Escherichia coli

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 the native toxin, indicating that ADP ribosylation is not critically involved . Whereas adjuvanticity of LTB was found to be directly related to GM1-binding activity, adjuvanticity of LT was found to be independent of GM1-binding affinity . Moreover, a mutant lacking both GM1-binding and ADP-ribosylation activity, also retained adjuvanticity . These results demonstrate that neither ADP-ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit.

Immunology, 1998 Jul, 94(3), 304 - 9
Functional immaturity of rat alveolar macrophages during postnatal development; Bakker JM et al.; Alveolar macrophages (AM) are important in the regulation of immune responses in the lung, through their role as scavenger cells and through the production of many bioactive factors . Because in early infancy pulmonary infections are a recurrent problem, we studied the postnatal functional maturation of AM in a rat model . AM were isolated from rat lungs by bronchoalveolar lavage at several time intervals after birth and tested for their ability to ingest Escherichia coli in the presence of surfactant protein A (SP-A) . Furthermore, their capacity to produce nitric oxide (NO) and interleukin-1 beta (IL-1 beta) after in vitro lipopolysaccharide (LPS) stimulation was analysed, as well as their capacity to downregulate proliferation of T cells from both mature and neonatal rats . SP-A-mediated phagocytosis of E . coli by AM was reduced in 14-day-old neonatal rats, as compared with mature rats (P < or = 0.05) . Also the IL-1 beta production by rat AM after LPS stimulation was impaired at 14 days of age, as compared with IL-1 beta production by AM from mature rats (P < or = 0.05) . In contrast, the LPS-induced NO production by rat AM as well as the capacity to inhibit T-cell proliferation were well developed at all ages tested . In conclusion, during postnatal development the rat AM is functionally immature, with respect to phagocytosis and secretion of inflammatory mediators . These differences may underly the enhanced susceptibility to pulmonary infections as found in human neonates.

Biochim Biophys Acta, 1998 Oct 15, 1375(1-2), 110 - 6
Unmyristoylated MARCKS-related protein (MRP) binds to supported planar phosphatidylcholine membranes; Michielin O et al.; We have recently shown that unmyristoylated MARCKS-related protein (MRP) does not bind to neutral phospholipid vesicles, unless negatively charged phospholipids are present . Similar behaviour has also been reported for MARCKS itself . Here we have compared the binding of MRP to neutral and negatively charged supported planar lipid bilayer membranes (SPLM) using two-mode waveguide spectroscopy . We find appreciable binding of unmyristoylated MRP to neutral SPLM . We propose that hydrophobic residues in the effector domain constitute an additional factor capable of mediating MRP-membrane interaction.

Biochim Biophys Acta, 1998 Oct 15, 1375(1-2), 101 - 9
Characterization of a heat modifiable protein, Escherichia coli outer membrane protein OmpA in binary surfactant system of sodium dodecyl sulfate and octylglucoside; Ohnishi S et al.; A membrane protein, OmpA of Escherichia coli, in the process of refolding from its heat-modified form in the presence of sodium dodecyl sulfate (SDS) to its non-heated one by the addition of systematic amounts of octylglucoside (OG) was characterized by means of dynamic light scattering and the size exclusion chromatography combined with low angle laser light scattering photometry . Upon heating in the presence of SDS only, the amount of SDS bound to OmpA was increased from 1.8 to 2.3 g/g of protein and its hydrodynamic radius increased from 3.7 to 4.7 nm . On the addition of OG, the once denatured OmpA regained its original size above the weight fraction of OG in the total amount of surfactants, 0.8 . During the process, the hydrodynamic radius was observed to decrease cooperatively at the weight fraction of 0.6, while no change took place in the molar mass of the protein . The refractive index increment of OmpA reflecting the amount of surfactant binding also regained the value before the heating in parallel with the change of size . Examination of the amount of surfactants bound to the membrane protein according to known properties of the binary surfactant micellar system of the surfactants showed that SDS was principally responsible for the denaturing phenomena of OmpA.

