be categorized as important partition proteins along with topoisomerases . E . coli strains, lacking mukB or seqA functions, are defective in segregation and cell division . The nucleoids in these mutant strains exhibit altered condensation and superhelicity as can be demonstrated by sedimentation analysis and by fluorescence microscopy . As the supercoiling of an extrachromosomal element (a plasmid DNA) was also influenced by the mukB and seqA mutations we concluded that the MukB and SeqA proteins are possibly involved in maintaining the general supercoiling activity in the cell . The segregation of E . coli chromosome might therefore be predominantly driven by factors that operate by affecting the superhelicity and condensation of the nucleoid (MukB, SeqA, topoisomerases and additional unknown proteins) . A picture thus emerges in which replication and partition are no longer compartmentalized into separable stages with clear gaps (S and M phases in eukaryotes) but are parallel processes that proceed concomitantly through a cell cycle continuum.

Biochimie, 2001 Jan, 83(1), 25 - 32
Defective initiation in an Escherichia coli dnaA(Cs,Sx) mutant; Boye E et al.; Mutations in the Escherichia coli gene for initiation of DNA replication, dnaA, which suppress the polymerization defect and growth inhibition caused by temperature-sensitive (Ts) mutations in the polymerization gene, dnaX, are called Cs,Sx . We show here that these mutations, on their own, also cause defects in initiation, including inhibition of initiation at both low (20 degrees C) and high (44 degrees C) temperatures and asynchronous initiation at both the permissive (34 degrees C) and suppression (39 degrees C) temperatures . These findings suggests a relationship between partially defective initiation and suppression of the polymerization defect, both of which occur at 39 degrees C.

Biochimie, 2001 Jan, 83(1), 19 - 23
Escherichia coli DnaA protein--phospholipid interactions: in vitro and in vivo; Crooke E; DNA replication in Escherichia coli is controlled at the initiation stage, possibly by regulation of the essential activity of DnaA protein . The cellular membrane has long been hypothesized to be involved in chromosomal replication . Accumulating evidence, both in vitro and in vivo, that supports the importance of membrane phospholipids influencing the initiation activity of DnaA is reviewed.

Biochimie, 2001 Jan, 83(1), 13 - 7
Multiple pathways regulating DnaA function in Escherichia coli: distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA; Katayama T et al.; Escherichia coli DnaA protein forms a multimeric complex at the chromosomal origin of replication (oriC), where a series of initiation reactions occurs and DNA polymerase III holoenzyme is loaded . The ATP-bound form of DnaA, which is active for initiation, is converted to the inactive ADP-bound form through interaction with the sliding clamp, the beta subunit of DNA polymerase III holoenzyme loaded on DNA . This negative regulation, termed RIDA, is required for preventing untimely initiations . Here, we asked if RIDA is functionally related to another negative regulation, DnaA titration by the datA site . The datA site can harbor hundreds of DnaA molecules, and is also required for preventing untimely initiations . We reveal here that, in growing cells of the datA(+) and datA-deleted strains, the ATP-DnaA levels were both maintained in a limited range of about 20-30% of the total ATP- plus ADP-DnaA molecules . This indicates that RIDA functions in the absence of datA . In synchronized datA-deleted cells, the ATP-DnaA level fluctuated in a manner similar to that observed in datA(+) cells . This suggests that RIDA operates independent from DnaA titration to datA . We suggest that these two mechanisms may play complementary roles during the cell cycle to prevent untimely initiations and thus ensure the scheduled initiation.

Mol Biochem Parasitol, 2001 Mar, 113(1), 35 - 43
Identification of Leishmania major cysteine proteinases as targets of the immune response in humans; Rafati S et al.; In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major . Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L . major cathepsin L-like gene family CPB, whereas the other gene codes for the L . major homologue of the cysteine proteinase a (CPA) of L . mexicana . By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique . Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle . Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes . Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients . Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB . This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

J Lipid Res, 2001 Mar, 42(3), 379 - 89
Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements; Cho KH et al.; The central region of apolipoprotein A-I (apoA-I), spanning residues 143--165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements . To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant . These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma . The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity . In the lipid-free state, the mutants self-associated similarly to wild-type protein . All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes . In the lipid-bound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96--98 A) . Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type . In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters . In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipid-free form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants . Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%) . Tested for their ability to rearrange into 78-A particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL) . Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II . These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143--165 region of apoA-I . The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects . Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved . -- Cho, K-H., D . M . Durbin, and A . Jonas . Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements . J . Lipid Res . 2001 . 42: 379--389.

