Nucleic Acids Res, 1998 Nov 1, 26(21), 4846 - 52 Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter; Rincon JC et al.; Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III . Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library . The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function . Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters . Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors . Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes . Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription. Nucleic Acids Res, 1998 Nov 1, 26(21), 4828 - 36 EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities; Davies GP et al.; For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR) . The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV . We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV . Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes . We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs . Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage . The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates . Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity. Nucleic Acids Res, 1998 Nov 1, 26(21), 4804 - 10 Role of the DNA ligase III zinc finger in polynucleotide binding and ligation; Taylor RM et al.; Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta . Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase . Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms . Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay . In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers . Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold . These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer . The implications of these results to the cellular role of the putative zinc finger are discussed. Nature, 1998 Oct 1, 395(6701), 516 - 21 Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins {see comment}; Hill K et al.; The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes . Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore . Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now . Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure . Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane . We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins. Nature, 1998 Oct 1, 395(6701), 511 - 6 Efficiency of signalling through cytokine receptors depends critically on receptor orientation; Syed RS et al.; Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells . It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade . The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM, 10(-2) of its Kd value for erythropoietin-EPOR binding site 1 (Kd approximately equal to nM), and 10(-5) of the Kd for erythropoietin-EPOR binding site 2 (Kd approximately equal to 1 microM) . Overall half-maximal binding (IC50) of cell-surface receptors is produced with approximately 0.18 nM erythropoietin, indicating that only approximately 6% of the receptors would be bound in the presence of 10 pM erythropoietin . Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses but much less efficiently, requiring concentrations close to their Kd values (approximately 0.1 microM) . The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 A from two crystal forms, shows that erythropoietin imposes a unique 120 degrees angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways. J Clin Lab Anal, 1998, 12(5), 263 - 7 An enzyme-linked immunosorbent assay and reference ranges for bisphosphoglycerate mutase in human erythrocytes; Takubo T et al.; We established an enzyme-linked immunosorbent assay (ELISA) system for the determination of human bisphosphoglycerate mutase (BPGM) protein content in human erythrocytes using a polyclonal anti-BPGM antibody, we determined reference ranges for BPGM protein content, synthase activity, and specific activity in human erythrocytes . We produced a recombinant human BPGM (rBPGM) by gene manipulation using E . coli and then obtained the polyclonal antibody by immunizing rabbits with purified rBPGM . The reproducibility of the ELISA was in an acceptable range with a coefficient of variation under 1.5% . The ELISA was reliable in the range of 0.1 to 10 ng/mL . The polyclonal anti-rBPGM antibody did not show any cross-reaction with recombinant human B type phosphoglycerate mutase, which is highly homologous to rBPGM . The ELISA was found to be practical for the determination of BPGM protein content in human erythrocytes . The mean BPGM protein content was 56.3+/-9.7 microg/mL in whole blood (mean+/-SD, n = 50) . The ELISA can be used to examine various hematologic disorders with abnormal red cell size and cell counts, and to detect BPGM enzymopathy in human erythrocytes. J Immunol Methods, 1998 Aug 1, 217(1-2), 1 - 10 F(ab')2 molecules made from Escherichia coli produced Fab' with hinge sequences conferring increased serum survival in an animal model; Humphreys DP et al.; Fab's with hinges based on the human gamma1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab')2 . We find that the F(ab')2 made with hinges containing 2 or 4 cysteines have a high level (approximately 70%) of multiple disulphide bonds . These F(ab')2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond . One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab')2 formed by chemical cross-linking . F(ab')2 made from the Fab' with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols . These hinge sequences provide a simple method for producing robust F(ab')2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates. Poult Sci, 1998 Oct, 77(10), 1531 - 3 A comparison of Escherichia coli levels at four sampling sites on turkey carcasses; Bodnaruk PW et al.; The objective of this study was to investigate the levels of Escherichia coli on different sites of turkey carcasses by sponge sampling using a 50 cm2 template . The breast, thigh, back, and cavity sites were sampled to determine which sites would be suitable for quantifying E . coli levels for the purpose of assessing control of the slaughtering and chilling processes . Results show that the breast and cavity sites have the lowest levels of E . coli (2.6 and 2.7 cfu/cm2, respectively), whereas thigh and back sites have the highest (6.7 and 7.6 cfu/cm2, respectively) . Data analyses indicate that E . coli levels at the breast and cavity site are lower (P < 0.05) than the other sites . A composite sample consisting of thigh and back sites for E . coli testing is recommended for assessing process control in turkey slaughtering facilities. Chem Pharm Bull (Tokyo), 1998 Sep, 46(9), 1490 - 2 Synthesis of new peptides with prolactin-releasing activity by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction; Nishimura O et al.; A newly isolated peptide from bovine hypothalamus with prolactin-releasing activity (prolactin-releasing peptide; PrRP) was synthesized by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction, together with rat and human homologs . The peptides were expressed in the form of fusion proteins with basic fibroblast growth factor mutein, which were purified by heparin-affinity chromatography . The fusion proteins were cleaved at the cysteine residues of the junction site by cyanylation, followed by treatment with ammonia for C-terminal amidation . Purification of the resulting crude peptides was performed using chromatography on a gel-filtration column, a cation-exchange column, and a reversed-phase column . As an example, about 90 mg of bovine PrRP (bPrRP) was obtained from 201 of culture bloth . The purified b PrRP showed full biological activities in binding to its receptor expressed on CHO cells and releasing arachidonic acid metabolite from the same cells, while the C-terminal acid form of bPrRP had little of these activities . These results indicate that the C-terminal amide structure is very important for expressing biological activity . The peptides obtained here might be very useful for studies on their biological significance and roles in vivo. Mol Cell, 1998 Sep, 2(3), 361 - 72 Crystal structure of an octameric RuvA-Holliday junction complex; Roe SM et al.; Holliday junctions occur as intermediates in homologous recombination and DNA repair . In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands . The crystal structure of a complex between the RuvA protein of M . leprae and a synthetic four-way junction has now been determined . Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer-tetramer interface . Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA. Biochim Biophys Acta, 1998 Oct 14, 1388(1), 273 - 8 Molecular characterization of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase; Seong C et al.; We have isolated and characterized the cDNA and the genomic DNA encoding Drosophila melanogaster pterin 4alpha-carbinolamine dehydratase (PCD) . The amino acid sequence deduced from the cDNA sequence was very similar to those of PCDs previously reported in other species (19-57% identity) . The protein coding region of the cDNA was expressed in E . coli as a histidine fusion protein, and the expressed protein proved to have PCD activity . The characterization of the Drosophila genomic clone revealed that the Drosophila PCD gene is interrupted by two introns . The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks the distinct TATAAA box consensus sequence. Biochim Biophys Acta, 1998 Oct 14, 1388(1), 77 - 83 Unique primary structure of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula; Inohara-Ochiai M et al.; The gene of the monomeric multimetal beta-galactosidase of Saccharopolyspora rectivirgula was cloned and sequenced . Although the enzyme could be assigned as a member of beta-galactosidases belonging to the glycosyl hydrolase family 2, it has unusual structural features for beta-galactosidase of this family; it contained a unique sequence which consists of approximately 200 amino acid residues with no similarity to known proteins . This 200-residue sequence exists as if it is inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme. Biochim Biophys Acta, 1998 Oct 14, 1388(1), 1 - 9 Glucosamine-6-phosphate deaminase from beef kidney is an allosteric system of the V-type; Lara-Lemus R et al.; The enzyme glucosamine-6-phosphate deaminase from beef kidney has been purified to homogeneity by allosteric-site affinity chromatography . Its amino acid composition and the N-terminal sequence (1-42), were obtained . The amino acid sequence of this segment is essentially identical to the corresponding regions of the human and hamster glucosamine-6-phosphate deaminases . The beef enzyme is a hexamer of 32.5 kDa subunits; this is nearly 2.5 kDa higher than the molecular mass of the homologous enzyme from Escherichia coli . Beef kidney deaminase exhibits a notable difference from the bacterial enzyme in its allosteric activation by N-acetylglucosamine 6-phosphate This metabolite, which is also is the allosteric activator of the bacterial glucosamine-6-phosphate deaminase, activates the enzyme by increasing its kcat without any change in the Km values for glucosamine 6-phosphate, over a wide range of activator concentration . This observation places beef kidney deaminase in the class of V-type allosteric systems. Mol Cell Biol, 1998 Nov, 18(11), 6826 - 38 Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores; Santos-Rosa H et al.; We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export . This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p . In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2 . In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export . At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts . Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p. Mol Cell Biol, 1998 Nov, 18(11), 6515 - 24 Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex; Dekker PJ et al.; The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP) . We have analyzed the molecular architecture of the Tom machinery . The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes . A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized . Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7 . In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5 . Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40 . The following conclusions are suggested . (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery . (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex . (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40. J Clin Microbiol, 1998 Nov, 36(11), 3337 - 41 Development of a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot for detection of CMV-specific IgM; Lazzarotto T et al.; We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera . The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli . These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a mu-specific conjugate . The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals . The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100% . We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history . Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests . Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring. J Clin Microbiol, 1998 Nov, 36(11), 3170 - 2 Diagnostic potential of Toscana virus N protein expressed in Escherichia coli; Valassina M et al.; The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector . The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA) . The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples . The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity. J Clin Microbiol, 1998 Nov, 36(11), 3143 - 8 Detection of Lassa virus antinucleoprotein immunoglobulin G (IgG) and IgM antibodies by a simple recombinant immunoblot assay for field use; Ter Meulen J et al.; The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein . Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane . A total of 1 microgram of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay . Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 microgram . A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay . The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples) . In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens . The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%) . Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea . On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay . One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah . Seven PCR-negative patients were reactive by immunoblotting . The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively . Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic. J Struct Biol, 1998 Sep, 123(1), 67 - 71 A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography; Boekema EJ et al.; Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli . Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography . A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density . J Biol Chem, 1998 Oct 23, 273(43), 28078 - 84 Mutant membrane protein toxicity; Stewart C et al.; This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12) . In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells . In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10(4)-fold . Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed . The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix . The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding . An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease. J Biol Chem, 1998 Oct 23, 273(43), 27927 - 33 A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L; Berkemeyer M et al.; We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1 . 1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82) . Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach . A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase . A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide . In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa . Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts . Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues . The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids. J Biol Chem, 1998 Oct 23, 273(43), 27873 - 8 Deletions in the second stalk of F1F0-ATP synthase in Escherichia coli; Sorgen PL et al.; In Escherichia coli F1F0-ATP synthase, the two b subunits form the second stalk spanning the distance between the membrane F0 sector and the bulk of F1 . Current models predict that the stator should be relatively rigid and engaged in contact with F1 at fixed points . To test this hypothesis, we constructed a series of deletion mutations in the uncF(b) gene to remove segments from the middle of the second stalk of the subunit . Mutants with deletions of 7 amino acids were essentially normal, and those with deletions of up to 11 amino acids retained considerable activity . Membranes prepared from these strains had readily detectable levels of F1-ATPase activity and proton pumping activity . Removal of 12 or more amino acids resulted in loss of oxidative phosphorylation . Levels of membrane-associated F1-ATPase dropped precipitously for the longer deletions, and immunoblot analysis indicated that reductions in activity correlated with reduced levels of b subunit in the membranes . Assuming the likely alpha-helical conformation for this area of the b subunit, the 11-amino acid deletion would result in shortening the subunit by approximately 16 A . Since these deletions did not prevent the b subunit from participating in productive interactions with F1, we suggest that the b subunit is not a rigid rodlike structure, but has an inherent flexibility compatible with a dynamic role in coupling. J Biol Chem, 1998 Oct 23, 273(43), 27841 - 7 Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression; Trevino RJ et al.; Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability . The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli . Rhodanese species with up to 9 residues deleted were expressed and purified . Kinetic parameters for the mutants were similar to those of the full-length enzyme . Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC . Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated . Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo . However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity . Truncations in the NH2 terminus affect the chemical stability of the distantly located active site . Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro. EMBO J, 1998 Oct 15, 17(20), 6076 - 85 Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems; Radnedge L et al.; The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells . It encodes two DNA sites and two proteins, ParA and ParB . Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific . Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules . The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition . Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts . We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA . Similarly, ParA acts in partition by making a specific contact with ParB bound at parS . Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB . As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition . The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules. EMBO J, 1998 Oct 15, 17(20), 6069 - 75 The Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking ribosome binding; Altuvia S et al.; OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli . This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded sigma(s) subunit of RNA polymerase . Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS . We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation. EMBO J, 1998 Oct 15, 17(20), 6061 - 8 The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein; Zhang A et al.; The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage . Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase . Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase . We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message . This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I) . Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity. EMBO J, 1998 Oct 15, 17(20), 5887 - 95 Voltage-generated torque drives the motor of the ATP synthase; Kaim G et al.; The mechanism by which ion-flux through the membrane-bound motor module (F0) induces rotational torque, driving the rotation of the gamma subunit, was probed with a Na+-translocating hybrid ATP synthase . The ATP-dependent occlusion of 1 (22)Na+ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22Na+ was added first and ATP second, but not if the order of addition was reversed . These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22Na+-loaded or a DCCD-modified rotor subunit reaches the Na+-impermeable stator . The ATP synthase with a Na+-permeable stator catalyzed 22Na+out/Na+in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (DeltaPsi) of 90 mV . We propose that in the idling mode of the motor, Na+ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator . This motional flexibility is arrested if either ATP or DeltaPsi induces the switch from idling into a directed rotation . The Propionigenium modestum ATP synthase catalyzed ATP formation with DeltaPsi of 60-125 mV but not with DeltapNa+ of 195 mV . These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor. J Pept Res, 1998 Sep, 52(3), 229 - 40 One peptide, two topologies: structure and interconversion dynamics of human uroguanylin isomers; Marx UC et al.; The peptide hormone uroguanylin stimulates chloride secretion via activation of intestinal guanylyl cyclase C (GC-C) . It is characterized by two disulfide bonds in a 1-3/2-4 pattern that causes the existence of two topological stereoisomers of which only one induces intracellular cGMP elevation . To obtain an unambiguous structure-function relationship of the isomers, we determined the solution structure of the separated uroguanylin isoforms using NMR spectroscopy . Both isomers adopt well-defined structures that correspond to those of the isomers of the related peptide guanylin . Furthermore, the structure of the GC-C-activating uroguanylin isomer A closely resembles the structure of the agonistic Escherichia coli heat-stable enterotoxin . Compared with guanylin isomers, the conformational interconversion of uroguanylin isomers is retarded significantly . As judged from chromatography and NMR spectroscopy, both uroguanylin isoforms are stable at low temperatures, but are subject to a slow pH-dependent mutual isomerization at 37 degrees C with an equilibrium isomer ratio of approximately 1:1 . The conformational exchange is most likely under the sterical control of the carboxy-terminal leucine . These results imply that GC-C is activated by ligands exhibiting the molecular framework corresponding to the structure of uroguanylin isomer A. Anticancer Drugs, 1998 Aug, 9(7), 653 - 7 The relationship between the antitumor activity and the ribonucleotide reductase inhibitory activity of (E)-2'-deoxy-2'-(fluoromethylene) cytidine, MDL 101,731; Kanazawa J et al.; (E)-2'-deoxy-2'-(fluoromethylene) cytidine (MDL101,731) is a new deoxycytidine analog which shows potent antitumor activity against several human tumor models . We previously showed that MDL101,731 inhibited human ribonucleotide reductase (RNR) in HeLa S3 human cervical carcinoma cells . Recently, it has been reported that another deoxycytidine analog, 2'-deoxy-2'-methylidenecytidine (DMDC) which also inhibits RNR from Escherichia coli, does not inhibit RNR in intact L1210 murine leukemia cells . MDL101,731 was designed as an inhibitor of RNR, so it is important to know the contribution of the RNR inhibitory activity of the drug on its antitumor efficacy in vivo . Therefore, we examined the relationship between the antitumor activity and RNR inhibitory activity of MDL101,731 using LX-1 human lung carcinoma which was highly sensitive to this drug . MDL101,731 showed strong inhibition of RNR activity in LX-1 lung carcinoma by both i.v . and p.o . administration . Administration of 15 mg/kg i.v . and 30 mg/kg p.o . of MDL101,731, doses which showed almost the same degree of antitumor activity against LX-1 lung carcinoma on a daily 5 day schedule, caused a similar degree and similar kinetics of inhibition of RNR in LX-1 lung carcinoma at least for 12 h after administration . On the other hand, DMDC as well as 1-beta-D-arabinofuranosyl-cytosine (ara-C), which is a well-known deoxycytidine analog and inhibits DNA polymerase alpha, did not inhibit RNR in LX-1 lung carcinoma at doses demonstrating antitumor activity . These results indicate that MDL101,731 exhibited antitumor activity through inhibition of RNR activity in tumor cells in vivo and the mechanism of antitumor action of MDL 101,731 might be different from those of DMDC and ara-C, at least in part. Acta Anaesthesiol Scand, 1998 Sep, 42(8), 966 - 73 Intravenous endotoxin does not increase tissue extravasation of albumin in rats; Metcalf K et al.; BACKGROUND: It is unclear whether activation of the inducible nitric oxide synthase (iNOS) increases or decreases the extravasation of plasma . METHODS: Chloralose anaesthetised male Wistar rats received E . coli lipopolysaccharide (LPS), 3 mg kg-1 i.v., or the corresponding volume of saline, 3 or 5 h before the end of the experiment . Mean arterial pressure (MAP) and heart rate (HR) were recorded . Tissue clearance of radio-labelled albumin, during the last 2 h of each experiment, was determined by a double-isotope method . In separate animals, the serum concentration of nitrite and nitrate was determined, 5 h after LPS or the solvent . MAIN RESULTS: LPS initially decreased MAP and lastingly increased HR . In the 3-h LPS animals (n = 8), tissue plasma clearance was lower in the heart and calf muscle and increased only in diaphragm, compared to corresponding control animals (n = 8) . In the 5-h LPS rats, clearance was lowered (n = 8) in the entire gastrointestinal tract and in testes, compared to controls (n = 8) . The serum nitrite/nitrate concentration was higher in animals given LPS (n = 6) than in controls (n = 6) . CONCLUSION: After LPS, tissue clearance of albumin was not increased in any major tissue, in spite of increased serum levels of NO end products . Apparently, after activation of iNOS, the augmented release of NO is not necessarily associated with increased albumin extravasation. Shi Yan Sheng Wu Xue Bao, 1996 Dec, 29(4), 385 - 93 {Experimental treatment of brain tumor cells using CD suicide gene}; Xu LF et al.; A negative selection system for glioma gene therapy was established in vitro . C 6 rat glioma cells were infected with recombined retrovirus which contain Escherichia coli cytosine deaminase (EC-CD) gene . The enzyme CD can transform the non-toxic prodrug 5-Fluorocytosine (5-FC) to the highly cellular toxic compound 5-Fluorouracil (5-FU) . The growth inhibition studies proved that CD-positive cells were highly sensitive to 5-FC, the IC50 about 3 mumol/L, compared with an IC50 of approximately 6000 mumol/L in parental C 6 cells . Both CD-positive and negative cells were sensitive to 5-FU at very low concentration (IC50 < 1 mumol/L) . Mixed cellular assay showed CD-positive cells had "bystander effect" on CD-negative cells when exposed to 5-FC . Our results demonstrate that EC-CD gene should be an efficient suicide gene for the treatment of glioma. Shi Yan Sheng Wu Xue Bao, 1996 Dec, 29(4), 357 - 63 {Vimentin and Nup 180 in vitro binding assay}; Cai ST et al.; In order to investigate relationship between vimentin and nuclear pore complex, we examined binding ability of vimentin, expressed in E . coli, with nucleoporin, isolated from rat liver nuclei, in vitro . Negative staining electron microscopy showed that the vimentin expressed in bacteria assembled 10 nm filament in vitro . SDS-PAGE and western blotting showed that Nup 180 bind to vimentin in binding assay in vitro . Combining immunogold labeling and negative staining electron microscopy techniques, we showed that Nup 180 bind on the 10 nm vimentin filaments . The experiment results indicated that vimentin filament may be anchored on nuclear pore complex in vivo by binding with Nup 180. Zhonghua Yi Xue Za Zhi, 1997 May, 77(5), 359 - 62 {The effects of removing circulated TNF by immunoadsorption on renal changes in rabbits with endotoxin shock}; Lin Y et al.; OBJECTIVES: To remove circulating tumor necrosis factor (TNF) by specific immunoadsorption column containing anti-TNF monoclonal antibody (McAb) and observe the effects of the therapy on renal changes in experimental endotoxin shock . METHODS: New Zealand white rabbits were injected with lethal dose of E . coli endotoxin (LPS, 8 x 10(9) cfu/kg) to produce endotoxin shock, then were divided into two groups: control (n = 7) and perfusion group (n = 10) . Perfusion was started at 1 hour after administration of endotoxin . Mean arterial pressure (MAP) was monitored continuously . Blood samples were collected for assay of TNF, urea nitrogen (BUN) and creation (Cr) . Six hours after infusion of LPS, rabbits were sacrificed, then the kidney was taken for pathologic observation . RESULTS: MAP was increased 30 minutes after perfusion in perfusion group, then kept at a level higher than that in the control group in the monitoring period (P < 0.01) . The plasma TNF activation was reduced significantly in the perfusion group (44 +/- 10) compared with the control group (1448 +/- 226, P < 0.05) one hour after perfusion . The levels of BUN and Cr were decreased in the perfusion group compared with the control group . The degree of glomerular congestion and infiltration of leukocytes in glomeruli was significantly lower in the perfusion group than in the control group . The degree of tubular necrosis was decreased significantly in the perfusion group compared to that in the control group (P < 0.05) . The mitochondrial ultrastructural changes were more severe in the control group than in the perfusion group . CONCLUSIONS: Specific immunoadsorptive column containing anti-TNFMcAb could remove effectively circulating TNF in experimental endotoxin shock . Reducing levels of TNF in the early phase of endotoxin shock may ameliorate the state of hypotension. Sci China C Life Sci, 1996 Oct, 39(5), 523 - 33 Urokinase mutant with better fibrin-specificity; Ma Z et al.; A 150-156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its recombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli . After denaturation and renaturation in vitro, the expressed products were both purified to a single silver-stained band by means of IgG affinity chromatography . After activation by plasmin, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion . In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA . Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA . It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fibrin specificity by means of mutation. Sci China C Life Sci, 1996 Dec, 39(6), 571 - 83 Variety of molecular conformation of plasmid pUC18 DNA and solenoidally supercoiled DNA; Huang X et al.; The plasmid pUC18 DNA isolated from Escherichia coli HB101 were analyzed by two-dimensional agarose gel electrophoresis and hybridization . The results show that the DNA sample can be separated into six groups of different structural components . The plectonemically and solenoidally supercoiled pUC18 DNA coexist in it . These two different conformations of supercoiled DNA are interchangeable with the circumstances (ionic strength and type, etc.) . The amount of solenoidally supercoiled pUC18 DNA in the samples can be changed by treatment of DNA topoisomerases . Under an electron microscope, the solenoidal supercoiling DNA has a round shape with an average diameter of 45 nm . The facts suggest that solenoidally supercoiled DNA be a structural entity independent of histones . The polymorphism of DNA structure may be important to packing of DNA in vivo. Int J Oncol, 1998 Nov, 13(5), 1061 - 7 NRG-3 in human breast cancers: activation of multiple erbB family proteins; Hijazi MM et al.; Ligands of the EGF/Heregulin family control the growth of epithelial cells by binding to receptors of the erbB family . By searching a large database of cDNA sequences at Human Genome Sciences Inc . we have identified a new encoded protein sequence containing all the conserved elements of the EGF/Heregulin family . The same sequence has recently been independently identified as NRG-3 . The EGF-like domain of NRG-3 was generated as a recombinant protein in E . coli and used to test the specificity of receptor binding . In human breast cancer cells and in 32D cells transfected by erbB family members, NRG-3 activated multiple erbB family members . These include EGF receptor (erbB1) and erbB4 when expressed individually and erbB2 and erbB3 when expressed together . Recombinant NRG-3 altered the growth of human breast cancer cells growing in vitro . NRG-3 was expressed in cell lines derived from breast cancer . These results indicate that NRG-3 is a potential regulator of normal and malignant breast epithelial cells in vivo. Toxicol Appl Pharmacol, 1998 Sep, 152(1), 166 - 74 Identification of amino acid residues essential for high aflatoxin B1-8,9-epoxide conjugation activity in alpha class glutathione S-transferases through site-directed mutagenesis; Van Ness KP et al.; Mice constitutively express glutathione S-transferase mGSTA3-3 in liver . This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity . In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure . Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO . To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site . Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues . Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity . Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes . When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2 . 8 nmol/mg/min . Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO . Biochemistry, 1998 Oct 13, 37(41), 14500 - 7 Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia; Geeganage S et al.; Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme) . Gln 168 in E . coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate {Wedekind, J . E., Frey, P . A., and Rayment, I . (1996) Biochemistry 35, 11560-11569} . In humans, the point variant Q188R accounts for 60% of galactosemia cases . The corresponding E . coli variant Q168R has been overexpressed and purified . In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme . At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P . Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent . The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites . Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively . In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively . The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value . The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R . The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R . The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits . The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme . These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions . Instead, the main defect is very slow uridylylation and deuridylation. Biochemistry, 1998 Oct 13, 37(41), 14484 - 90 Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli; Catanzano F et al.; Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements . The process proved reversible when the protein concentration was lower than 0.01 mg mL-1 . Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1 . Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species . These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits . A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5 . This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins. Biochemistry, 1998 Oct 13, 37(41), 14477 - 83 SecB binds only to a late native-like intermediate in the folding pathway of barstar and not to the unfolded state; Panse VG et al.; SecB is a cytosolic, tetrameric chaperone of Escherichia coli which maintains precursor proteins in a translocation competent state . We have investigated the effect of SecB on the refolding kinetics of the small protein barstar in 1 M guanidine hydrochloride at pH 7.0 and 25 degreesC using fluorescence spectroscopy . We show that SecB does not bind either the native or the unfolded states of barstar but binds to a late near-native intermediate along the folding pathway . For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB . SecB does not change the apparent rate constant of barstar refolding . The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models. Biochemistry, 1998 Oct 13, 37(41), 14369 - 75 Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate catabolic pathway in Escherichia coli; Hubbard BK et al.; The genes encoding the enzymes in the (D)-glucarate/galactarate catabolic pathway have been identified in the Escherichia coli genome . These encode, in three transcriptional units, (D)-glucarate dehydratase (GlucD), galactarate dehydratase, 5-keto-4-deoxy-(D)-glucarate aldolase, tartronate semialdehyde reductase, a glycerate kinase that generates 2-phosphoglycerate as product, and two hexaric acid transporters . We also have identified a gene proximal to that encoding GlucD that encodes a protein that is 72% identical in primary sequence to GlucD (GlucD-related protein or GlucDRP) . However, whereas GlucD catalyzes the efficient dehydration of both (D)-glucarate and (L)-idarate as well as their epimerization, GlucDRP is significantly impaired in both reactions . Perhaps GlucDRP is an example of gene duplication and evolution in progress in the E . coli chromosome. Nat Genet, 1998 Oct, 20(2), 123 - 8 A new logic for DNA engineering using recombination in Escherichia coli; Zhang Y et al.; A straightforward way to engineer DNA in E . coli using homologous recombination is described . The homologous recombination reaction uses RecE and RecT and is transferable between E . coli strains . Several target molecules were manipulated, including high copy plasmids, a large episome and the E . coli chromosome . Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA. J Nutr, 1998 Oct, 128(10), 1760 - 6 Lipopolysaccharide-induced reductions in food intake do not decrease the efficiency of lysine and threonine utilization for protein accretion in chickens; Webel DM et al.; Exposure of animals to infectious agents induces immune responses that result in reductions in food consumption and weight gain . The effect of these changes on amino acid requirements and utilization remains unclear . Three assays were conducted with young chicks with Escherichia coli lipopolysaccharide (LPS) used to stimulate the immune system . An initial study was conducted to evaluate the effects of LPS on animal performance . In a daily or alternate day injection regimen for 9 d, chicks were given intraperitoneal injections of sterile saline containing 0, 100 or 400 microgram LPS . Administration of 100 or 400 microgram LPS daily, or every other day, decreased both weight gain and food consumption . In two subsequent growth assays, chicks were fed graded levels of lysine or threonine and injected with either 0 or 400 microgram LPS every other day to evaluate the effect of LPS administration on the efficiency of amino acid utilization . At the three lowest amino acid doses, whole-body protein accretion was a linear function of supplemental lysine or threonine intake, and slopes of the accretion curves were not altered by LPS administration . The dietary lysine concentration required to maximize protein accretion was unaffected by LPS, but the absolute lysine intake required to maximize chick performance was lower in LPS-injected chicks than in saline-injected chicks . These results show that LPS administration reduces weight gain, food intake, efficiency of food utilization and the absolute quantity of lysine required to maximize these criteria . However, LPS administration does not affect the efficiency of amino acid utilization, nor does it affect the concentration of dietary lysine required to maximize performance. J Nutr, 1998 Oct, 128(10), 1657 - 60 Pretreatment of young pigs with vitamin E attenuates the elevation in plasma interleukin-6 and cortisol caused by a challenge dose of lipopolysaccharide; Webel DM et al.; The effect of a short-term, high-dose intramuscular injection of d-alpha-tocopherol was studied in pigs given a challenge dose of lipopolysaccharide (LPS) . Twenty-four pigs surgically fitted with jugular catheters were used in a 2 x 2 factorial design . Pigs received either 0 or 600 mg d-alpha-tocopherol by intramuscular injection for 3 d before receiving an intraperitoneal injection of saline containing either 0 or 5 microgram/kg body weight Escherichia coli LPS . Blood was collected from indwelling jugular catheters at 0, 1, 2, 4, 6, 8, 12 and 24 h after injection of LPS . Plasma alpha-tocopherol levels were 13-fold greater (P < 0.01) at time 0 in pigs pretreated with 600 mg d-alpha-tocopherol (9.9 +/- 1.3 mg/L) than in those not treated with d-alpha-tocopherol (0.74 +/- 0.09 mg/L) . Injection of LPS increased (P < 0.05) plasma levels of interleukin-6 (IL-6) and cortisol at 2-h postinjection, regardless of vitamin E treatment . However, pigs that received alpha-tocopherol before the LPS challenge had substantially lower (P < 0.05) peak levels of IL-6 and cortisol than pigs not receiving alpha-tocopherol . These results suggest that supplementation with a surfeit level of vitamin E reduces the response of pigs to endotoxin. Cancer Chemother Pharmacol, 1998, 42(5), 357 - 62 Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells; Parsels LA et al.; PURPOSE: We have reported previously that the expression of E . coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity . In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors . In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity . METHODS: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones . HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity . RESULTS: Following a 24-h treatment with 100 microM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells . DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h . However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line . In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity . CONCLUSIONS: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line . The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12462 - 7 DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription; Majdalani N et al.; DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress sigma factor, RpoS . These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation . The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function . The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation . Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation . We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12456 - 61 Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci; Lease RA et al.; DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (sigmas) . We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the hns locus to antagonize H-NS translation . We also give evidence that supports a role for RNA:RNA interactions at the rpoS locus to enhance RpoS translation . Negative regulation of hns by DsrA is achieved by the RNA:RNA interaction blocking translation of hns RNA . In contrast, results suggest that positive regulation of rpoS by DsrA occurs by formation of an RNA structure that activates a cis-acting translational operator . Sequences within DsrA complementary to three additional genes, argR, ilvIH, and rbsD, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12158 - 62 Polyadenylation of stable RNA precursors in vivo; Li Z et al.; Polyadenylation at the 3' terminus has long been considered a specific feature of mRNA and a few other unstable RNA species . Here we show that stable RNAs in Escherichia coli can be polyadenylated as well . RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length . The polyadenylation process depends on the presence of poly(A) polymerase I . A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs . These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12141 - 6 Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments; Pelletier JN et al.; Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity . We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme . We show that active murine dihydrofolate reductase (E.C . 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners . Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro . The effects of mutations that disrupt fragment affinity or enzyme activity were assessed . The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant . The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization . Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2. Virology, 1998 Oct 10, 250(1), 220 - 9 Construction and characterization of E3-deleted bovine adenovirus type 3 expressing full-length and truncated form of bovine herpesvirus type 1 glycoprotein gD; Zakhartchouk AN et al.; Using the homologous recombination machinery of E . coli, a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid . Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3 . E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replication in vitro . Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3 . E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1 . Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells . Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses . These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals . FEMS Microbiol Lett, 1998 Sep 15, 166(2), 369 - 75 The CcmE protein from Escherichia coli is a haem-binding protein; Reid E et al.; We previously reported that a 17.5-kDa haem-binding polypeptide accumulates in Escherichia coli K-12 mutants defective in an essential gene for cytochrome c assembly, ccmF, and speculated that this polypeptide is either CcmE or CcmG . The haem-containing polypeptide, which is associated with the cytoplasmic membrane, has now been identified by N-terminal sequencing to be CcmE . The haem-dependent peroxidase activity of CcmE is clearly visible not only in a ccmF mutant, but also in ccmG and ccmH mutants, implying that CcmE functions either before or in the same step as CcmF, CcmG and CcmH in cytochrome c maturation . A trxA mutant, like the dipZ mutant, was unable to assemble c-type cytochromes or catalyse formate-dependent nitrite reduction: both activities were restored in the trxA and dipZ, but not ccmG, mutants by the reducing agent, 2-mercaptoethanesulphonic acid . Our data suggest that haem transferred across the cytoplasmic membrane by the CcmABCD complex becomes associated with CcmE, possibly by a labile covalent bond, before it is transferred to the cytochrome c apoproteins by the periplasmic haem lyase encoded by ccmF and ccmH . We further propose that CcmG is essential to reduce the disulphide bonds formed in cytochrome c apoproteins by DsbA, before haem is attached by the haem lyase . Electrons for disulphide bond reduction are supplied from thioredoxin in the cytoplasm via DipZ in the membrane, but can be replaced by the chemical reductant, 2-mercaptoethanesulphonic acid . According to this model, CcmG is the last protein in the reducing pathway which interacts stereospecifically with the apoprotein. FEMS Microbiol Lett, 1998 Sep 15, 166(2), 333 - 9 Control of metabolic interconversion of isocitrate dehydrogenase between the catalytically active and inactive forms in Escherichia coli; el-Mansi EM; The enzymic interconversion of Escherichia coli isocitrate dehydrogenase (ICDH) between the catalytically active and inactive forms is mediated through the activities of ICDH-kinase/phosphatase in response to changes in the metabolic environment . In this study, the use of mutant strains devoid of isocitrate lyase (aceA:: Tn10) and pyruvate dehydrogenase activities revealed that the signal which triggers the reversible inactivation of ICDH in vivo is not directly related to acetate itself, but rather to the need to maintain high intracellular levels of isocitrate and free co-enzyme A . The use of these mutants also revealed, rather unexpectedly, that acetate grown cells contain more ICDH protein than those grown with other carbon sources and that the catalytic activity of ICDH kinase/phosphatase is in excess of cellular demands . Furthermore, this study also revealed the presence of a 50-kDa (+/- 2 kDa) acetate-specific polypeptide, the identity of which has yet to be established. FEMS Microbiol Lett, 1998 Sep 15, 166(2), 257 - 65 Salicylate inhibits fimbriae mediated HEp-2 cell adherence of and haemagglutination by enteroaggregative Escherichia coli; Kang G et al.; Enteroaggregative Escherichia coli (EAggEC) are associated with both acute and persistent diarrhoea in children . Bowel colonisation due to fimbrial adherence factors appears to play a major role in the disease process . In this study, we investigated the effect of sodium salicylate and 5-aminosalicylic acid on adherence of a type strain and 40 clinical isolates of EAggEC to HEp-2 cells and erythrocytes from different species . Growth in the presence of 10 mM salicylate resulted in markedly decreased adherence to tissue culture cells with 33/40 (82.5%) isolates, and was also associated with inhibition of haemagglutination in 20/33 (60.6%) isolates . Complete or partial inhibition of adherence was also seen in two of five isolates showing localised adherence and three of five isolates with diffuse adherence . Decrease in adherence was associated with decreased or absent expression of fimbriae in 28/40 (70%) of the EAggEC isolates, although production of outer membrane proteins was not affected . Salicylates appear to inhibit adherence mediated by fimbrial adhesins. FEMS Microbiol Lett, 1998 Sep 15, 166(2), 219 - 23 Response of the NAD(P)H-oxidising flavohaemoglobin (Hmp) to prolonged oxidative stress and implications for its physiological role in Escherichia coli; Anjum MF et al.; The Escherichia coli flavohaemoglobin (Hmp) has a globin-like N-terminal domain and a ferredoxin-NADP-reductase-like C-terminal domain . We show here that purified Hmp oxidises both NADH and NADPH with Km values of 1.8 and 19.6 microM, respectively . Prolonged incubation of a hmp-lacZ fusion strain with the redox cycling agent paraquat resulted in a 28-fold induction of hmp gene expression, nearly 3-fold higher than after short periods of exposure . A strain overproducing Hmp was significantly more sensitive to paraquat than was the wild-type strain but, in vitro, purified Hmp was not an effective NADPH-paraquat diaphorase . Prolonged incubation of a wild-type strain with paraquat increased intracellular Hmp to spectrally detectable levels. J Clin Invest, 1998 Oct 1, 102(7), 1421 - 30 Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene; Factor P et al.; Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na+ transport and lung edema clearance . We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs . To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat alpha1 and beta1 Na,K-ATPase subunits (adMRCMValpha1 and adMRCMVbeta1, respectively) . As compared to controls, adMRCMVbeta1 increased beta1 subunit expression and Na,K-ATPase function by 2 . 5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers . No change in Na,K-ATPase function was noted after infection with adMRCMValpha1 . Rat lungs infected with adMRCMVbeta1, but not adMRCMValpha1, had increased beta1 protein levels and lung liquid clearance 7 d after tracheal instillation . Alveolar epithelial permeability to Na+ and mannitol was mildly increased in animals infected with adMRCMVbeta1 and a similar Escherichia coli lacZ-expressing virus . Our data shows, for the first time, that transfer of the beta1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs . Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema. J Clin Invest, 1998 Oct 1, 102(7), 1360 - 8 Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption; Choi SJ et al.; Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy . However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited . Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity . A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined . This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide . Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein . Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures . In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types . These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption. Am J Respir Crit Care Med, 1998 Oct, 158(4), 1109 - 13 L-2-Oxothiazolidine-4-carboxylic acid prevents endotoxin-induced cardiac dysfunction; Poon BY et al.; We tested the hypothesis that treatment with the glutathione repleting agent, L-2-oxothiazolidine-4-carboxylic acid (OTZ), could prevent endotoxin-induced ventricular dysfunction . Rabbits were treated with OTZ 2.4 g/kg (10% solution subcutaneously), or an equal volume and osmolality of saline, 24 h prior to, and again (intravenously) just prior to, infusion of 1 mg/kg E . coli endotoxin (or vehicle control) . Ventricular contractility was measured in isolated hearts perfused by support rabbits . Contractility did not change in control groups (Saline/Control {n = 7} or OTZ/Control {n = 7}) over 6 h . However, Emax decreased in the Saline/Endotoxin group (-16.1 +/- 4.5% from baseline, n = 7, p < 0.05) and this was prevented by pretreatment with OTZ in the OTZ/ Endotoxin group (+6.3 +/- 4.1%, n = 7, p < 0.05 by analysis of variance) . To better understand the mechanism of this effect we measured myocardial glutathione concentration and found it to be greater in OTZ/Endotoxin animals (104 +/- 4 ng/g) than in the Saline/Endotoxin animals (80 +/- 3 ng/g, p < 0.05) . OTZ did not appreciably alter the endotoxin-induced increase in serum concentration of tumor necrosis factor (TNF) or the endotoxin-induced increase in myocardial leukocyte content . We conclude that oxygen radicals contribute to the early decrease in left ventricular contractility after endotoxin infusion and this decrease may be prevented by OTZ. Exp Parasitol, 1998 Oct, 90(2), 175 - 80 Trichomonas vaginalis: expression and characterisation of recombinant S-adenosylhomocysteinase; Minotto L et al.; The gene encoding S-adenosylhomocysteinase activity (S-adenosylhomocysteine hydrolase, SAHH; EC 3.3.1.1) in Trichomonas vaginalis has been expressed in Escherichia coli to facilitate the characterisation of the enzyme . Expression of this gene using the pQE-30 (6xHis N-terminal tag) expression system (QIAGEN) has enabled the one-step purification of 6 mg of active recombinant enzyme from a 100-ml bacterial culture by affinity chromatography using a nickel-NTA matrix . The recombinant enzyme has a molecular weight of approximately 56,000 and identification of tryptic peptides by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry has shown that the purified recombinant protein is identical in primary structure to the predicted sequence . The presence of the N-terminal 6xHis tag in the recombinant enzyme did not appear to affect its kinetic and other properties, which are similar to those exhibited by the "native" enzyme present in cell-free extracts of T . vaginalis . These properties include a similar apparent Km for adenosine (20-25 microM for the recombinant and 5-10 microM for the native enzymes, respectively) and similar inhibition/inactivation patterns exhibited by adenosine analogues such as arabinosyl adenine (ara-A) . J Mol Biol, 1998 Oct 23, 283(2), 409 - 17 Involvement of two novel chaperones in the assembly of mitochondrial NADH:Ubiquinone oxidoreductase (complex I); Kuffner R et al.; The respiratory complex I of mitochondria consists of some 40 different subunits which form an L-shaped structure . Perpendicular to a hydrophobic arm embedded in the inner mitochondrial membrane a peripheral arm protrudes into the matrix . Assembly of the complex as studied in the fungus Neurospora crassa involves the formation of discrete intermediates . The matrix arm and the membrane arm are formed independently of each other and are joined in the course of assembly . The membrane arm itself is formed by association of two assembly intermediates, a smaller of 200 kDa and a larger of 350 kDa . The latter is associated with two extra proteins of 84 and 30 kDa which are not constituent parts of mature complex I . Their primary structures show no similarity to known proteins . Mutants generated by disrupting the genes of either of the two proteins accumulate the matrix arm of complex I and the small membrane arm assembly intermediate, but are incapable of forming the large intermediate . In the wild-type, the extra proteins exclusively associate with the large membrane arm assembly intermediate . Pulse-chase labelling experiments showed that the two proteins are repeatedly involved in many assembly cycles of the intermediate . These results indicate that the two proteins are novel chaperones specific for complex I membrane arm assembly . J Mol Biol, 1998 Oct 23, 283(2), 395 - 407 Reproducing the natural evolution of protein structural features with the selectively infective phage (SIP) technology . The kink in the first strand of antibody kappa domains; Spada S et al.; The beta-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer beta-sheet (away from the VH-VL interface) and the second part to the inner beta-sheet . This kink differs in length and sequence between the Vkappa, Vlambda and VH domains and yet is involved in several almost perfectly conserved interactions with framework residues . We have used the selectively infective phage (SIP) system to select the optimal kink region from several defined libraries, using an anti-hemagglutinin single-chain Fv (scFv) fragment as a model system . Both for the kink with the Vkappa domain length and that with the Vlambda length, a sequence distribution was selected that coincides remarkably well with the sequence distribution of natural antibodies . The selected scFv fragments were purified and characterized, and thermodynamic stability was found to be the prime factor responsible for selection . These data show that the SIP technology can be used for optimizing protein structural features by evolutionary approaches . J Mol Biol, 1998 Oct 23, 283(2), 331 - 8 Conformational flexibility in a highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin and antibody binding; Feliu JX et al.; The G-H loop of foot-and-mouth disease virus VP1 protein is a highly mobile peptide, that extends from the capsid surface and that in native virions is invisible by X-ray crystallography . In serotype C, this segment contains a hypervariable region with several continuous, overlapping, B-cell epitopes that embrace the conserved Arg-Gly-Asp (RGD) cell attachment motif . The solvent-exposed positioning of this peptide by selective insertion into different structural frameworks of E . coli beta-galactosidase, generates a spectrum of antigenic variants which react distinctively with a panel of anti-VP1 monoclonal antibodies and exhibit different efficiencies as cell ligands . The cell attachment efficiency is much less restricted by the different positioning of the viral segment at the insertion sites . A molecular model of an inserted stretch reveals a highest flexibility of the RGD tripeptide segment compared with the flanking sequences, that could allow a proper accommodation to integrin receptors even in poorly antigenic conformations . The non-converging structural requirements for RGD-mediated integrin binding and antibody recognition, explains the dynamism of the generation of neutralisation-resistant antigenic variants in the viral quasi-species, arising from a conformational space of integrin-binding competent peptides . This might be of special relevance for foot-and-moth disease virus evolution, since unlike in other picornaviruses, the cell binding motif and the major neutralising B-cell epitopes overlap in a solvent-exposed peptide accessible to the host immune system, in a virion lacking canyons and similar hiding structures . Int J Cancer, 1998 Oct 29, 78(3), 346 - 52 DNA interaction and cytostatic activity of the new liver organotropic complex of cisplatin with glycocholic acid: Bamet-R2; Marin JJ et al.; The aim of this study was to investigate the ability of the new liver organotropic complex of cisplatin with glycocholate (GC), Bamet-R2, to interact with DNA, inhibit its replication and hence reduce tumor-cell proliferation . Changes in the electrophoretic mobility of the open and covalently closed circular forms of the pUC18 plasmid DNA from Escherichia coli, a shift in the denaturation temperature of double-stranded DNA, and ethidium-bromide displacement from DNA binding, were induced by Bamet-R2 and cisplatin, but not by GC . Neutral-red retention was used to measure the number of living cells in culture after long-term (72-hr) exposure to these compounds and to evaluate the effect on cell viability after short-term (6-hr) exposure . Bamet-R2 and cisplatin, but not GC, induced significant inhibition of cell growth . This effect ranged from mild to strong, depending upon the sensitivity of the different cell types as follows: cisplatin, rat hepatocytes in primary culture < rat hepatoma McA-RH7777 cells (rH) < human colon carcinoma LS 174T cells (hCC) < mouse hepatoma Hepa 1-6 cells (mH); Bamet-R2, rat hepatocytes < mH approximately equal to hCC < rH . DNA synthesis was measured by radiolabeled-thymidine incorporation into DNA . Bamet-R2 and cisplatin, but not GC, significantly inhibited the rate of DNA synthesis by these cells . After short-term exposure to Bamet-R2 or GC, no acute cell toxicity was observed, except on hCC cells . By contrast, acute toxicity was induced by cisplatin for all cell types studied . The in vivo anti-tumoral effect was investigated in 3 different strains of mice following s.c . implantation of tumor cells (mouse sarcoma S-18011 cells in Swiss and B6 mice and hCC cells in nude mice) . In all 3 models, tumor growth was inhibited by Bamet-R2 and cisplatin to a similar degree . However, signs of toxicity (increases in blood urea concentrations and decreases in packed blood cell volume and in liver, kidney and body weight) and a reduction in survival rate were observed only during cisplatin administration . In sum, these results indicate that this bile-acid derivative can be considered as a cytostatic drug whose potential usefulness deserves further investigation. Plant Physiol, 1998 Oct, 118(2), 471 - 82 Manipulation of glutathione and amino acid biosynthesis in the chloroplast Noctor G, Arisi AC, Jouanin L, Foyer CH. Poplars (Populus tremula x Populus alba) were transformed to overexpress Escherichia coli gamma-glutamylcysteine synthetase (gamma-ECS) or glutathione synthetase in the chloroplast . Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product . Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity . Enhanced chloroplastic gamma-ECS activity markedly increased gamma-glutamylcysteine and GSH levels . These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol . Similar to cytosolic gamma-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes . Light was required for maximal accumulation of GSH in poplars overexpressing gamma-ECS in the chloroplast . High chloroplastic, but not cytosolic, gamma-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast . We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased gamma-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast. Curr Microbiol, 1998 Nov, 37(5), 356 - 8 Buchnera aphidicola (Aphid endosymbiont) contains genes encoding enzymes of histidine biosynthesis; Clark MA et al.; Buchnera aphidicola is an endosymbiont of aphids . One of its functions appears to be the synthesis of essential amino acids for the aphid host . A 12.8-kilobase B . aphidicola DNA fragment has been cloned and sequenced . It contains genes encoding all of the enzymes required for the biosynthesis of the essential amino acid histidine . The order of the genes, hisGDCBHAFI, is the same as that found in Escherichia coli and is consistent with their constituting a single transcription unit . The DNA fragment also contained genes involved in aromatic amino acid biosynthesis (aroC), the oxidative pentose pathway (gnd), and 2'-deoxyribonucleotide metabolism (dcd), as well as a tRNA synthase (metG). Curr Microbiol, 1998 Nov, 37(5), 341 - 6 A genomically modified marker strain of Escherichia coli; Ammons D et al.; Contamination of the environment with human sewage represents a serious public health concern in which Escherichia coli plays a central role, either directly as a human pathogen or indirectly through its use as an indicator organism . There is thus an ongoing effort to better understand the behavior of E . coli within such environments . Useful to such studies is the ability to readily detect a specific E . coli population and distinguish it from similar indigenous bacteria . Herein, we report the construction of an E . coli strain (PCPHR) that expresses a Stable Artificial RNA (SAR) from the chromosomal rrnH operon . The SAR product is present in large numbers of copies/cell and thus provides an enhanced detection signal without significant effect on the wild-type growth rate . Detection can be accomplished by any of several routine molecular methods . Preliminary studies suggest SAR expression levels correlate positively with growth . PCPHR is immediately available for use as a marker strain for E . coli in application in the arena of public health or environmental studies. J Mol Evol, 1998 Oct, 47(4), 385 - 93 Biased usages of arginines and lysines in proteins are correlated with local-scale fluctuations of the G + C content of DNA sequences; Nishizawa M et al.; Amino acid residues arginine (R) and lysine (K) have similar physicochemical characteristics and are often mutually substituted during evolution without affecting protein function . Statistical examinations on human proteins show that more R than K residues are used in the proximity of R residues, whereas more K than R are used near K residues . This biased use occurs on both a global and a local scale (shorter than approximately 100 residues) . Even within a given exon, G + C-rich and A + T-rich short DNA segments preferentially encode R and K, respectively . The biased use of R and K on a local scale is also seen in Saccharomyces cerevisiae and Caenorhabdidtis elegans, which lack global-scale mosaic structures with varying GC%, or isochores . Besides R and K, several amino acids are also used with a positive or negative correlation with the local GC% of third codon bases . The local-, or "within-gene"-, scale heterogeneity of the DNA sequence may influence the sequence of the encoded protein segment. Parasite Immunol, 1998 Aug, 20(8), 377 - 85 Immunological properties of recombinant proteins of the transmission blocking vaccine candidate, Pfs48/45, of the human malaria parasite Plasmodium falciparum produced in Escherichia coli; Milek RL et al.; A precondition for the development of a transmission blocking vaccine based on the sexual stage-specific surface antigen Pfs48/45 of Plasmodium falciparum is its heterologous synthesis in a native state . Here we describe the production of recombinant Pfs48/45 in Escherichia coli . Two recombinant proteins, of which one is a glutathione-S-transferase fusion protein, were produced . Enzyme-linked immunosorbent assays showed that at least a subfraction of the recombinant proteins had a conformation capable of binding transmission blocking monoclonal antibodies . However, despite the fact that both proteins were very immunogenic, they did not induce transmission blocking immunity in mice or rabbits . Immunological studies with congenic mouse strains demonstrated that immune responses could be boosted with gametocyte extracts and were not restricted to a particular class II major histocompatibility complex haplotype. Parasite Immunol, 1998 Aug, 20(8), 351 - 7 Identification, characterization and expression of Toxocara canis nematode polyprotein allergen TBA-1; Yahiro S et al.; We have cloned the cDNA of TBA-1, the Nematode polyprotein allergen (NPA) of Toxocara canis and found it to be most similar to ABA-1, the Ascaris NPA, on the basis of amino acid sequence . We could study the antigenic properties of an E-coli synthesized fusion protein prepared with the cloned gene since no glycosylation site was expected from the deduced amino acid sequence . Although no IgE responses to TBA-1 were detected, recombinant TBA-1 was differently recognized by serum IgG antibodies when the recombinant TBA-1 was directly adsorbed vs when immobilized via a streptavidin linkage on polystyrene microtitre wells . One group of sera recognized TBA-1 directly immobilized while the second only recognized TBA-1 immobilized via streptavidin linkage . The former were from rodents immunized with a Toxocara sp . adult worm extract while the latter were obtained from rodents infected with T . canis larva or immunized with a Anisakis simplex L3 larval extract . These observations suggest that the two in vivo forms of TBA-1 are expressed, but during different stages of the parasite's life cycle. Mol Microbiol, 1998 Sep, 29(5), 1297 - 306 Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli; Hedlund M et al.; Uropathogenic Escherichia coli attach to epithelial cells through P fimbriae that bind Galalpha1-4Galbeta-oligosaccharide sequences in cell surface glycosphingolipids . The binding of P-fimbriated E . coli to uroepithelial cells causes the release of ceramide, activation of the ceramide signalling pathway and a cytokine response in the epithelial cells . The present study examined the molecular source of ceramide in human kidney A498 cells exposed to P-fimbriated E . coli . Agonists such as TNF-alpha and IL-1beta released ceramide from sphingomyelin by the activation of endogenous sphingomyelinases and hydrolysis of sphingomyelin, and triggered an IL-6 response . P-fimbriated E . coli caused a slight increase in endogenous sphingomyelinase activity, but there was no associated sphingomyelin hydrolysis . Instead, the concentration of galactose-containing glycolipids decreased . We propose that P-fimbriated E . coli differ from other activators of the ceramide pathway, in that release of ceramide is from receptor glycolipids and not from sphingomyelin . Receptor breakdown may be an efficient host defence strategy, as it reduces the concentration of cell surface receptors, releases soluble receptor analogues and activates an inflammatory response. Mol Microbiol, 1998 Sep, 29(5), 1225 - 36 Crl stimulates RpoS activity during stationary phase; Pratt LA et al.; RpoS, an alternative primary sigma factor, has been shown to be regulated at multiple levels, including transcription, translation and protein stability . Here, we present evidence that suggests that RpoS is regulated at yet another level by the product of the crl gene . The crl gene was first thought to encode the major curlin subunit of curli (curli are surface structures that are induced by growth into stationary phase under conditions of low osmolarity and low temperature) . Later, it was determined that crl actually contributes in a positive fashion to stimulate transcription of csgBA, the true locus encoding for the major subunit of curli . RpoS is also required for normal stationary-phase induction of csgBA . We found that lesions in crl, like lesions in rpoS, cause increased transcription of ompF during stationary phase . Taken together, these observations prompted us to analyse the effects of crl on an additional RpoS-regulated phenomenon . We found that a crl null allele influences expression of RpoS-regulated genes in a fashion similar to an rpoS null allele . Genetic evidence suggests that crl and rpoS function in a single pathway and that Crl functions upstream, or in concert with, RpoS . Although the effects of Crl on RpoS-regulated genes is entirely dependent on the integrity of RpoS, the presence of a crl null allele does not decrease the level of RpoS protein . Thus, we propose that Crl stimulates the activity of the RpoS regulon by stimulating RpoS activity during stationary phase. Mol Microbiol, 1998 Sep, 29(5), 1179 - 90 Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA; Fekkes P et al.; In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB . SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins . The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation . Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA . The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (delta8proOmpA) bearing a non-functional signal sequence . Delta8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prIA4 strain . SecB reduces the translocation of delta8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed . These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA . It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase. Mol Microbiol, 1998 Sep, 29(5), 1147 - 54 A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli; Gardina PJ et al.; The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity . Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP) . To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer . Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes . The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (delta tar) . However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled delta tar cells to respond to aspartate or maltose . The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP . When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other . In the 'opposite' orientation, such inhibitory effects were negligible . These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation . When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit . In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar+ cells. Mol Microbiol, 1998 Sep, 29(5), 1137 - 45 Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability; Summers D; Multicopy plasmids of Escherichia coli are distributed randomly at cell division and, as long as copy number remains high, plasmid-free cells arise only rarely . Copy number variation is minimized by plasmid-encoded control circuits, and the limited data available suggest that deviations are corrected efficiently under most circumstances . However, plasmid multimers confuse control circuits, leading to copy number depression . To make matters worse, multimers out-replicate monomers and accumulate clonally within the culture, creating a subpopulation of cells with a significantly increased rate of plasmid loss . Multimers of natural multicopy plasmids, such as ColE1, are resolved to monomers by a site-specific recombination system (Xer-cer) whose activity is limited to intramolecular recombination . Recombination requires the heterodimeric XerCD recombinase plus two accessory proteins (ArgR and PepA), which activate recombination and prevent intermolecular events . Evidence is accumulating that Xer-cer recombination is relatively slow, and there is a risk that cells might divide before multimer resolution is complete . The Rcd transcript encoded within cer may solve this problem by preventing the division of multimer-containing cells . Working in concert, the triumvirate of copy number control, multimer resolution and cell division control achieve an extremely high fidelity of plasmid maintenance. Mol Microbiol, 1998 Aug, 29(4), 1113 - 23 Downregulation of Escherichia coli yfiD expression by FNR occupying a site at -93.5 involves the AR1-containing face of FNR; Green J et al.; The promoter of the FNR-activated yfiD gene of Escherichia coli has an unusual architecture because it contains two FNR sites, an arrangement usually associated with FNR-mediated repression . Investigation of yfiD promoter derivatives with altered FNR sites revealed that occupation of the far upstream FNR site (FNR II) downregulated expression, despite the presence of a FNR dimer activating expression from the promoter proximal site (FNR I) . Transcript mapping by primer extension, and mutagenesis of potential -10 elements, indicated that yfiD expression is driven from a single FNR-dependent promoter with FNR sites at -40.5 (FNR I) and -93.5 (FNR II) . However, yfiD mRNA is processed in stationary-phase cultures independently of rne, rpoS, ihfA and fis to yield transcripts lacking 12 and 21 bases from their respective 5' ends . Single amino acid substitutions (G74-->C, F92-->S, A95-->P, R184-->P, P188-->A or L193-->P) in the surface of FNR that contains activating region 1 (AR1 contacts the alpha-subunit of RNA polymerase to promote transcription activation) reduced the inhibitory effect of FNR at FNR II, indicating that this region of the protein may have a role in repression as well as activation . The FNR variant F92-->S was notable because, although it activated transcription of yfiD (two FNR sites), it was unable to activate transcription from model Class I and II promoters, which contain only a single FNR site. Mol Microbiol, 1998 Aug, 29(4), 1101 - 12 A novel mechanism for upregulation of the Escherichia coli K-12 hmp (flavohaemoglobin) gene by the 'NO releaser', S-nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA-hmp intergenic region; Membrillo-Hernandez J et al.; The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS-independent manner . We now show that hmp expression is also upregulated by S-nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor) . Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression . Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression . Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy . We propose that the previously documented modulation by Hcy of MetR binding to the glyA-hmp intergenic regulatory region regulates hmp transcription . Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP . GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S-nitrosohomocysteine, although in the latter case this would be co-ordinated to the Fe in SNP as a stable species . The free S-nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO . As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation. Mol Microbiol, 1998 Aug, 29(4), 1077 - 90 The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation; Sacerdot C et al.; The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translational level . ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine-Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3) . Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region -12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single-stranded sequence in domain 3 . These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2) . This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded region of domain 3 is present . A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation . As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance. Mol Microbiol, 1998 Aug, 29(4), 1053 - 63 Expression of ptsG, the gene for the major glucose PTS transporter in Escherichia coli, is repressed by Mlc and induced by growth on glucose; Plumbridge J; The gene for the glucose-specific transporter of the phosphotransferase system, ptsG, is expressed from two promoters separated by 141 bp . The expression of the major, shorter transcript is very strongly dependent upon cAMP/CAP . However, unlike other CAP-activated genes, the expression of ptsG is higher in glucose media than in glycerol, implying that ptsG is controlled by a glucose-inducible regulator . A mutation in the mlc gene greatly enhances ptsG expression in a glycerol-grown culture but has no effect on ptsG expression during growth on glucose . The mlc gene encodes a transcriptional regulator that has been shown to affect the expression of manXYZ and malT . ptsG mRNA levels are lower in the mlc strain grown on glucose than in the same strain grown on glycerol . This is presumably because of the greater catabolite repression in the glucose culture than in glycerol . The final level of expression of ptsG in a mlc+ strain in glucose is a compromise between specific induction by glucose and generalized catabolite repression . The result is that ptsG expression is very similar in glucose-grown cultures of wild-type and mlc strains . The Mlc protein binds to two sites centred at -6 and -175 upstream of the major ptsG transcript . CAP binds at -40.5 compared with this site, typical of class II CAP-regulated promoters, and the binding of CAP and Mlc is co-operative. Mol Microbiol, 1998 Aug, 29(4), 1039 - 51 Negative regulation by RpoS: a case of sigma factor competition; Farewell A et al.; A mutation in the Escherichia coli gene encoding the stationary phase-inducible sigma factor (sigmaS, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase-induced genes . We have examined this phenomenon of repression by sigmaS using as a model system the divergently transcribed stationary phase-inducible genes, uspA and uspB . uspA is transcribed by sigma70-programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is sigmaS dependent . The data suggest that the superinduction of uspA is caused by an increased amount of sigma70 bound to RNA polymerase in the absence of the competing sigmaS . Increasing the ability of sigma70 to compete against sigmaS by overproducing sigma70 mimics the effect of an rpoS mutation by causing superinduction of sigma70-dependent stationary phase-inducible genes (uspA and fadD), silencing of sigmaS-dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of sigmaS-dependent phenotypes, such as hydrogen peroxide resistance in stationary phase . In addition, overproduction of sigmaS markedly reduced stationary phase expression of a sigma70-dependent promoter . Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase . The implications of this competition in the passive control of promoter activity is discussed. Mol Microbiol, 1998 Aug, 29(4), 1019 - 27 Probing the active site of mitogillin, a fungal ribotoxin; Kao R et al.; Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and alpha-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond . The site of cleavage occurs between G4325 and A4326, which are present in a 14-base sequence (the alpha-sarcin loop) conserved among the large subunit rRNAs of all living species . The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone . These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae . Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity . Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays . These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin . We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin. Immunology, 1998 Jul, 94(3), 424 - 30 Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat-labile enterotoxin and its B subunit; de Haan L et al.; Escherichia coli (E . coli) heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co-administered antigens . LT is composed of one copy of the A subunit, which has ADP-ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1 . Both the ADP-ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation . We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP-ribosylation activity, GM1-binding affinity, or both . Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated . With respect to the immunogenicity of LT and LTB, we found that GM1-binding activity is essential for effective induction of anti-LTB antibodies . On the other hand, an LT mutant lacking ADP-ribosylation activity retained the immunogenic properties of