Biochim Biophys Acta, 1998 Oct 2, 1394(1), 57 - 64
Expression and characterization of human group V phospholipase A2; Chen Y et al.; Group V phospholipase A2 (GV-PLA2) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear . We report the over-expression and characterization of GV-PLA2 . The GV-PLA2 cDNA was synthesized from human heart polyA+ mRNA by RT-PCR, and an expression construct containing the GV-PLA2 was established . After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography . The purified GV-PLA2 protein was folded to form active enzyme . The recombinant GV-PLA2 has an absolute requirement for Ca2+ for enzymatic activity . The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate . GV-PLA2 preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles . However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids . The fatty acid preference of GV-PLA2 is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles . 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA2, strongly inhibits GV-PLA2 with an IC50 value of about 36 nM which is comparable to its inhibition of group IIA PLA2.

Biochim Biophys Acta, 1998 Oct 23, 1442(1), 39 - 48
Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli; Banecki B et al.; DnaA protein of Escherichia coli is a sequence-specific DNA binding protein required for the initiation of DNA replication from the chromosomal origin, oriC, and of several E . coli plasmids . At a moderate ionic strength, purified DnaA protein has a strong tendency to aggregate; the self-aggregate form is inactive in DNA replication . Binding of ATP or ADP to DnaA protein protected it from aggregation to maintain its replication activity . AMP or cyclic AMP had no protective effect . The molecular chaperone DnaK protected DnaA protein from aggregation with or without ATP . DnaJ and GrpE were not stimulatory . Chaperonins GroEL and GroES were also able to prevent aggregation but only in the presence of ATP . The studies presented here show that for DnaA protein to be active in the initiation of DNA replication, it must be prevented from forming a self-aggregate by the binding of adenine nucleotides, and/or by the action of molecular chaperones.

Genetica, 1998, 102-103(1-6), 569 - 80
Genetic measurement of theory of epistatic effects; Wagner GP et al.; Epistasis is defined as the influence of the genotype at one locus on the effect of a mutation at another locus . As such it plays a crucial role in a variety of evolutionary phenomena such as speciation, population bottle necks, and the evolution of genetic architecture (i.e., the evolution of dominance, canalization, and genetic correlations) . In mathematical population genetics, however, epistasis is often represented as a mere noise term in an additive model of gene effects . In this paper it is argued that epistasis needs to be scaled in a way that is more directly related to the mechanisms of evolutionary change . A review of general measurement theory shows that the scaling of a quantitative concept has to reflect the empirical relationships among the objects . To apply these ideas to epistatic mutation effects, it is proposed to scale A x A epistatic effects as the change in the magnitude of the additive effect of a mutation at one locus due to a mutation at a second locus . It is shown that the absolute change in the additive effect at locus A due to a substitution at locus B is always identical to the absolute change in B due to the substitution at A . The absolute A x A epistatic effects of A on B and of B on A are identical, even if the relative effects can be different . The proposed scaling of A x A epistasis leads to particularly simple equations for the decomposition of genotypic variance . The Kacser Burns model of metabolic flux is analyzed for the presence of epistatic effects on flux . It is shown that the non-linearity of the Kacser Burns model is not sufficient to cause A x A epistasis among the genes coding for enzymes . It is concluded that non-linearity of the genotype-phenotype map is not sufficient to cause epistasis . Finally, it is shown that there exist correlations among the additive and epistatic effects among pairs of loci, caused by the inherent symmetries of Mendelian genetic systems . For instance, it is shown that a mutation that has a larger than average additive effect will tend to decrease the additive effect of a second mutation, i.e., it will tend to have a negative (canalizing) interaction with a subsequent gene substitution . This is confirmed in a preliminary analysis of QTL-data for adult body weight in mice.

J Chromatogr B Biomed Sci Appl, 1998 Sep 4, 714(2), 223 - 35
Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis; Colangeli R et al.; Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens . Since purification of protein from M . tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M . tuberculosis from E . coli cells . Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays . The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography . The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E . coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells . The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M . tuberculosis) counterparts . Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.