J Immunol, 2001 Apr 1, 166(7), 4644 - 9
The alpha 4 beta 1 (very late antigen (VLA)-4, CD49d/CD29) and alpha 5 beta 1 (VLA-5, CD49e/CD29) integrins mediate beta 2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation; Burns JA et al.; The beta(2) integrin cell adhesion molecules (CAM) mediate polymorphonuclear leukocyte (PMNL) emigration in most inflamed tissues, but, in the lung, other yet to be identified CAMs appear to be involved . In Lewis rats, the intratracheal injection of Escherichia coli-LPS induced acute (6-h) PMNL accumulation in the lung parenchyma (280 x 10(6) by myeloperoxidase assay; PBS control = 35 x 10(6)) and bronchoalveolar lavage fluid (BALF = 27 x 10(6); PBS = 0.1 x 10(6)) . Parenchymal accumulation was not inhibited by a blocking Ab to beta(2) integrins and only minimally inhibited (20.5%; p < 0.05) in BALF . We examined the role of alpha(4)beta(1) and alpha(5)beta(1) integrins and of selectins in this PMNL recruitment . Treatment with mAbs to alpha(4)beta(1) or alpha(5)beta(1), even in combination, had no effect on PMNL accumulation induced by intratracheal LPS . However, anti-alpha(4) combined with anti-beta(2) mAbs inhibited PMNL recruitment to the parenchyma by 56% (p < 0.001) and to BALF by 58% (p < 0.01) . The addition of anti-alpha(5) mAb to beta(2) plus alpha(4) blockade inhibited PMNL accumulation further (by 79%; p < 0.05) . In contrast, blockade of L-, P-, and E-selectins in combination or together with beta(2), alpha(4), and alpha(5) integrins had no effect . LPS-induced BALF protein accumulation was not inhibited by treatment with anti-beta(2) plus alpha(4) mAbs, but was prevented when alpha(5)beta(1) was also blocked . Thus, while selectins appear to play no role, alpha(4)beta(1) and alpha(5)beta(1) function as major alternate CAMs to the beta(2) integrins in mediating PMNL migration to lung and to pulmonary vascular and epithelial permeability.

Infect Immun, 2001 Apr, 69(4), 2318 - 27
Novel molecular variants of allele I of the Escherichia coli P fimbrial adhesin gene papG; Johnson JR et al.; P fimbriae of extraintestinal pathogenic Escherichia coli mediate digalactoside-specific adherence via the tip adhesin molecule PapG, which occurs in three known variants (I to III), which are encoded by the corresponding three alleles of papG . In the present study, newly discovered variants of papG allele I and the respective wild-type source strains were characterized . One of the new papG allele I variants conferred a unique agglutination phenotype that combined the phenotypes associated with papG alleles I, II, and III . Comparative hydrophilicity analysis of predicted PapG peptides revealed regions that might explain the observed phenotypic similarities and differences between the PapG variants . The new papG allele I variants occurred either as the sole papG allele or together with both papG alleles II and III, rather than with only papG allele III, as in archetypal strains J96 and CP9 . They also occurred in the absence of the usual F13 papA allele . One of the new papG allele I variants occurred in a serogroup O6 strain that, according to random amplified polymorphic DNA analysis, was phylogenetically distant from the "J96-like" clonal group of E . coli O4:H5, which includes all previously identified examples of papG allele I . Cluster analysis of nucleotide and predicted peptide sequences suggested that papG allele I represents the earliest evolutionary branch from a common papG ancestor . These results demonstrate unexpected diversity within papG allele I and, together with previous findings, suggest that the J96-like clonal group of E . coli O4:H5 may represent the original source of papG within the species.

Infect Immun, 2001 Apr, 69(4), 2162 - 71
DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T . brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide; Shoda LK et al.; The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition . We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W . C . Brown, D . M . Estes, S . E . Chantler, K . A . Kegerreis, and C . E . Suarez, Infect . Immun . 66:5423-5432, 1998) . However, activation of macrophages by DNA from protozoan parasites has not been demonstrated . The present study was therefore conducted to determine whether DNA from the protozan parasites B . bovis, Trypanosoma cruzi, and T . brucei activates macrophages to secrete inflammatory mediators associated with protective immunity . DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) . Regulation of IL-12 and NO production occurred at the level of transcription . The amounts of IL-12, TNF-alpha, and NO induced by E . coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E . coli > or = T . cruzi > T . brucei > B . bovis . Induction of inflammatory mediators by E . coli, T . brucei, and B . bovis DNA was dependent on the presence of unmethylated CpG dinucleotides . However, at high concentrations, E . coli and T . cruzi DNA-mediated macrophage activation was not inhibited following methylation . The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

Infect Immun, 2001 Apr, 69(4), 2066 - 74
Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli; Meysick KC et al.; Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton . Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation . Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin . Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity . To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies . Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains . All MAbs showed reactivity to CNF1 by Western and dot blot analyses . However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2 . Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity) . Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a {IgG2a}), BF8 (IgA), and NG8 (IgG2a), were more precisely defined . MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain . MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164 . Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2 . MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain . Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.

