Chem Biol, 1998 Nov, 5(11), 661 - 7 Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae; Xue Y et al.; Background: . Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin . Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring . In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain . Results: . Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S . venezuelae . Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium . The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells . Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro . Conclusions: . These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S . venezuelae . This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system . The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics. Eur J Gastroenterol Hepatol, 1998 Oct, 10(10), 837 - 41 Cancer-associated strains of Helicobacter pylori stimulate DNA synthesis in IEC-6 cells; Bark J et al.; OBJECTIVE: To determine whether water extracts of Helicobacter pylori strains, which express CagA, can influence DNA synthesis in untransformed intestinal epithelial cells in vitro . DESIGN: We used water extracts produced from H . pylori strains (A, B, C), collected from gastric mucosa of gastric cancer patients . Strain A was CagA+/VacA+ whereas strains B and C were CagA+/VacA- . Water extracts from Helicobacter mustelae and Escherichia coli were used as controls . METHODS: IEC-6 cells (small intestinal epithelial cell line from germ-free rats) were incubated with various concentrations of the bacterial extracts for 24 h . The cells were labelled with {3H}methylthymidine for 4 h and thereafter processed for autoradiography . DNA synthesis was evaluated by the labelling index (LI%) . RESULTS: Water extracts from CagA-positive strains of H . pylori, with or without the capacity to produce vacuolating toxins, increased the LI in a dose-related manner (P < 0.05) . The water extracts of E . coli significantly increased the LI (P < 0.001), whereas the water extracts of H . mustelae did not affect DNA synthesis . CONCLUSIONS: Cancer-associated, CagA-positive strains of H . pylori stimulate DNA synthesis in epithelial cells in vitro, independently of their ability to produce VacA toxin . Our findings suggest that unknown mitogenic components of H . pylori may contribute to the increased cell proliferation observed in the histological stages preceding gastric cancer. J Immunol Methods, 1998 Oct 1, 219(1-2), 119 - 29 Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent--application to a human single-chain Fv fragment; Tsumoto K et al.; An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed . Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies . Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv . The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95% . The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments. Eur Cytokine Netw, 1998 Sep, 9(3), 279 - 88 Biological activity and brain actions of recombinant rat interleukin-1alpha and interleukin-1beta; Anforth HR et al.; IL-1alpha and IL-1beta have potent effects on the central nervous system resulting in fever, activation of the hypothalamic-pituitary-adrenal axis and behavioural depression . These effects have mainly been studied in rats, using recombinant human and mouse IL-1 . Because IL-1alpha and IL-1beta show some species specificity in the potency of their biological activities, the objective of the present work was to directly compare the effects of recombinant rat IL-1alpha and IL-1beta in the rat system as a first step to dissect out the mechanisms that are involved in these effects . In vitro, recombinant rat IL-1alpha and IL-1beta bound with the same affinity as human IL-1 to the rat insulinoma Rin m5F cell line that mainly expresses type I IL-1 receptors . This binding activated IL-1 receptors, as shown by induction of the synthesis of TNF-alpha mRNA . In vivo, recombinant rat IL-1alpha and IL-1beta enhanced body temperature, increased plasma levels of corticosterone and ACTH, and depressed social behaviour . All these effects were obtained at doses 100-1,000 fold lower when IL-1 was injected centrally than when it was administered peripherally, indicating that they are centrally mediated . The relative potencies of recombinant rat IL-1alpha and IL-1beta were not the same depending on the endpoint and the route of injection, indicating that different mechanisms are likely to be involved in the various effects of IL-1 on the brain. Lett Appl Microbiol, 1998 Nov, 27(5), 297 - 301 Rapid evaluation in Escherichia coli of antisense RNAs and ribozymes; Tatout C et al.; The characterization of a simple bacterial system using commercially available plasmids and strains, developed to assess the effectiveness of trans-acting (antisense RNA and ribozymes) RNA in Escherichia coli, is reported . This system was used to test various trans-acting RNA molecules against the expression of the I factor, a functional transposable element from Drosophila melanogaster . For this target, results indicate that antisense RNA efficiency depends on the hybridization length between sense and antisense RNA . The introduction of a single hammerhead ribozyme within the antisense RNAs does not increase its inhibitory activity . These predictions were subsequently confirmed in Drosophila melanogaster. Lett Appl Microbiol, 1998 Nov, 27(5), 279 - 82 Survival and biological activity of heat damaged DNA; Masters CI et al.; The thermal degradation of plasmid pUC18 held at temperatures between 100 and 135 degrees C was examined by measuring the ability of heat-treated plasmid preparations to transform Escherichia coli to ampicillin resistance using electroporation . Substantial protection against loss of transforming ability during heating was provided by concentrations of NaCl between 0.25 and 2.0 mol l-1 . For example, the addition of 1.0 mol l-1 NaCl to samples heated at 100 degrees C for 15 min increased transformation frequency about 200-fold compared with samples heated without NaCl . In the presence of 0.5-2.0 mol l-1 NaCl, transforming capacity was not destroyed even by heating at 121 degrees C for 15 min, i.e . after a typical sterilization treatment . These findings may have implications for the safe disposal of genetically modified micro-organisms and recombinant DNA preparations. J Appl Microbiol, 1998 Nov, 85(5), 883 - 90 Biotransformation of D(+)-carnitine into L(-)-carnitine by resting cells of Escherichia coli O44 K74; Castellar MR et al.; L(-)-carnitine was produced from D(+)-carnitine by resting cells of Escherichia coli O44 K74 . Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process . Aerobic conditions led to higher product yield than anaerobic conditions . The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4.30 g l-1 cells and 100 mmol l-1 D(+)-carnitine . Under these conditions the L(-)-carnitine production rate was 0.55 g l-1 h-1, the process yield was 44%, and the productivity was 0.22 g l-1 h-1 after a 30 h incubation period . Crotonobetaine production, besides L(-)-carnitine, showed that the action of more than one enzyme occurred during the biotransformation process . On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l-1) led to a higher metabolic activity, which meant an increment of L(-)-carnitine production. Can J Microbiol, 1998 Aug, 44(8), 707 - 17 Regulation in the rpoS regulon of Escherichia coli; Loewen PC et al.; In Escherichia coli, the transcription factor sigma s, encoded by rpoS, controls the expression of a large number of genes involved in cellular responses to a diverse number of stresses, including starvation, osmotic stress, acid shock, cold shock, heat shock, oxidative DNA damage, and transition to stationary phase . A list of over 50 genes under the control of rpoS has been compiled . The transcription factor sigma s acts predominantly as a positive effector, but it does have a negative effect on some genes . The synthesis and accumulation of sigma s are controlled by mechanisms affecting transcription, translation, proteolysis, and the formation of the holoenzyme complex . Transcriptional control of rpoS involves guanosine 3',5'-bispyrophosphate (ppGpp) and polyphosphate as positive regulators and the cAMP receptor protein-cAMP complex (CRP-cAMP) as a negative regulator . Translation of rpoS mRNA is controlled by a cascade of interacting factors, including Hfq, H-NS, dsrA RNA, LeuO, and oxyS RNA that seem to modulate the stability of a region of secondary structure in the ribosome-binding region of the gene's mRNA . The transcription factor sigma s is sensitive to proteolysis by ClpPX in a reaction that is promoted by RssB and inhibited by the chaperone DnaK . Despite the demonstrated involvement of so many factors, arguments have been presented suggesting that sensitivity to proteolysis may be the single most important modulator of sigma s levels . The activity of sigma s may also be modulated by trehalose and glutamate, which activate holoenzyme formation and promote holoenzyme binding to certain promoters. Proteins, 1998 Nov 15, 33(3), 430 - 43 Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase; Ha Y et al.; Tyr 165 in the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition . Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function . We have determined the crystal structure of Y165F at 2.4 A resolution by molecular replacement, using a wild-type T state structure as the probe, and refined it to an R value of 25.2% . The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state . The two catalytic trimers move closer by approximately 0.14 A and rotate by approximately 0.2 degrees , in the opposite direction to the T-->R rotation; the two domains of each catalytic chain rotate by approximately 2.1 degrees, also in the opposite direction to the T-->R transition; and the 240s loop adopts a new conformation . Residues 229 to 236 shift by approximately 2.4 A so that the active site is more open . Residues 237 to 244 rotate by approximately 24.1 degrees, altering interactions within the 240s loop and at the C1-C4 and C1-R4 interfaces . Arg 167, a key residue in domain closure and interactions with L-Asp, swings out from the active site to interact with Tyr 197 . This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme. J Biol Chem, 1998 Dec 4, 273(49), 33064 - 72 Partial functional deficiency of E160D flap endonuclease-1 mutant in vitro and in vivo is due to defective cleavage of DNA substrates; Frank G et al.; To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene . The mutants were then expressed in Escherichia coli and purified using a His-tag . Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant . This mutant retained wild type-like binding ability, specificity, and partial catalytic activity . Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage . When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity. J Biol Chem, 1998 Dec 4, 273(49), 32995 - 3001 Conformational changes of Escherichia coli RNA polymerase sigma70 factor induced by binding to the core enzyme; Callaci S et al.; Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared . The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase . Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme . Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1) . Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70 . Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains. J Biol Chem, 1998 Dec 4, 273(49), 32927 - 33 A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis . Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization; Reverter D et al.; A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis . Amino acid sequence analysis provided a nearly complete primary structure . which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques . The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues . It does not show sequence similarity to any other protein except at its C-terminal end . In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner . This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme . Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity . Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B . Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions. J Biol Chem, 1998 Dec 4, 273(49), 32864 - 9 Signal decay through a reverse phosphorelay in the Arc two-component signal transduction system; Georgellis D et al.; Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system . Under those conditions, the tripartite sensor kinase ArcB undergoes autophosphorylation at the expense of ATP and subsequently transphosphorylates its cognate response regulator ArcA through a His --> Asp --> His --> Asp phosphorelay pathway . In this study we used various combinations of wild-type and mutant ArcB domains to analyze in vitro the pathway for signal decay . The results indicate that ArcA-P dephosphorylation does not occur by direct hydrolysis but by transfer of the phosphoryl group to the secondary transmitter and subsequently to the receiver domain of ArcB . This reverse phosphorelay involves both the conserved His-717 of the secondary transmitter domain and the conserved Asp-576 of the receiver domain of ArcB but not the conserved His-292 of its primary transmitter domain . This novel pathway for signal decay may generally apply to signal transduction systems with tripartite sensor kinases. J Biol Chem, 1998 Dec 4, 273(49), 32806 - 11 Site-directed chemical modification of cysteine-scanning mutants as to transmembrane segment II and its flanking regions of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a transmembrane water-filled channel; Kimura-Someya T et al.; Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H+ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II . None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation . {14C}N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated . The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A . (1997) J . Biol . Chem . 272, 580-585) . However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with {14C}NEM . The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment . Positions reactive to NEM appear periodically in the second half . They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel . The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half . These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway. J Biol Chem, 1998 Dec 4, 273(49), 32753 - 62 On the active site of Old Yellow Enzyme . Role of histidine 191 and asparagine 194; Brown BJ et al.; Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes . The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover . Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K . M., and Karplus, P . A . (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group . His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site . Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1 . It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e . decreased binding affinity and decreased charge-transfer absorbance . Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased . The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15 . The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations . The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme. J Biol Chem, 1998 Dec 4, 273(49), 32739 - 45 Isoform-dependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosynthesis from Arabidopsis thaliana; Noji M et al.; Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the formation of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine, plays a regulatory role in the biosynthesis of cysteine by its property of feedback inhibition by cysteine in bacteria and certain plants . Three cDNA clones encoding SATase isoforms (SAT-c, SAT-p, and SAT-m) have been isolated from Arabidopsis thaliana . However, the significance of the feedback regulation has not yet been clear in these different isoforms of SATase from A . thaliana . We constructed the overexpression vectors for cDNAs encoding three SATase isoforms of A . thaliana and analyzed the inhibition of SATase activity by cysteine using the recombinant SATase proteins . In the case of SAT-c, the activity was feedback-inhibited by a low concentration of cysteine (the concentration that inhibits 50% activity; IC50 = 1.8 microM) . By contrast, SAT-p and SAT-m were feedback inhibition-insensitive isozymes . We also determined the subcellular localization of three SATase isozymes by the transient expression of fusion proteins of each SATase N-terminal region with jellyfish green fluorescent protein (GFP) in 4-week-old Arabidopsis leaves . The SAT-c-GFP fusion protein was stayed in cytosol, whereas SAT-p-GFP and SAT-m-GFP fusion proteins were localized in chloroplasts and in mitochondria, respectively . These results suggest that these three SATase isoforms, which are localized in the different organelles, are subjected to different feedback regulation, presumably so as to play the particular roles for the production of OAS and cysteine in Arabidopsis cells . Regulatory circuit of cysteine biosynthesis in the plant cells is discussed. J Biol Chem, 1998 Dec 4, 273(49), 32587 - 94 GroEL under heat-shock . Switching from a folding to a storing function; Llorca O et al.; Chaperonin GroEL from Escherichia coli, together with its cochaperonin GroES, are proteins involved in assisting the folding of polypeptides . GroEL is a tetradecamer composed of two heptameric rings, which enclose a cavity where folding takes place through multiple cycles of substrate and GroES binding and release . GroEL and GroES are also heat-shock proteins, their synthesis being increased during heat-shock conditions to help the cell coping with the thermal stress . Our results suggest that, as the temperature increases, GroEL decreases its protein folding activity and starts acting as a "protein store." The molecular basis of this behavior is the loss of inter-ring signaling, which slows down GroES liberation from GroEL and therefore the release of the unfolded protein from the GroEL cavity . This behavior is reversible, and after heat-shock, GroEL reverts to its normal function . This might have a physiological meaning, since under thermal stress conditions, it may be inefficient for the cell to fold thermounstable proteins that are prone to denaturation. J Biol Chem, 1998 Dec 4, 273(49), 32576 - 81 LolA-dependent release of a lipid-modified protein from the inner membrane of Escherichia coli requires nucleoside triphosphate; Yakushi T et al.; The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone . The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal . To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system . The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system . An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation . L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts . Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P . In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP) . A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release . The outer membrane-specific sorting signal was essential for the in vitro release of L10P . Furthermore, L10P released in vitro was specifically incorporated into the outer membrane . These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy. J Biol Chem, 1998 Dec 4, 273(49), 32547 - 53 Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase; Kitada S et al.; Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides . The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage . To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat beta-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed . None of mutant proteins analyzed was impaired in dimer formation with the alpha-subunit . Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency . Together with sequence alignment data, glutamate 136 appears to be involved in metal binding . Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in kcat value by the mutation . Mutation of glutamate 79 led to decrease in kcat value and increase in Km values . Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding . Thus, some glutamate residues required for substrate binding and cleavage activity have been identified. J Biol Chem, 1998 Dec 4, 273(49), 32467 - 74 Structural, functional, and genetic characterization of Gastrophilus hemoglobin; Dewilde S et al.; Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized . At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting . Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h) . The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level . The open reading frame codes for a polypeptide of 150 amino acids . Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates . Freshly isolated hemoglobin was crystallized from polyethylene glycol . Crystals contain two hemoglobin molecules per asymmetric unit . Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals . In order to determine its three-dimensional structure, G . intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity . The globin gene contains two introns at positions D7.0 and G7.0 . The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought. J Biol Chem, 1998 Dec 4, 273(49), 32393 - 9 Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit; Schrattenholz A et al.; The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector . Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure . The fragment bound alpha-{3H}bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding) . The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR . Binding of small ligands was demonstrated by competition with alpha-{3H}bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM . The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor. J Biol Chem, 1998 Dec 4, 273(49), 32384 - 7 Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex; Rehrauer WM et al.; The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions . Normally, RecA protein mediates homologous pairing of DNA . We show that purified Umu(D')2C blocks this recombination function . Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers . Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament . This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function . Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function . This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication. J Bacteriol, 1998 Dec, 180(23), 6429 - 32 mraY is an essential gene for cell growth in Escherichia coli; Boyle DS et al.; The synthesis of the murein precursor lipid I is performed by MraY . We have shown that mraY is an essential gene for cell growth . Cells depleted of MraY first swell and then lyse . The expression of mraY DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol, 1998 Dec, 180(23), 6424 - 8 Role of the C terminus of FtsK in Escherichia coli chromosome segregation; Yu XC et al.; FtsK is essential for Escherichia coli cell division . We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions . Combining the corresponding truncated ftsK gene with a mukB null mutation resulted in a synthetic lethal phenotype . When the truncated ftsK was combined with a minCDE deletion, chains of minicells were generated, many of which contained DNA . These results suggest that the C terminus of FtsK has an important role in chromosome partitioning. J Bacteriol, 1998 Dec, 180(23), 6419 - 23 secG and temperature modulate expression of azide-resistant and signal sequence suppressor phenotypes of Escherichia coli secA mutants; Ramamurthy V et al.; SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the Escherichia coli inner membrane . To understand more about this process, azide-resistant (azi) and signal sequence suppressor (prlD) alleles of secA were studied . We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable . secG function is required for expression of azide-resistant and signal sequence suppressor activities of azi and prlD alleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a secG null mutant . These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation. J Bacteriol, 1998 Dec, 180(23), 6412 - 4 Identification and characterization of the Myxococcus xanthus argE gene; Harris BZ et al.; The chromosomal acetylornithine deacetylase (argE) gene of Myxococcus xanthus was identified via homology to acetylornithine deacetylases from other bacterial species . A mutant carrying a disruption in argE was unable to grow on minimal media lacking supplemental arginine and formed fruiting bodies and spores in response to arginine starvation at high cell density. J Bacteriol, 1998 Dec, 180(23), 6408 - 11 Sequence analysis of Tn10 insertion sites in a collection of Escherichia coli strains used for genetic mapping and strain construction; Nichols BP et al.; The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined . In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E . coli chromosome sequence . Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB. J Bacteriol, 1998 Dec, 180(23), 6396 - 9 Molecular analysis of a gene encoding a cell-bound esterase from Streptomyces chrysomallus; Berger R et al.; A gene (estA) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (cypA) of Streptomyces chrysomallus . Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of S . chrysomallus X2 and Streptomyces lividans . EstA specifically hydrolyzes short-chain p-nitrophenyl esters . EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase . Expression of estA from the melanin (mel) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes. J Bacteriol, 1998 Dec, 180(23), 6364 - 74 Effects of chromosome underreplication on cell division in Escherichia coli; Botello E et al.; The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth . We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain . In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle . We used underreplication conditions to study the response of cell division to a delayed initiation of replication . The bacteria were grown exponentially at 39 degreesC (normal DNA/mass ratio) and shifted to 38 and 37 degreesC . In the last two cases, new, stable, lower DNA/mass ratios were obtained . The rate of replication elongation was not affected under these conditions . At increasing degrees of underreplication, increasing proportions of the cells became elongated . Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big . The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell . These results favor a model in which cell division takes place at only distinct cell sizes . Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids . This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value . Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion. J Bacteriol, 1998 Dec, 180(23), 6306 - 15 RecA-independent pathways of lambdoid prophage induction in Escherichia coli; Rozanov DV et al.; Two Escherichia coli genes, expressed from multicopy plasmids, are shown to cause partial induction of prophage lambda in recA mutant lysogens . One is rcsA, which specifies a positive transcriptional regulator of the cps genes, which are involved in capsular polysaccharide synthesis . The other is dsrA, which specifies an 85-nucleotide RNA that relieves repression of the rcsA gene by histone-like protein H-NS . Genetic contexts known to increase Cps expression also cause RecA-independent lambda induction: the rcsC137 mutation, which causes constitutive Cps expression, and the lon and rcsA3 mutations, which stabilize RcsA . Lambdoid phages 21, phi80, and 434 are also induced by RcsA and DsrA overexpression in recA lysogens . Excess lambda cI repressor specifically blocks lambda induction, suggesting that induction involves repressor inactivation rather than repressor bypass . RcsA-mediated induction requires RcsB, the known effector of the cps operon, whereas DsrA-mediated induction is RcsB independent in stationary phase, pointing to the existence of yet another RecA-independent pathway of prophage induction. J Bacteriol, 1998 Dec, 180(23), 6269 - 75 Sister chromatid exchange frequencies in Escherichia coli analyzed by recombination at the dif resolvase site; Steiner WW et al.; Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site . Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described . To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions . Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD . It was also increased by a fur mutation, which increased oxidative DNA damage . Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E . coli . Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present . This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells. J Bacteriol, 1998 Dec, 180(23), 6260 - 8 Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli; Hussein MJ et al.; An Escherichia coli strain deficient in p-aminobenzoate synthesis was mutagenized, and derivatives were selected for growth on folic acid . Supplementation was shown to be due to p-aminobenzoyl-glutamate present as a breakdown product in commercial folic acid preparations . Two classes of mutations characterized by the minimum concentration of p-aminobenzoyl-glutamate that could support growth were obtained . Both classes of mutations were genetically and physically mapped to about 30 min on the E . coli chromosome . A cloned wild-type gene from this region, abgT (formerly ydaH) could confer a similar p-aminobenzoyl-glutamate utilization phenotype on the parental strain . Interruption of abgT on the plasmid or on the chromosome of the mutant strain resulted in a loss of the phenotype . abgT was the third gene in an apparent operon containing abgA, abgB, abgT, and possibly ogt and might be regulated by a divergently transcribed LysR-type regulator encoded by abgR . Two different single-base-pair mutations that gave rise to the p-aminobenzoyl-glutamate utilization phenotype lay in the abgR-abgA intercistronic region and appeared to allow the expression of abgT . The second class of mutation was due to a tandem duplication of abgB and abgT fused to fnr . The abgA and abgB gene products were homologous to one another and to a family of aminoacyl aminohydrolases . p-Aminobenzoyl-glutamate hydrolysis could be detected in extracts from several of the mutant strains, but intact abgA and abgB were not essential for p-aminobenzoyl-glutamate utilization when abgT was supplied in trans. J Bacteriol, 1998 Dec, 180(23), 6207 - 14 Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon; Haruki M et al.; We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli . This gene was expressed in an rnh mutant strain of E . coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E . coli RNases HI and HII . RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer . Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E . coli RNase HII proteins, respectively . The enzymatic activity was determined at 30 degreesC and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate . Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E . coli RNase HII, and Mg2+ for E . coli RNase HI . The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6 . 8-fold higher than that of E . coli RNase HII and 4.5-fold lower than that of E . coli RNase HI . Like E . coli RNase HI, RNase HIIPk and E . coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond . In addition, these enzymes cleave oligomeric substrates in a similar manner . These results suggest that RNase HIIPk and E . coli RNases HI and HII are structurally and functionally related to one another. J Bacteriol, 1998 Dec, 180(23), 6203 - 6 The general stress sigma factor sigmaS of Escherichia coli is induced during diauxic shift from glucose to lactose; Fischer D et al.; The general stress sigma factor sigmaS (RpoS) of Escherichia coli is strongly induced in response to glucose starvation . This increase in the cellular sigmaS level is due to stabilization of sigmaS, which under non-stress conditions is subject to rapid proteolysis . In the present study, it is demonstrated that sigmaS is also induced during the diauxic shift from glucose to lactose, i.e., under conditions of glucose exhaustion in the presence of another, less-preferred carbon source that eventually gets utilized . This sigmaS induction, which is due to stabilization, is transient and precedes the induction of beta-galactosidase . In parallel, sigmaS-dependent genes are transiently activated, as was shown here for osmY . Although sigmaS can mediate transcription of lacZ in vitro, sigmaS does not contribute to the induction of beta-galactosidase during the diauxic lag phase . Rather, the induction of sigmaS and the general stress response during the diauxic shift plays the role of a rapidly activated emergency system, which is shut off again as soon as the cells are able to cope with the stress situation by utilizing a more specific and more economical system. J Bacteriol, 1998 Dec, 180(23), 6187 - 92 Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro; Rosenheck S et al.; Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II) . It associates with the polymerase preferentially in stationary phase and is essential for some stress responses . Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts . We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35 degreesC) . In contrast, at moderate temperature (23 degreesC) Pol II activity is independent of Rpb4 . These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures . The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay . This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits . Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases . Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells . This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase. J Bacteriol, 1998 Dec, 180(23), 6140 - 7 uspB, a new sigmaS-regulated gene in Escherichia coli which is required for stationary-phase resistance to ethanol; Farewell A et al.; The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions . UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains . Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase . In contrast to uspA, stationary-phase induction of uspB requires the sigma factor sigmaS . The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering sigmaS levels . Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon's previously known role in thermotolerance, osmotolerance, and oxidative stress resistance. J Bacteriol, 1998 Dec, 180(23), 6117 - 25 H-NS and StpA proteins stimulate expression of the maltose regulon in Escherichia coli; Johansson J et al.; The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in Escherichia coli . Its role in gene regulation is manifested by the increased expression of several gene products in hns mutant strains . Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon . In studies with hns mutant strains and derivatives also deficient in the stpA gene, we found that expression of the LamB porin was decreased . Our results showed that the amounts of both LamB protein and lamB mRNA were greatly reduced in hns and hns-stpA mutant strains . The same results were obtained when we monitored the amount of transcription from the malEFG operon . The lamB gene is situated in the malKlamBmalM operon, which forms a divergent operon complex together with the malEFG operon . The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein . Using a malT-lacZ translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in hns and hns-stpA mutant strains in comparison with the wild-type strain . Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon. J Bacteriol, 1998 Dec, 180(23), 6090 - 100 Expression of lacZ from the promoter of the Escherichia coli spc operon cloned into vectors carrying the W205 trp-lac fusion; Liang ST et al.; The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of beta-galactosidase by standard enzymatic assay . Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon . The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt . In the presence of trpt, lacZ expression was temperature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity . The frequency of transcript termination at trpt was estimated to be near zero at 20 degreesC and at about 45% at 37 degreesC . The amount of Pspc-derived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between Pspc and lacZ . These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation . One particular construct without trpt did not seem to contain fortuitous transcription or translation signals generated at the fusion junction . In this strain, lacZ expression from Pspc was compared at the enzyme activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon . At any given growth rate, the different activities of beta-galactosidase in these two strains were found to reflect the same differences in their amounts of lacZ mRNA . Assuming that the promoter-lacZ fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of Pspc and the relative translation efficiency of Pspc-lacZ mRNA at different growth rates . Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h . The translation frequency of lacZ mRNA expressed from Pspc was unaffected by growth rates. Mol Gen Genet, 1998 Oct, 260(1), 75 - 80 A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12; Nakano H et al.; A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division . One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42 degrees C but not at 32 degrees C . The Kohara genomic library was screened for complementing clones . Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148 . DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme . When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32 degrees C but not at 42 degrees C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele . A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant. J Biotechnol, 1998 Oct 27, 65(2-3), 183 - 90 Expression and purification of a mutant human growth hormone that is resistant to proteolytic cleavage by thrombin, plasmin and human plasma in vitro; Alam KS et al.; The region having a sequence from amino acid 134 to 150 in human growth hormone (hGH) is known to be cleaved by proteases in human plasma, plasmin and thrombin . In this study, oligonucleotide primer-directed mutagenesis was used to produce recombinant mutant hGHs resistant to limited proteolysis by these proteases . Substitution of Arg134 and Thr135 of hGH with Asp134 and Pro135 yielded a thrombin-resistant hGH mutant, and substitution of Arg134, Thr135 and Lys140 with Asp134, Pro135 and Ala140 yielded a plasmin-resistant hGH mutant . The latter mutant hGH was also insensitive to in vitro proteolysis by human plasma incubated for 7 days . These alterations in amino acid residues of hGH did not disrupt its biological conformation and retained full growth promoting activities on rat Nb2 cells and human T-47D breast cancer cells. Biosens Bioelectron, 1998 Oct 1, 13(7-8), 741 - 56 Fully integrated biocatalytic electrodes based on bioaffinity interactions; Katz E et al.; Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers . A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems . NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer . The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer . The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer . The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate . The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH . Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C . 1.9.6.1 from E . coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode . The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules . The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer . The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively . Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode . Vectorial electron transfer proceeds in the synthetic heme-protein monolayer . Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate. Hepatology, 1998 Dec, 28(6), 1655 - 62 Endotoxin suppresses the oltipraz-mediated induction of major hepatic glutathione transferases and cytochromes P450 in the rat; Maheo K et al.; The effect of Escherichia coli lipopolysaccharide (LPS), a classic inducer of the acute-phase response, has been analyzed on both constitutive and oltipraz (a chemoprotective agent)-inducible messenger RNAs (mRNAs) and enzyme activities of major cytochromes P450 (CYPs) and glutathione transferases (rGSTs) in rat liver . At the dose administered (1 mg/kg) and the time studied (6 and 24 hours), endotoxin had no effect on the expression of either CYPs and GSTs with the exception of CYP1A2, which was reduced at both mRNA and activity levels . A strong increase of rGSTA1/2, rGSTM1, rGSTP1, CYP1A2, CYP2B1/2, and CYP2E1 was observed after 3 days of treatment with oltipraz (0.075%, wt/wt) . Oltipraz induction of these enzymes (with the exception of CYP2E1) was found to be suppressed at both mRNA, protein, and activity levels during the acute-phase response to endotoxin . Moreover, it is shown that oltipraz induction of CYP1A2 and CYP2B1/2 and its suppression by E . coli LPS occurred at a transcriptional level . These data support the idea that the chemoprotective effect of oltipraz is altered in the course of inflammation and that adaptation in chemoprotective strategies should be considered in certain physiopathologic situations. Protein Sci, 1998 Nov, 7(11), 2413 - 20 Prediction by a neural network of outer membrane beta-strand protein topology; Diederichs K et al.; An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) beta-strand proteins . Specifically, the NN predicts the z-coordinate of Calpha atoms in a coordinate frame with the outer membrane in the xy-plane, such that low z-values indicate periplasmic turns, medium z-values indicate transmembrane beta-strands, and high z-values indicate extracellular loops . To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z-axis . The relationship between Calpha z-values and topology was thereby established . To predict the topology of other OM proteins, all seven porins were used for the training set . Z-values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set . The results of topology prediction compare favorably with experimental topology data. Protein Sci, 1998 Nov, 7(11), 2384 - 90 The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding; Randall LL et al.; The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli . It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures . The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands . A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions . Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist. Protein Sci, 1998 Nov, 7(11), 2314 - 23 Toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of LP-130 complexed with proteases from HIV-1, FIV, and EIAV; Kervinen J et al.; One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance . The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of HIV-1 PR . The statine-based inhibitor LP-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral PRs . We solved the crystal structures of LP-130 in complex with retroviral PRs from HIV-1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active-site residues of the enzymes . This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules . The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3' positions, which might be responsible for the variation in the Ki values . These findings indicate that successful inhibition of different retroviral PRs by LP-130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active-site region . Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound . Hence, the ability of potential anti-AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance . These studies may also contribute to the development of a small-animal model for preclinical testing of antiviral compounds. Cell, 1998 Nov 13, 95(4), 553 - 62 Architecture of the gamma delta resolvase synaptosome: oriented heterodimers identity interactions essential for synapsis and recombination; Murley LL et al.; Gammadelta resolvase catalyzes recombination within a complex nucleoprotein structure containing at least 12 resolvase subunits bound to two 114 bp res sites . The 2,3' interaction between resolvase dimers is essential for synapsis and recombination . Using oriented resolvase heterodimers, we have identified half sites of res that must be occupied by a 2,3'-proficient protomer . For synapsis, only four of the eight subunits bound to sites II and III, those at II-L and III-L, require a 2,3'-proficient interface . This 2,3' interaction, apparently between protomers bound to adjacent sites, may nucleate assembly of the core synaptic complex . For recombination, 2,3'-proficient subunits are also required at sites I-R and III-R, suggesting an important communication between the crossover site and the core of the synapse. Cell, 1998 Nov 13, 95(4), 541 - 52 Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis; Ban C et al.; MutL and its homologs are essential for DNA mismatch repair . Mutations in genes encoding human homologs of MutL cause multiorgan cancer susceptibility . We have determined the crystal structure of a 40 kDa N-terminal fragment of E . coli MutL that retains all of the conserved residues in the MutL family . The structure of MutL is homologous to that of an ATPase-containing fragment of DNA gyrase . We have demonstrated that MutL binds and hydrolyzes ATP to ADP and Pi . Mutations in the MutL family that cause deficiencies in DNA mismatch repair and a predisposition to cancer mainly occur in the putative ATP-binding site . We provide evidence that the flexible, yet conserved, loops surrounding this ATP-binding site undergo conformational changes upon ATP hydrolysis thereby modulating interactions between MutL and other components of the repair machinery. FEBS Lett, 1998 Nov 6, 438(3), 267 - 71 Distinct mechanisms of antibody-mediated enzymatic reactivation in beta-galactosidase molecular sensors; Feliu JX et al.; The antibody-mediated reactivation of engineered Escherichia coli beta-galactosidases {Benito et al . (1996) J . Biol . Chem . 271, 21251-21256} has been thoughtfully investigated in three recombinant molecular sensors . Proteins M278VP1, JX772A and JX795A display the highly antigenic G-H loop peptide segment of foot-and-mouth disease virus VP1 protein, accommodated in different solvent-exposed loops of the assembled tetramer . These chimaeric enzymes exhibit a significant increase in enzymatic activity upon binding of either monoclonal antibodies or sera directed against the inserted viral peptide . In JX772A but not in M278VP1, the Fab 3E5 antibody fragment promotes reactivation to the same extent as the complete antibody . On the other hand, M278VP1 Km is reduced by more than 50% in the presence of activating serum, this parameter remains invariable in JX772A and it is only slightly modified in JX795A . In these last two proteins, significant k(cat) variations can account for the increased enzymatic activity . Alternative reactivation mechanisms in the different beta-galactosidase probes are discussed in the context of the bacterial enzyme structure and its tolerance to antibody-induced conformational modifications. FEBS Lett, 1998 Nov 6, 438(3), 159 - 60 Oxidative injury and survival during endotoxemia; Basu S et al.; This study investigates the plasma levels of 8-iso-PGF2alpha, a non-enzymatic, and 15-K-DH-PGF2alpha, a cyclooxygenase catalyzed oxidation product of arachidonic acid in an experimental porcine endotoxemic shock model . A significant (P < 0.001) and rapid appearance and disappearance of PGF2alpha metabolite after endotoxin infusion was very similar in both non-survival and survival groups indicating an acute progression and recession of inflammation . When oxidative injury was assessed by measuring free 8-iso-PGF2alpha the levels in plasma increased significantly up to 2 h and remained at this level until death among the non-survivors . This was apparently different from the survivors where the 8-iso-PGF2alpha levels increased to its height at 1 h, then decreased to the basal levels after 5 h . Thus, free radical and cyclooxygenase catalyzed oxidation of arachidonic acid occurs during endotoxemia . Free radical dependent oxidative injury following endotoxin induced inflammation may be the major cause of organ failure and increased mortality. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1998 Oct, 120(3), 351 - 6 Production of a polyclonal antibody raised against recombinant flounder p53 protein; Cachot J et al.; The cDNA encoding the wild type p53 protein from flounder, Platichthys flesus, was expressed in Escherichia coli using the GST fusion protein system . Several milligrams of recombinant p53 protein were purified . This protein displayed an apparent molecular weight of 45,000 Da, a value which is very similar to Xenopus p53, but significantly greater than was expected based on the length of the open reading frame . Immunization of rabbits against purified p53 protein allowed the production of high titre polyclonal antiserum . This new polyclonal antibody recognized recombinant flounder p53 protein in Western blot . Cross reaction was also observed with recombinant Xenopus p53 protein but not with human p53 protein . Immunoblotting of the total protein extract from normal flounder ovaries did not reveal any p53 expression. Res Microbiol, 1998 Oct, 149(9), 645 - 51 Colicin N interaction with sensitive Escherichia coli cells: in situ and kinetic approaches; el Kouhen R et al.; The kinetics of colicin N binding to the Escherichia coli surface, comprising the recognition of and association with cell-surface-exposed sites of OmpF porin, is a rapid event taking place during the first seconds of incubation . Immunogold labelling demonstrates the membrane localization of the colicin N bound to sensitive cells . Analyses of colicin-induced efflux indicate a short lag before the onset of cytoplasmic K+ release . This delay reflects the time necessary for translocation from the external side and pore-forming insertion into the cytoplasmic membrane. Nucleic Acids Res, 1998 Dec 1, 26(23), 5486 - 91 At least three linear regions but not the zinc-finger domain of U1C protein are exposed at the surface of the protein in solution and on the human spliceosomal U1 snRNP particle; Dumortier H et al.; No structural information on U1C protein either in its free state or bound to the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle is currently available . Using rabbit antibodies raised against a complete set of 15 U1C overlapping synthetic peptides (16-30 residues long) in different immunochemical tests, linear regions exposed at the surface of free and U1 snRNP-bound U1C were identified . Epitopes within at least three regions spanning residues 31-62, 85-103 and 116-159 were recognized on free and plastic-immobilized recombinant human U1C expressed in Escherichia coli, on in vitro translated U1C protein and on U1C bound to the U1 snRNP particle present in HeLa S100 extract . Using a zinc affinity labeling method, we further showed that the N-terminal U1C peptide containing a zinc-finger motif (peptide 5-34) effectively binds65Zn2+ . The N-terminal region of U1C, which is functional in U1 snRNP assembly, is apparently not located at the surface of the U1 snRNP particle. Nucleic Acids Res, 1998 Dec 1, 26(23), 5351 - 7 Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein); Sidorkina OM et al.; The Escherichia coli Fpg protein is involved in the repair of oxidized residues . We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein . Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG) . While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently . FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA . The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold . The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation . The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg . In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells . These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14326 - 31 Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors; Vales-Gomez M et al.; Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins . The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess . We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2 . NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates . However, the p50 receptors bind only very weakly, if at all, to HLA-C . The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14290 - 5 Tsetse thrombin inhibitor: bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans; Cappello M et al.; The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae) . A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes . The full-length cDNA contains a 26-bp untranslated segment at its 5' end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide . A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein . Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors . Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity . The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14142 - 6 A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma; Kendrick BS et al.; Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering . Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention . We have chosen recombinant human interferon-gamma (rhIFN-gamma) as a model protein for a mechanistic study of aggregation . In the presence of 0.9 M guanidinium hydrochloride, rhIFN-gamma aggregates with first order kinetics, a process that is inhibited by addition of sucrose . We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-gamma and the effect of sucrose . In this pathway, aggregation proceeds through a transient expansion of the native state . Sucrose shifts the equilibrium within the ensemble of rhIFN-gamma native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-gamma against aggregation . This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface . In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation . The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14130 - 5 Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries; Hanes J et al.; Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain . After three rounds of ribosome display, positive scFvs were isolated and characterized . Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor . The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution . One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor . It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method . The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding . Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14118 - 23 Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability; Vytvytska O et al.; The stability of the ompA mRNA depends on the bacterial growth rate . The 5' untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation . An RNA-binding protein with affinity for the 5' untranslated region ompA was purified and identified as Hfq, a host factor initially recognized for its function in phage Qbeta replication . The ompA RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the ompA mRNA . In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost . Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate . Hfq has no affinity for the lpp transcript whose degradation, like that of bulk mRNA, is not affected by bacterial growth rate . Compatible with our results, we found that the intracellular concentration of RNase E and its associated degradosome components is independent of bacterial growth rate . Thus our results suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14112 - 7 Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1; Lopez MC et al.; The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites . Rap1p has a modular domain structure . In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein . In the carboxyl terminus of Rap1p lie its silencing and putative activation domains . We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p . We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay . The ability to detect ternary complexes containing Rap1p.DNA . Gcr1p depended on the presence of binding sites for both proteins in the probe DNA . The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p . Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site . Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA . When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p.DNA.Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14106 - 11 The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme; Tomer G et al.; The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion . In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins . We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid . Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate . Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min . When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80% . DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts . This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction . These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14045 - 50 Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase; Lavie A et al.; The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5'-adenosyl)-P5-(5'-thymidyl)pentaphosphate and P1-(5'-adenosyl)P5-{5'-(3'-azido-3'-deoxythymidine)} pentaphosphate have been solved to 2.0-A and 2.2-A resolution, respectively . The overall structure of the bacterial TmpK is very similar to that of yeast TmpK . In contrast to the human and yeast TmpKs, which phosphorylate 3'-azido-3'-deoxythymidine 5'-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme . The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E . coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate . Yeast TmpK interacts with the 3'-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent . In E . coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3'-hydroxyl of dTMP . This different mode of interaction between the P loop carboxylic acid with the 3' substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E . coli enzyme without significant P loop movement . In addition, although the yeast enzyme uses Arg-15 (a glycine in E . coli) to stabilize the transition state, E . coli seems to use Arg-153 from a region termed Lid instead . Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase. Biophys J, 1998 Dec, 75(6), 3057 - 63 Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations; Hammermann M et al.; Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O . For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model . Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM . The position of the undulation corresponds to a distance of approximately 10-20 nm . This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA . From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA . The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers . We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix . The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration . Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations . It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration . This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies. Biophys J, 1998 Dec, 75(6), 2794 - 800 Weak substrate binding to transport proteins studied by NMR; Spooner PJ et al.; The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays . Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection . Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes . The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose . FucP appeared to bind both anomers of its substrates equally well . An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein . Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system. Biophys J, 1998 Dec, 75(6), 2712 - 20 Visualization of trp repressor and its complexes with DNA by atomic force microscopy; Margeat E et al.; We used tapping mode atomic force microscopy to visualize the protein/protein and the protein/DNA complexes involved in transcriptional regulation by the trp repressor (TR) . Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged . In the presence of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mica surface . We observed the expected decrease in specificity of TR for its operators with increasing protein concentration (1-5 nM) . This loss of DNA-binding specificity is accompanied by the formation of large protein assemblies of varying sizes on the mica surface, consistent with the known tendency of the repressor to oligomerize in solution . When the co-repressor is omitted, no repressor molecules are seen, either on the plasmid fragments or free on the mica surface, probably because of the formation of larger aggregates that are removed from the surface upon washing . All these findings support a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor. Biochem Biophys Res Commun, 1998 Nov 18, 252(2), 465 - 71 Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits: a functional role for the N-terminus of the factor; Moreno JM et al.; In the initiation step of bacterial protein synthesis initiation factor IF2 has to join the 30S ribosomal subunit in order to promote the binding of the fMet-tRNAMetf . In order to identify regions within IF2 which may be involved in the primary ribosome-factor interaction, we have constructed several C-terminal and N-terminal truncated forms of the factor as well as isolated structural domains, and tested them in a 30S ribosomal binding assay in vitro . Monoclonal antibodies with epitopes located within the two N-terminal domains of IF2 were used in these experiments . Hitherto, no function has been allocated to the N-terminal region of IF2 . Here we show that a mutant consisting of the two N-terminal domains has intrinsic affinity to the ribosomal subunit . Furthermore, a deletion mutant of IF2 which is lacking the two N-terminal domains shows negligible affinity . Moreover mAb with epitopes located within domain II strongly inhibits the binding capacity of IF2 to the 30S ribosomal subunit, whereas mAb with epitopes mapped within domain I do not affect the binding of the factor . The C-terminal domain of IF2 shows no affinity for the small ribosomal subunit . In addition, mutants with C-terminal deletions are not significantly affected in this interaction . Therefore, we conclude that the N-terminus of IF2 has affinity per se to bind the ribosomal subunit, with domain II being directly involved in the interaction . J Mol Biol, 1998 Dec 4, 284(3), 633 - 46 Primer-template misalignments during leading strand DNA synthesis account for the most frequent spontaneous mutations in a quasipalindromic region in Escherichia coli; Rosche WA et al.; Spontaneous mutant sequences which differ from the starting DNA sequence by the specific correction of quasipalindromic to perfect palindromic sequence are hallmarks of mutagenesis mediated by misalignments directed by palindromic complementarity . The mutant sequences are specifically predicted by templated, but ectopic, DNA polymerization on a misaligned DNA substrate . In a previous study, we characterized a spontaneous frameshift hotspot near a 17 bp quasipalindromic DNA sequence within the mutant chloramphenicol acetyl transferase (CAT) gene of plasmid pJT7 . A one base-pair insertion hotspot, ectopically templated by misalignment mediated by palindromic complementarity, was shown to occur more frequently during synthesis of the leading than the lagging DNA strand . Here we analyze the misalignment mechanisms that can account for the DNA sequences of 123 additional spontaneous frameshift mutations (22 distinct genotypes) occurring in the same quasipalindromic DNA region in plasmids pJT7 and p7TJ (a pJT7 derivative with the CAT gene in the inverse orientation) . Approximately 80% of the small frameshift mutants in each plasmid are predicted by palindromic misalignments of the leading strand . Smaller numbers of mutations are consistent with other DNA misalignments, including those predicted by simple slippage of the nascent DNA strand on its template . The results show that remarkable changes in the mutation spectra of a reporter gene may not be revealed by measurements of mutation frequency . J Mol Biol, 1998 Dec 4, 284(3), 621 - 31 The modification of the wobble base of tRNAGlu modulates the translation rate of glutamic acid codons in vivo; Kruger MK et al.; In Escherichia coli, uridine in the wobble position of tRNAGlu and tRNALys is modified to mnm5s2U34 . This modification is believed to restrict the base-pairing capability, i.e . to prevent misreading of near-cognate codons and reduce the efficiency of cognate codon reading, especially of codons ending in G . We have determined the influence of the 5-methylaminomethyl and the 2-thio modifications of mnm5s2U34 in tRNAGlu on the translation rate of the glutamate codons GAA and GAG in vivo . In wild-type cells, GAG is translated slower (7 . 7 codons/second) and GAA faster (18 codons/second) than the average codon (13 codons/second) . Surprisingly, tRNAGlu lacking the 5-methylaminomethyl group, thus containing s2U34, translated GAA twofold faster (47 codons/second) and GAG fourfold slower (1.9 codons/second) than fully modified tRNAGlu . In contrast, tRNAGlu that contains mnm5U34 instead of mnm5s2U34 translated GAA fourfold slower (4.5 codons/second) and GAG only 20% slower (6.2 codons/second) . Clearly, the 5-methylaminomethyl group of mnm5s2U34 facilitates base-pairing with G while decreasing base-pairing with A, resulting in rates of translation of GAG and GAA that approach that of the average codon . The 2-thio group increases the recognition of GAA and has only a minor effect on the decoding of GAG . Furthermore, the 2-thio group is important for aminoacylation (see the accompanying paper) . These data imply that the function of mnm5s2U34 may be different from what has been suggested previously . J Mol Biol, 1998 Dec 4, 284(3), 609 - 20 Aminoacylation of hypomodified tRNAGlu in vivo; Kruger MK et al.; The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition . All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus . Thus, the recognition by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA . The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm5s2U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro . Here, we have determined the aminoacylation level in vivo of tRNAGlu, tRNALys, and tRNA1GIn in Escherichia coli strains containing undermodified derivatives of mnm5s2U34 . Lack of the 5-methylaminomethyl group did not reduce charging levels for any of the three tRNAs . Lack of the s2U34 modification caused a 40% reduction in the charging level of tRNAGlu . Charging of tRNALys and tRNA1Gln were less affected . There was no compensating regulation of expression of glutamyl-tRNA synthetase because the relative synthesis rate was the same in the wild-type and mutant strains . These results indicate that the mnm5U34 modification is not an important recognition element in vivo for the glutamyl-tRNA synthetase . In contrast, lack of the s2U34 modification reduced the efficiency of charging by at least 40% . This is the minimal estimate because the turn-over rate of Glu-tRNAGlu was also reduced in the absence of the 2-thio group . Lack of either modification did not affect