J Leukoc Biol, 1998 Oct, 64(4), 459 - 66
Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: role of TNF-alpha; McKinney LC et al.; Macrophages are activated to become cytotoxic by a highly coordinated set of cytokine signals . Ionizing radiation can mimic cytokine signals and lead to enhanced states of activation . We tested the ability of gamma-radiation, alone and with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS), to induce nitric oxide (NO) production in J774.1 and RAW264.7 murine macrophages . NO was induced weakly, moderately, or strongly by IFN-gamma alone, LPS alone, or IFN-gamma + LPS, respectively . Radiation alone (0.5-50 Gy) did not induce NO, but enhanced NO production in a dose-dependent manner (0.5-5 Gy) when cells were exposed to IFN-gamma or LPS 24 h post-irradiation . Immunoblots showed parallel induction of nitric oxide synthase (NOS2) . Application of anti-tumor necrosis factor alpha (TNF-alpha) antibody before irradiation blocked induction of NO by IFN-gamma . We conclude (1) that irradiated cells produce more NO in response to either IFN-gamma or LPS and (2) that the increase is mediated by induction of TNF-alpha.

Res Microbiol, 1998 Apr, 149(4), 265 - 75
Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG; Freer G et al.; A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8 . This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents . In the present work, SA-5K was purified from BCG CFs by affinity chromatography . A protein that could be detected in Western blot but not by standard protein staining techniques was obtained . When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M . tuberculosis . The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage . The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions . After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions . The latter MM was close to that estimated from the ORF of M . tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge . The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M . tuberculosis . The gene coding for SA-5K was cloned by PCR from BCG and M . tuberculosis DNA and was expressed in Escherichia coli.

Res Microbiol, 1998 Apr, 149(4), 235 - 45
Homologues of helicase genes priA and ruvAB of Borrelia burgdorferi, the Lyme borreliosis agent; Boursaux-Eude C et al.; A DNA library of strain HB19 from Borrelia burgdorferi sensu stricto, an agent of Lyme borreliosis, was constructed in the cosmid pLA2917 . Genes involved in initiation of DNA replication and resolution of recombination intermediates (Holliday junctions) were found on a 23-kbp region up to 0.7 kbp of the "left" extremity of the linear chromosome in representative species of B . burgdorferi sensu lato . The potential ruvB gene, located at 22 kbp from the left telomere, was identified by the similarity of its deduced amino acid sequence to RuvB (helicases) of other bacteria . B . burgdorferi ruvB is part of an operon which comprises the homologues of ruvA, queA and pfbB . Expression of the B . burgdorferi ruvB and ruvA genes renders a wild-type Escherichia coli sensitive to UV light and mitomycin, indicative of negative complementation . priA, which encodes the potential recognition factor for the primosome assembly site, was found at 15 kbp from the left telomere . RuvB and PriA sequences have motifs characteristic of helicases: a DExH box and an ATP binding site.

Res Microbiol, 1998 Mar, 149(3), 177 - 88
Cloning and sequence determination of a phi AAU2 gene whose product aborts the phage lytic cycle; Le Marrec C et al.; This communication describes the cloning of a 1.8-kb fragment from the genome of the corynephage phi AAU2, which aborts the phage lytic cycle when cloned on a high-copy shuttle vector . The associated phenotype, called Apld (aborting phage lytic development), was revealed by noting the reduced plaque size and lower efficiencies of plaquing of phi AAU2 cp, a virulent derivative of phi AAU2, on "Arthrobacter aureus"-C70 Apld+ cells . Adsorption and phage DNA transfection experiments showed evidence that Apld acted once the phage DNA had entered into the cell; apld was confined to a single open reading frame (ORF), encoding a putative 63-aa polypeptide which did not show any homology to proteins contained in the databanks; apld is followed by an ORF the product of which shows homology with a protein expressed by the early region of the Streptomyces phage phi C31.