Am J Respir Crit Care Med, 2001 Mar, 163(3 Pt 1), 762 - 9
Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice; Kitamura Y et al.; To determine the possible contribution of apoptosis in the pathogenesis of acute lung injury (ALI), we investigated Fas antigen (Fas), Fas ligand (FasL), perforin, granzyme A, and granzyme B expressions in a murine model of ALI after intratracheal instillation of Escherichia coli lipopolysaccharide (LPS: 0.3-30 microg) into the left lung . Lung injury, examined by water-to-dry weight ratio and albumin leakage, demonstrated maximal epithelial injury 1 d after 30 microg LPS instillation . Expressions of the proapoptosis molecules' mRNA were dose-dependently up-regulated, with maximal expression in the early phase in the instilled lung and most apparent 1 d after LPS instillation . Negligible mRNA expression of proapoptosis molecules was observed in noninstilled lungs . The terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) demonstrated positive signals in neutrophils and macrophages as well as in alveolar wall cells of the instilled lung 1 d after LPS instillation . Immunohistochemistry demonstrated that Fas was up-regulated in alveolar and inflammatory cells and FasL-positive inflammatory cells migrated into the air spaces in the LPS-instilled lung . Intratracheal administration of P2 antibody, which is an anti-Fas blocking antibody, attenuated the lung injury after 30 microg LPS instillation without attenuating mRNA expressions of proapoptosis molecules and neutrophil accumulation in the lung . In contrast, concanamycin A, which inhibits the function of perforin, did not alter the outcome after LPS instillation . These results indicate that the Fas/FasL system could be important in the pathogenesis of LPS-induced ALI, and proper regulation of the FasL/Fas system might be important for potential treatment of ARDS.

Am J Respir Crit Care Med, 2001 Mar, 163(3 Pt 1), 753 - 61
Protective role of heme oxygenases against endotoxin-induced diaphragmatic dysfunction in rats; Taille C et al.; Reactive oxygen species are strongly implicated in diaphragmatic dysfunction during sepsis . We investigated whether the heme oxygenase (HO) pathway, which is a powerful protective cellular system, protects the diaphragm against oxidative stress and contractile failure during sepsis . A basal expression of both the inducible and constitutive HO protein isoforms (HO-1 and HO-2, respectively) was found in the diaphragm . Enhanced HO-1 expression in diaphragmatic myocytes was observed 24 h after Escherichia coli endotoxin (lipopolysaccharide, LPS) inoculation and remained elevated for at least 96 h . Enhanced HO-1 expression was also observed in the rectus abdominis and soleus muscles and in the left ventricular myocardium of endotoxemic animals . Diaphragmatic HO-2 expression was not modified by endotoxin . Diaphragmatic HO activity exhibited a biphasic time course characterized by a transient decrease during the first 12 h followed by a significant increase at 24 h, corresponding to HO-1 induction . Diaphragmatic force was significantly reduced 24 h after LPS, concomitantly with muscular oxidative stress . Administation of an inhibitor of heme oxygenase activity, zinc protoporphyrin IX (ZnPP-IX), further impaired muscular oxidative stress and contractile failure . By contrast, increased levels of HO-1 expression obtained by pretreatment of rats with hemin, a powerful inducer of HO-1, completely prevented LPS-mediated diaphragmatic oxidative stress and contractile failure . This protective effect was reversed by ZnPP-IX . These results show an important protective role for the HO pathway against sepsis-induced diaphragmatic dysfunction, which could be related to its antioxidant properties.

J Mol Biol, 2001 Mar 23, 307(2), 683 - 706
Predicting the functional consequences of non-synonymous single nucleotide polymorphisms: structure-based assessment of amino acid variation; Chasman D et al.; We have developed a formalism and a computational method for analyzing the potential functional consequences of non-synonymous single nucleotide polymorphisms . Our approach uses a structural model and phylogenetic information to derive a selection of structure and sequence-based features serving as indicators of an amino acid polymorphim's effect on function . The feature values can be integrated into a probabilistic assessment of whether an amino acid polymorphism will affect the function or stability of a target protein . The method has been validated with data sets of unbiased mutations in the lac repressor and lysoyzyme . Applying our methodology to recent surveys of genetic variation in the coding regions of clinically important genes, we estimate that approximately 26-32 % of the natural non-synonymous single nucleotide polymorphisms have effects on function . This estimate suggests that a typical person will have about 6240-12,800 heterozygous loci that encode proteins with functional variation due to natural amino acid polymorphism .

J Mol Biol, 2001 Mar 23, 307(2), 587 - 603
The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives; Stillman TJ et al.; The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe(3+) and Zn(2+) derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin.The binding of both metals is accompanied by small changes in the amino acid ligand positions . Mean Fe(A)(3+)-Fe(B)(3+) and Zn(A)(2+)-Zn(B)(2+) distances are 3.24 A and 3.43 A, respectively . In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation . The Fe(3+) derivative alone shows a third metal site (Fe( C)( 3+)) joined to Fe(B)(3+) by a long anti-anti bidentate bridge through Glu130 (mean Fe(B)(3+)-Fe(C)(3+) distance 5.79 A) . The third metal site is unique to the non-haem bacterial ferritins.The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water . Models representing the possible binding mode of dioxygen to the dinuclear Fe(3+) pair suggest that a gauche micro-1,2 mode may be preferred stereochemically.Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles . The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s . deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %) . Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions .

Mol Gen Genet, 2001 Feb, 264(6), 931 - 5
Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli; Lee H et al.; The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation . The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase . In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type . These results suggest that the HU protein functions as a negative modulator of seqA expression.