mischarging or mistranslation . J Mol Biol, 1998 Dec 4, 284(3), 579 - 90 A direct estimation of the context effect on the efficiency of termination; Pavlov MY et al.; An in vitro assay in which terminating Escherichia coli ribosomes with different stop signals in the A-site compete for a limited amount of a release factor (RF1 or RF2) has been used to estimate the relative termination efficiencies at stop codons with different adjacent downstream nucleotides . The assay allows direct measurements of relative kcat/Km parameters for the productive association of release factors to ribosomes . The kcat/Km parameter is larger for UAA(U) than for UAA(C) programmed ribosomes and the difference in kcat/Km is much larger for RF2 (about 80%) than for RF1 (about 30%) . These differences in the kcat/Km parameter are not affected by the addition of release factor RF3 . The only discernible effect of RF3 is a considerable acceleration of RF1/2 recycling.The estimated kcat/Km parameters correlate well with the affinities of release factors for ribosomes programmed with different stop signals . These affinities were estimated from the extent of inhibition of ribosomal recycling by high concentrations of release factors in the absence of release factor RF3 . The affinity for RF2 depends on the immediate downstream context of the stop codon in the translated mRNA and is about three times higher for UAA(U) than for UAA(C) . The corresponding difference in affinities for RF1 is twofold . For all stop signals studied, the estimated affinity of RF2 for terminating ribosomes is much lower than that of RF1 . It is also striking that the affinity of ribosomes for a chromosomally expressed RF2 is at least three times higher than for RF2 isolated from an overproducing E . coli strain . Gynecol Oncol, 1998 Nov, 71(2), 278 - 87 High-efficacy thymidine kinase gene transfer to ovarian cancer cell lines mediated by herpes simplex virus type 1 vector; Wang M et al.; In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro . The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines . The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK . Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro . Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy . Infect Immun, 1998 Dec, 66(12), 6049 - 53 Enteropathogenic Escherichia coli virulence genes encoding secreted signalling proteins are essential for modulation of Caco-2 cell electrolyte transport; Collington GK et al.; The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhea remains uncertain . In vitro, EPEC stimulates a rapid increase in short-circuit current (Isc) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion . This study has examined the roles of specific EPEC virulence proteins in this Isc response . EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport. Infect Immun, 1998 Dec, 66(12), 5915 - 20 Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection; McGill SL et al.; Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis . Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen . Bartonella-negative control sera were used to determine baseline antibody activity . Heterogeneous B . henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed . Though individual banding patterns were variable, one approximately 83-kDa B . henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection . Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident . Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B . quintana-infected patients did react to Bh83 . This cross-reactivity suggests epitope conservation during infection with B . henselae or B . quintana . Western blot analysis further revealed similar banding patterns when B . henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA . Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis . Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1 . These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B . henselae or B . quintana. Infect Immun, 1998 Dec, 66(12), 5882 - 8 Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii; Macellaro A et al.; A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by {35S}methionine labeling, autoradiography, and immunoblotting . The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies . The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed . A single open reading frame (ORF) with a size of 1,968 bp was identified . The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800 . The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence . The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment . The gene was expressed in Escherichia coli as an intact protein . The C . burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family . A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E . coli DnaK, is more than 80% . The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins . The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy . The dual localization was verified by Western blot analysis of proteins in C . burnetii cell fractions, using purified antibodies directed to the 71-kDa protein. Infect Immun, 1998 Dec, 66(12), 5692 - 7 Environmental growth conditions influence the ability of Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum resistance; Badger JL et al.; A major limitation to advances in prevention and therapy of neonatal meningitis is our incomplete understanding of the pathogenesis of this disease . In an effort to understand the pathogenesis of meningitis due to Escherichia coli K1, we examined whether environmental growth conditions similar to those that the bacteria might be exposed to in the blood could influence the ability of E . coli K1 to invade brain microvascular endothelial cells (BMEC) in vitro and to cross the blood-brain barrier in vivo . We found that the following bacterial growth conditions enhanced E . coli K1 invasion of BMEC 3- to 10-fold: microaerophilic growth, media buffered at pH 6.5, and media supplemented with 50% newborn bovine serum (NBS), magnesium, or iron . Growth conditions that significantly repressed invasion (i.e., 2- to 250-fold) included iron chelation, a pH of 8.5, and high osmolarity . More importantly, E . coli K1 traversal of the blood-brain barrier was significantly greater for the growth condition enhancing BMEC invasion (50% NBS) than for the condition repressing invasion (osmolarity) in newborn rats with experimental hematogenous meningitis . Of interest, bacterial growth conditions that enhanced or repressed invasion also elicited similar serum resistance phenotype patterns . This is the first demonstration that bacterial ability to enter the central nervous system can be affected by environmental growth conditions. Infect Immun, 1998 Dec, 66(12), 5684 - 91 Cloning and sequencing of yajC and secD homologs of Brucella abortus and demonstration of immune responses to YajC in mice vaccinated with B . abortus RB51; Vemulapalli R et al.; To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B . abortus RB51 . One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs) . The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species . Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP) . In Western blots, sera from mice vaccinated with B . abortus RB51 recognized YajC but not SecD . Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B . abortus 19 or 2308 or B . melitensis RM1 also produced antibodies to YajC . In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B . abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4 . This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent. Infect Immun, 1998 Dec, 66(12), 5650 - 8 Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b; Rousset E et al.; Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure . STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively . STb exhibited the highest binding affinity for sulfatide . STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms . The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide . The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions . Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb . Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC) . Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard . The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide . In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard . Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E . Rousset, J . Harel, and J . D . Dubreuil, Microb . Pathog . 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid . Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb . In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin. Infect Immun, 1998 Dec, 66(12), 5643 - 9 Identification of immunodominant regions within the C-terminal cell binding domain of intimin alpha and intimin beta from enteropathogenic Escherichia coli; Adu-Bobie J et al.; Enteropathogenic Escherichia coli (EPEC) strains are a common cause of infantile diarrhea in developing countries . EPEC strains induce a characteristic attaching and effacing (A/E) lesion on epithelial cells . A/E lesion formation requires intimin, an outer membrane adhesin protein . The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of the polypeptide (Int280) . So far, four distinct Int280 types (alpha, beta, gamma, and delta) have been identified . The aim of this study was to identify immunodominant regions within the Int280alpha and Int280beta domains . Recombinant DNA was used to construct and express overlapping polypeptides spanning these domains . Rabbit anti-Int280 antisera and human colostral immunoglobulin A were reacted with these polypeptides in Western blots and en