Res Microbiol, 1998 Feb, 149(2), 119 - 35
Computer identification of Escherichia coli rRNA gene restriction patterns; Machado J et al.; A total of 191 strains of Escherichia coli comprising 164 serovar reference strains and 28 clinical strains were characterized by rRNA gene restriction patterns (ribotypes) generated after cleavage of total DNA with MluI, ClaI or HindIII restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16 + 23S rRNA . A wide diversity of ribotypes was observed with endonucleases MluI (104 patterns), ClaI (90 patterns) and HindIII (98 patterns) . When MluI was used, 85% of patterns (11 to 15 fragments) shared five fragments 17.09, 3.94, 3.06, 2.23 and 1.76 kb in size . When these fragments were used as internal standards, the percent errors in fragment length determination was half of that obtained with an external standard . Two fragment size databases of MluI and ClaI ribotypes were built . Automatic identification was obtained after setting the percent fragment size variation tolerance (error) at 5% . MluI ribotyping is recommended as a primary epidemiological marker . Strains with similar MluI ribotype should then be submitted to ClaI ribotyping . Ribotyping with HindIII can only be the third choice, since the patterns were often uncertain due to the frequent occurrence of faint bands . Most of the studied serovars gave discrete patterns and these data provide the basis for a molecular typing system for E . coli which could possibly substitute for serotyping when the latter is not available.

Res Microbiol, 1998 Jul-Aug, 149(7), 457 - 72
Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli; Bastian SN et al.; The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes . Systems designed for the detection of genes of the stx1 type did not detect any variant genes of the stx2 type and conversely, no stx2 type-specific systems detected stx1 variant genes . Among five stx2 type-specific systems, none detected the stx2ev gene, and two detected the stx2e gene . Among systems designed for screening genes of the both stx1 and stx2 types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains . Shiga-toxin-producing E . coli frequently carry more than one stx variant gene . Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size . The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx1, stx2, stx2c, stx2e and stx2ev . Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.

Res Microbiol, 1997 Jun, 148(5), 413 - 25
Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization; Girjes AA et al.; We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus) . In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response . Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C . psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain . The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5) . DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C . pneumoniae, C . psittaci 6BC and C . trachomatis, and the kdtA gene of E . coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A . Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis . Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E . coli.

Res Microbiol, 1997 Jun, 148(5), 389 - 95
Activity of protein MalE (maltose-binding protein) fused to cytoplasmic and periplasmic regions of an Escherichia coli inner membrane protein; Dassa E et al.; We analysed the properties of mature MBP (maltose-binding protein or MalE protein) fused to an integral cytoplasmic membrane protein of Escherichia coli . Fusion of MalE to the first MalG periplasmic loop enabled a strain defective in the malE gene to utilize maltose . In contrast, fusion of MalE to a cytoplasmic loop did not complement the malE delta 444 deletion . We obtained results highly correlated with those obtained by using alkaline phosphatase as a reporter for the topology of MalG . We discuss the possibility of genetically determining the topology of cytoplasmic membrane proteins by a method based on engineered fusions to MBP.

Res Microbiol, 1997 Jun, 148(5), 375 - 87
Further genetic analysis of the C-terminal external loop region in Escherichia coli maltoporin; Klebba PE et al.; LamB specifically facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and acts as a general (i.e . non-specific) porin for small hydrophilic molecules (< 600 daltons) . We reported previously that deletion of the last predicted external domain near the C-terminus of the Eschirichia coli LamB protein (residues 376 to 405), affected in vivo the binding and transport of maltodextrins (specific pore functions), and also increased bacterial sensitivity to large antibiotics . The residues covered by this deletion correspond almost exactly to the major cell surface loop of LamB on the structural model based on X-ray crystallography (loop L9, residues 375 to 405) . The L9 loop comprises a large central portion, which varies in size and sequence between the LamB proteins from different species . This variable region is flanked by two highly charged and conserved portions, which overlap with the adjacent beta strands . To identify subregions in L9 that influence the pore properties of LamB, we constructed and analysed nine mutants in loop L9 and its flanking sequences . Deletion of the 23-amino-acids central variable portion of the loop (residues 379 to 401), and deletion of the downstream conserved region (residues 402 to 409), only moderately affected specific maltoporin function . In contrast, deletion of the conserved region (residues 372 to 378) upstream of the variable portion strongly decreased specific maltoporin function and also increased sensitivity to large antibiotics, accounting for most, if not all, of the effects of the complete deletion of L9.