Mol Gen Genet, 2001 Feb, 264(6), 924 - 30
DNA photolyase homologs are the major UV resistance factors in the cyanobacterium Synechocystis sp . PCC 6803; Ng WO et al.; In this study, the unicellular photosynthetic cyanobacterium Synechocystis sp . PCC 6803 was used as a model phototroph to study the contribution of enzymatic photoreactivation to the overall protection against UV irradiation . We have isolated genes encoding two DNA photolyase homologs, phrA and phrB, from Synechocystis 6803 . phrA encodes an 8-hydroxy-5-deazariboflavin (HDF) type, Class I DNA photolyase . By complementing a photolyase-deficient mutant strain of Escherichia coli, we demonstrated that PhrA is a DNA photolyase . Analysis of a phrA knockout mutant strain suggested that this gene is responsible for the majority of the observed UV resistance in Synechocystis 6803 . Similar studies on phrB demonstrated that it also contributes to photoreactivation, but to a much lesser degree . Based on these findings, we conclude that enzymatic photoreactivation is the primary process used for repairing UV-induced damage in Synechocystis 6803.

Mol Gen Genet, 2001 Feb, 264(6), 902 - 12
Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti; Ono Y et al.; We identified two rpoH-related genes encoding sigma32-like proteins from Sinorhizobium meliloti, a nitrogen-fixing root-nodule symbiont of alfalfa . The genes, rpoH1 and rpoH2, are functionally similar to rpoH of Escherichia coli because they partially complemented an E . coli rpoH null mutant . We obtained evidence indicating that these genes are involved in the heat shock response in S . meliloti . Following an increase in temperature, synthesis of several putative heat shock proteins (Hsps) was induced in cultures of wild-type cells: the most prominent were 66- and 60-kDa proteins, both of which are suggested to represent GroEL species . The other Hsps could divided into two groups based on differences in synthesis kinetics: synthesis of the first group peaked 5-10 min, and expression of the other group 30 min, after temperature upshift . In the rpoH1 mutant, inducible synthesis of the former group was markedly reduced, whereas that of the latter group was not affected . Synthesis of both the 66- and 60-kDa proteins was partially reduced . While no appreciable effect was observed in the rpoH2 single mutant, the rpoH2 mutation had a synergistic effect on the 60-kDa protein in the rpoH1- background . The results indicate that two distinct mechanisms are involved in the heat shock response of S . meliloti: one requires the rpoH1 function, while rpoH2 can substitute in part for the rpoH1 function . Moreover, the rpoH1 mutant and rpoH1 rpoH2 double mutant exhibited Nod+ Fix- and Nod- phenotypes, respectively, on alfalfa.

Mol Gen Genet, 2001 Feb, 264(6), 836 - 41
Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli; Watanabe T et al.; We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis . The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication . Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%) . The distributions of frequently mutated sites of G:C-->T:A and A:T-->C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors . These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.

Mol Gen Genet, 2001 Feb, 264(6), 796 - 808
The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli; El Hage A et al.; It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo . Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature . GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly . Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.

Mol Gen Genet, 2001 Feb, 264(6), 763 - 72
Multiple cellular processes affected by the absence of the Rpb4 subunit of RNA polymerase II contribute to the deficiency in the stress response of the yeast rpb4(delta) mutant; Bourbonnais Y et al.; We previously described the isolation of yeast mutants (sex mutants) that secrete reduced amounts of mature alpha-factor when it is synthesized as part of a fusion with prosomatostatin . In the present study we show that the sex3-1 mutant displays pleiotropic phenotypes . These include an abnormal morphology, an osmoremediable caffeine sensitivity, reduced secretion of mature alpha-factor, a weakened cell wall and a marked deficiency in halotolerance . Cloning of the SEX3 gene revealed that it is identical to the RPB4 gene . This gene encodes the fourth largest subunit of yeast RNA polymerase II, which has been postulated to play a major role in the response to stress . We show that transcriptional activation in response to either a cell wall stress or to growth in the presence of elevated salt concentrations is minimally affected by the loss of RPB4 function . However, whereas the levels of several mRNAs are similarly reduced (by about 30%) in rpb4 mutants grown in rich medium at moderate temperature, some transcripts, in particular ZDS1, are more abundant . An increase dosage of ZDS1, or of genes involved in cell wall assembly and in secretion (RHO1 and SR077, respectively), partially suppresses the sensitivity of rpb4delta cells to high temperature, heat shock and stationary phase . Collectively, our results indicate that the loss of Rpb4p perturbs several cellular functions that contribute to the inappropriate stress response of rpb4delta yeast . We therefore conclude that this RNA poiymerase II subunit is not specifically involved in the stress response.

Comp Biochem Physiol A Mol Integr Physiol, 1998 Jan, 119(1), 219 - 24
Effects of L-NAME and L-arginine on diaphragm contraction in a septic animal model; Shindoh C et al.; The effects of nitric oxide on diaphragm contraction after endotoxin administration were studied in Wistar rats . The animals were divided into seven treatments: a saline-injected group as control, three groups injected with L-NAME (0.01, 0.1, 1 mg/kg) and three groups injected with L-arginine (1, 10, 100 mg/kg) . Escherichia coli endotoxin was injected into the peritoneal cavity 15 min later . Twitch kinetics and force-frequency curves were measured 0, 2, and 4 hr after endotoxin injection . In the control group, the force-frequency curves significantly decreased from 0 hr to 4 hr . In the L-NAME group, the force-frequency curves at 4 hr showed significant increases in a dose-dependent manner . In the L-arginine group, the force-frequency curve with 100 mg/kg at 4 hr showed a significant increase . There was no consistent change in the contraction time, half relaxation time, or fatiguability . NADPH diaphorase histochemistry performed on diaphragm muscle samples 4 hr after endotoxin injection showed positive in the control and L-arginine group, but was only weakly observed in L-NAME group . These data suggest that nitric oxide contributes to the endotoxin induced diaphragm contractile deterioration.

Comp Biochem Physiol A Mol Integr Physiol, 1998 Jan, 119(1), 167 - 75
Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats; Sambe A et al.; Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis . Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines . The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction . Rats were inoculated intravenously (IV) with 10 mg/kg of E . coli endotoxin (LPS animals) or saline (C animals) . Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005) . Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals . LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro . In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction . We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E . coli endotoxemic rats.

Biotechniques, 2001 Mar, 30(3), 612 - 6, 618-9
Simultaneous insertion of two expression cassettes into adenovirus vectors; Danthinne X; We have designed AdenoQuick, a fast and versatile method to construct first-generation adenoviral vectors that contain one or two transgenes in the E1 and/or the E3 region . The method is based on the reconstitution of the entire genome of the desired recombinant virus in E . coli and the subsequent transfection of the DNA in a helper cell line . Since the construction of large adenoviral plasmids is generally difficult and therefore rebuffing for inexperienced researchers, we have optimized the cloning strategy by using bacterial positive-selection markers and a set of specific restriction enzymes that allow for directional cloning . The system is 99% efficient and allows one to insert simultaneously two expression cassettes into the E1 and E3 regions of the adenovirus genome.

Biotechniques, 2001 Mar, 30(3), 544 - 6, 548, 550 passim
Large-scale purification of a stable form of recombinant tobacco etch virus protease; Lucast LJ et al.; Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro . We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time . Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E . coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis . An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per liter of bacterial cell culture . NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.

Biotechniques, 2001 Mar, 30(3), 524 - 6, 528-9
Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants; Galindo I et al.; Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells . Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system . To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype . Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E . coli and vaccinia expression plasmids . Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.

EMBO Rep, 2001 Jan, 2(1), 17 - 20
The final cut . The importance of tRNA 3'-processing; Morl M et al.; To generate functional tRNA molecules, precursor RNAs must undergo several processing steps . While the enzyme that generates the mature tRNA 5'-end, RNase P, has been thoroughly investigated, the 3'-processing activity is, despite its importance, less understood . While nothing is known about tRNA 3'-processing in archaea, the phenomenon has been analysed in detail in bacteria and is known to be a multistep process involving several enzymes, including both exo- and endonucleases . tRNA 3'-end processing in the eukaryotic nucleus seems to be either exonucleolytic or endonucleolytic, depending on the organism analysed, whereas in organelles, 3'-end maturation occurs via a single endonucleolytic cut . An interesting feature of organellar tRNA 3'-processing is the occurrence of overlapping tRNA genes in metazoan mitochondria, which presents a unique challenge for the mitochondrial tRNA maturation enzymes, since it requires not only the removal but also the addition of nucleotides by an editing reaction.

Physiol Res, 2000, 49(6), 725 - 8
Inhibitory effect of FK 506 and cyclosporin A on nitric oxide production by LPS-treated cultured rat macrophages; Strestikova P et al.; We analyzed the effect of FK 506 on the production of nitric oxide by macrophages . Isolated rat peritoneal macrophages were cultured for 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 (0.1 and 1 microg/ml) . The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide production . FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively . The impact of cyclosporin A (CsA) was studied in order to compare their effects . CsA (0.1 and 1 microg/ml) decreased the levels of nitrites by 39% and 69%, respectively . The results obtained suggest that both immunosuppressive drugs exhibit a dose-dependent inhibitory effect on nitric oxide production and that FK 506 is a more potent agent than CsA in this respect.

Mol Microbiol, 2001 Mar, 39(5), 1199 - 211
Anaerobic acquisition of {4FE 4S} clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions; Ralph ET et al.; The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia . The anaerobic incorporation of oxygen-sensitive {4Fe 4S} clusters promotes dimerization, which in turn enhances DNA binding . Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo . Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen-sensitive {4Fe 4S} clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway . The C122 variant failed to assemble iron-sulphur clusters and to bind DNA . Second-site substitutions that partially restore activity to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122 . Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined . Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect . It was concluded that {4Fe 4S} cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.

Mol Microbiol, 2001 Mar, 39(5), 1153 - 65
What makes an Escherichia coli promoter sigma(S) dependent? Role of the -13/-14 nucleotide promoter positions and region 2.5 of sigma(S); Becker G et al.; The sigmaS and sigma70 subunits of Escherichia coli RNA polymerase recognize very similar promoter sequences . Therefore, many promoters can be activated by both holoenzymes in vitro . The same promoters, however, often exhibit distinct sigma factor selectivity in vivo . It has been shown that high salt conditions, reduced negative supercoiling and the formation of complex nucleoprotein structures in a promoter region can contribute to or even generate sigmaS selectivity . Here, we characterize the first positively acting sigmaS-selective feature in the promoter sequence itself . Using the sigmaS-dependent csiD promoter as a model system, we demonstrate that C and T at the -13 and -14 positions, respectively, result in strongest expression . We provide allele-specific suppression data indicating that these nucleotides are contacted by K173 in region 2.5 of sigmaS . In contrast, sigma70, which features a glutamate at the corresponding position (E458), as well as the sigmaS(K173E) variant, exhibit a preference for a G(-13) . C(-13) is highly conserved in sigmaS-dependent promoters, and additional data with the osmY promoter demonstrate that the K173/C(-13) interaction is of general importance . In conclusion, our data demonstrate an important role for region 2.5 in sigmaS in transcription initiation . Moreover, we propose a consensus sequence for a sigmaS-selective promoter and discuss its emergence and functional properties from an evolutionary point of view.

Mol Microbiol, 2001 Mar, 39(5), 1109 - 15
The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons; Opel ML et al.; Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling) . The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties . As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose . Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions . As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

Mol Microbiol, 2001 Feb, 39(4), 1069 - 79
The Escherichia coli histone-like protein HU regulates rpoS translation; Balandina A et al.; Escherichia coli HU protein is a major component of the bacterial nucleoid . HU stabilizes higher order nucleoprotein complexes and belongs to a family of DNA architectural proteins . Here, we report that HU is required for efficient expression of the sigma S subunit of RNA polymerase . This rpoS-encoded alternative sigmaS factor induces a number of genes implicated in cell survival in stationary phase and in multiple stress resistance . By analysis of rpoS-lacZ fusions and by pulse-chase experiments, we show that the efficiency of rpoS translation is reduced in cells lacking HU, whereas neither rpoS transcription nor protein stability is affected by HU . Gel mobility shift assays show that HU is able to bind specifically an RNA fragment containing the translational initiation region of rpoS mRNA 1000-fold more strongly than double-stranded DNA . Together with the in vivo data, this finding strongly suggests that, by binding to rpoS mRNA, HU directly stimulates rpoS translation . We demonstrate here that HU, an abundant DNA-binding, histone-like protein, is able specifically to recognize an RNA molecule and therefore play a role in post-transcriptional regulation.

Mol Microbiol, 2001 Feb, 39(4), 994 - 1009
The cold-shock stress response in Mycobacterium smegmatis induces the expression of a histone-like protein; Shires K et al.; The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism . The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37 degrees C to 10 degrees C having the most widespread effect on growth, metabolism and protein composition . This 27 degrees C temperature reduction was associated with a lag period of 21-24 h before increases were seen in all the measured cellular activities . The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate . The synthesis of at least 15 proteins was induced during the lag period . Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins . One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M . smegmatis, which is also induced during anaerobic-induced dormancy . The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9-12 h after temperature shift . Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.

Mol Microbiol, 2001 Feb, 39(4), 904 - 13
Interplay between recombination, cell division and chromosome structure during chromosome dimer resolution in Escherichia coli; Perals K et al.; Chromosome dimers form in bacteria by recombination between circular chromosomes . Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ) . We analyse here how these phenomena collaborate . We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers . Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.

Mol Microbiol, 2001 Feb, 39(4), 890 - 903
Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis; Karata K et al.; We have built a homology model of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli based on the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein . The resulting model of the hexameric ring of the ATP-bound form of the AAA ATPase suggests a plausible mechanism of ATP binding and hydrolysis, in which invariant residues of Walker motifs A and B and the second region of homology, characteristic of the AAA ATPases, play key roles . The importance of these invariant residues was confirmed by site-directed mutagenesis . Further modelling suggested a mechanism by which ATP hydrolysis alters the conformation of the loop forming the central hole of the hexameric ring . It is proposed that unfolded polypeptides are translocated through the central hole into the protease chamber upon cycles of ATP hydrolysis . Degradation of polypeptides by FtsH is tightly coupled to ATP hydrolysis, whereas ATP binding alone is sufficient to support the degradation of short peptides . Furthermore, comparative structural analysis of FtsH and a related ATPase, HslU, reveals interesting similarities and differences in mechanism.

Clin Exp Pharmacol Physiol, 2001 Apr, 28(4), 315 - 20
The lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock; Lee RP et al.; 1 . The present study was undertaken to determine the locus of nitric oxide (NO) production that is toxic to the lung and produces acute pulmonary oedema in endotoxin shock, to examine and compare the effects of changes in lung perfusate on endotoxin-induced pulmonary oedema (EPE) and to evaluate the involvement of constitutive and inducible NO synthase (cNOS and iNOS, respectively) . 2 . Experiments were designed to induce septic shock in anaesthetized rats with the administration of Escherichia coli lipopolysaccharide (LPS) . Exhaled NO, lung weight (LW)/bodyweight (BW) ratio, LW gain (LWG) and lung histology were measured and observed to determine the degree of EPE 4 h following LPS . The EPE was compared between groups in which LPS had been injected either into the systemic circulation or into the isolated perfused lung . The lung perfusate was altered from whole blood to physiological saline solution (PSS) with 6% albumin to test whether different lung perfusions affected EPE . Pretreatment with various NOS inhibitors was undertaken 10 min before LPS to investigate the contribution of cNOS and iNOS to the observed effects . 3 . Endotoxin caused profound systemic hypotension, but little change in pulmonary arterial pressure . The extent of EPE was not different between that induced by systemic injection and that following administration to isolated lungs preparations . Replacement of whole blood with PSS greatly attenuated (P < 0.05) EPE . In blood-perfused lungs, pretreatment with NOS inhibitors, such as Nomega-nitro-L-arginine methyl ester, aminoguanidine and dexamethasone, significantly prevented EPE (P < 0.05) . 4 . The major site of NO production through the whole blood is in the lung . The NO production mediated by the iNOS system is toxic to the endothelium in the pulmonary microvasculature . Inhalation of NO for patients with sepsis may be used with clinical caution . Therapeutic consideration of lung extracorporeal perfusion with PSS and pharmacological pretreatment with iNOS inhibitors may be warranted.

Clin Exp Allergy, 2001 Feb, 31(2), 331 - 41
Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen; Schramm G et al.; The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy . Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen . We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E . coli as MBP fusion proteins . Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity . Specificity of antibody binding was confirmed by competitive blot inhibition assays . At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation . The fragments were differentially recognized by individual patients' sera . By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment . It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule . Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5 . rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients . Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.

Chem Biol, 2001 Feb, 8(2), 133 - 45
Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin; Macmillan D et al.; BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures . The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples . Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins . The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed . This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites . Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity . RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli . Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS . Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed . Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography . Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein . CONCLUSIONS: Erythropoietin expressed in E . coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds . The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.

Int J Biol Macromol, 2001 Mar 14, 28(3), 227 - 34
Heteronuclear nuclear magnetic resonance assignments, structure and dynamics of SUMO-1, a human ubiquitin-like protein; Jin C et al.; The structure of a ubiquitin-like protein, small ubiquitin-related modifier-1 (SUMO-1), was earlier determined using homonuclear nuclear magnetic resonance (NMR) spectroscopy, since the spectral quality of the protein was not suitable for heteronuclear NMR data collection . In this study, a slightly different construct of the SUMO-1 gene was used for protein over-expression . The protein purified from this construct showed high spectral qualities, therefore, multi-dimensional heteronuclear NMR data for a dynamic study and structural determination were acquired . The structure of SUMO-1 obtained in this study differs in several respects from the structure obtained from homonuclear NMR data . Furthermore, structural differences were observed between the new SUMO-1 and ubiquitin structures . These differences may be important for SUMO-1-specific recognition in cells . Additionally, relaxation parameters indicate that SUMO-1 undergoes highly anisotropic tumbling in solution and that the long amino (N)-terminal sequence of SUMO-1 is highly dynamic with increasing flexibility towards the end.

Plant Cell, 2001 Mar, 13(3), 695 - 705
Identification of a mannitol transporter, AgMaT1, in celery phloem; Noiraud N et al.; A celery petiole phloem cDNA library was constructed and used to identify a cDNA that gives Saccharomyces cerevisiae cells the ability to grow on mannitol and transport radiolabeled mannitol in a manner consistent with a proton symport mechanism . This cDNA was named AgMaT1 (Apium graveolens mannitol transporter 1) . The expression profile in source leaves and phloem was in agreement with a role for mannitol in phloem loading in celery . The identification in eukaryotes of a mannitol transporter is important because mannitol is not only a primary photosynthetic product in species such as celery but is also considered a compatible solute and antioxidant implicated in resistance to biotic and abiotic stress.

Plant Cell, 2001 Mar, 13(3), 681 - 93
Gene duplication in the diversification of secondary metabolism: tandem 2-oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis; Kliebenstein DJ et al.; Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions . The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis . However, little is known about the evolution of enzymes involved in secondary metabolism . We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism . Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates . In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation . These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form . By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates . Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates . No ecotype coexpressed both genes . Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates . A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined . However, its catalytic role is still uncertain.

Mol Biol Cell, 2001 Mar, 12(3), 551 - 63
Positive regulation of Wee1 by Chk1 and 14-3-3 proteins; Lee J et al.; Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15 . The activity of Wee1 is highly regulated during the cell cycle . In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase . We report that Xwee1 is also regulated by association with 14-3-3 proteins . Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549 . A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2 . This mutation also affects the intranuclear distribution of Xwee1 . In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549 . The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response . Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1 . Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

EMBO J, 2001 Mar 15, 20(6), 1469 - 76
Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA; Speck C et al.; The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5'-TT(A/T)TNCACA) . If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP-DnaA box (5'-AGatct) . Using DNase footprinting and surface plasmon resonance we show that binding to ATP-DnaA boxes in the AT-rich region of oriC of E.coli requires binding to the 9mer DnaA box R1 . Cooperative binding of ATP-DnaA to the AT-rich region results in its unwinding . ATP-DnaA subsequently binds to the single-stranded region, thereby stabilizing it . This demonstrates an additional binding specificity of DnaA protein to single-stranded ATP-DnaA boxes . Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were approximately 1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA boxes, respectively . We propose that sequential recognition of high- and low-affinity sites, and binding to single-stranded origin DNA may be general properties of initiator proteins in initiation complexes.

EMBO J, 2001 Mar 15, 20(6), 1462 - 8
The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel; Barcena M et al.; Replicative helicases are motor proteins that unwind DNA at replication forks . Escherichia coli DnaB is the best characterized member of this family of enzymes . We present the 26 A resolution three-dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo-electron microscopy . Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC . The complex is arranged on the basis of interactions among DnaC and DnaB dimers . DnaC monomers are observed for the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase . This could be responsible for the freezing of DnaB in a C(3) architecture by its loading partner . The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through . We propose that the DnaB N-terminal domain is located at this face.

EMBO J, 2001 Mar 15, 20(6), 1425 - 38
The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation; Qiu H et al.; GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2 . GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS) . The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2 . Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation . HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function . The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V) . These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding . We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.

EMBO J, 2001 Mar 15, 20(6), 1245 - 58
Enteropathogenic Escherichia coli mediates antiphagocytosis through the inhibition of PI 3-kinase-dependent pathways; Celli J et al.; The extracellular pathogen enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inhibit its uptake by macrophages . We show that EPEC antiphagocytosis is independent of the translocated intimin receptor Tir and occurs by preventing F-actin polymerization required for bacterial uptake . EPEC-macrophage contact triggered activation of phosphatidylinositol (PI) 3-kinase, which was subsequently inhibited in a type III secretion-dependent manner . Inhibition of PI 3-kinase significantly reduced uptake of a secretion-deficient mutant, without affecting antiphagocytosis by the wild type, suggesting that EPEC blocks a PI 3-kinase-dependent phagocytic pathway . EPEC specifically inhibited Fc gamma receptor- but not CR3-receptor mediated phagocytosis of opsonized zymosan . We showed that EPEC inhibits PI 3-kinase activity rather than its recruitment to the site of bacterial contact . Phagocytosis of a secretion mutant correlated with the association of PI 3-kinase with tyrosine-phosphorylated proteins, which wild-type EPEC prevented . These results show that EPEC blocks its uptake by inhibiting a PI 3-kinase-mediated pathway, and translocates effectors other than Tir to interfere with actin-driven host cell processes . This constitutes a novel mechanism of phagocytosis avoidance by an extracellular pathogen.

Asian J Androl, 2001 Mar, 3(1), 49 - 53
Introduction of DT40 cells into chick embryos; Toba M et al.; AIM: To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds . METHODS: The DT40 cells incorporated with exogenous gene ( lacZ constructs encoding Escherichia coli beta-galactosidase: beta-gal) were introduced into chick embryos by the injection of cells into stage X blastoderm . Manipulated eggs were incubated for 3 (trial 1) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of beta-galactosidase and polymerase chain reaction (PCR) analysis . RESULTS: The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively . The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos . CONCLUSION: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.

Gene, 2001 Feb 21, 264(2), 225 - 31
Monitoring promoter activity and protein localization in Mycobacterium spp . using green fluorescent protein; Cowley SC et al.; Two green fluorescent protein (Gfp) fusion vectors were constructed for use in Mycobacterium spp . The first plasmid facilitates quantification of mycobacterial promoter activity . The second vector permits construction of translational fusions of mycobacterial proteins to Gfp in order to study subcellular localization including protein secretion . Using this translational fusion construct, we verify that a Gfp fusion to the putative secreted M . tuberculosis protein ChoD is translocated to the extracellular milieu when cloned and expressed in Mycobacterium smegmatis.

Gene, 2001 Feb 21, 264(2), 215 - 24
Characterization of the mouse liver fructose-1,6-bisphosphatase gene; Stein S et al.; A cDNA encoding fructose-1,6-bisphosphatase (FBPase) was isolated from mouse liver RNA . The cDNA encodes a polypeptide of 338 amino acids (36.9 kDa) . The liver and muscle FBPase isoenzymes of the mouse show positional identities of 69% at the cDNA level and 72% at the protein primary structure level . Starting from genomic YAC libraries and based upon the cDNA sequence all functional parts of the mouse liver FBPase gene (including exon-intron boundaries) were PCR-amplified and sequenced . The 5'-flanking regions of the liver and muscle FBPase genes were compared and showed no sequence similarity . Both genes are co-localized at chromosome 13B3-C1 . The transcriptional start site was assigned to a guanine 118 bases before the start codon in the liver FBPase gene . An analysis of the steady state mRNA levels of liver and muscle FBPase in various mouse tissues was performed by Northern blotting and RT/PCR.

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