Res Microbiol, 1997 Mar-Apr, 148(3), 251 - 61
Emergence of Cu(++)-tolerant mutants defective in gellan synthesis in Cu(++)-stressed cultures of Sphingomonas paucimobilis; Richau JA et al.; Cells defective in gellan synthesis appeared during cultivation of the gellan gum-producing strain Sphingomonas paucimobilis R40 with inhibitory concentrations of copper, supplied as CuCl2 . The percentage of less mucoid colonial variants dramatically increased with the increase in Cu++ supplementation, reaching 85% of total viable cells at the maximal concentration for growth . Results reported in this work indicate that emergence of colonial variants defective in gellan synthesis results from Cu(++)-induced mutation and the growth advantage of these mutants in Cu(++)-stressed cultures . In fact, DNA homologous recombination strongly increased with the increase in copper supplementation as indicated by the regeneration of kanamycin-resistant cells of R40 harbouring plasmid pBX404-7, which carries two non-overlapping truncated genes derived from a gene conferring kanamycin resistance . The four major groups of colonial mutants that emerged from Cu(++)-stressed cultures of R40 exhibited reduced growth rate and biomass yield in the absence of Cu++ stress and produced decreased levels of exopolysac-charide (EPS) which yielded solutions of lower or negligible viscosity . The level of increased Cu++ tolerance of these mutants, assessed by the inhibitory effect of Cu++ on growth, correlated with the degree of loss of the ability to secrete high-molecular-mass EPS . Consistent with the growth advantage of gellan-defective mutants in Cu(++)-stressed cultures, the non-producing strain RP10, spontaneously obtained during extended cultivation of R40, also exhibited a higher tolerance to Cu++ . In addition, its non-mucoid phenotype was stably maintained during Cu(++)-stressed cultivation despite the stimulation of homologous recombination by Cu++.

Radiats Biol Radioecol, 1998 Jul-Aug, 38(4), 488 - 94
{Proteins induction by cysteamine in Escherichia coli cells}; Suslov AV et al.; It was shown, what in presence of oxygen in broad interval time of treatment in E . coli cells was induced line proteins by cysteamine which are proteins SOS--repair system and heat--shock system . There are quantitative defined induction level RecA, GroEL and DnaK proteins which are members this systems . It was allowed to propose what radioprotective action of cysteamine in higher degree is defined by its characteristics as induction agent for stress systems of cells.

Biochim Biophys Acta, 1998 Aug 20, 1399(2-3), 219 - 24
Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana; Day IS et al.; We cloned and characterized a full-length cDNA that encodes a glutamyl-tRNA synthetase (GluRSAt) from Arabidopsis . The GluRSAt is coded by a single gene . A transcript of about 2.3 kb hybridized with the cDNA . The deduced protein from the cDNA contained 719 amino acids with an estimated molecular mass of 81 kDa . Expression of the GluRSAt in E . coli resulted in a protein of the expected size . Comparison of the amino acid sequence GluRSAt to other glutamyl-tRNA synthetases showed strong sequence similarity to cytoplasmic GluRS proteins.

Biochim Biophys Acta, 1998 Aug 20, 1399(2-3), 173 - 80
Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae; Beer HD et al.; A lipase from Rhizopus oryzae DSM 853 (ROL) was cloned from a chromosomal gene bank, sequenced and overexpressed in Escherichia coli . ROL and its precursors ProROL and PreProROL were purified and their pH and temperature profile was determined . In contrast to ROL, ProROL and PreProROL had considerably higher thermostability and a slightly higher pH optimum . Moreover, it could be demonstrated by in vitro experiments that the natural leader sequence of ROL is able to inhibit the folding supporting properties of the prosequence, resulting in a retardation of folding . In addition, there is strong evidence that all different lipase forms derived from Rhizopus sp . described in the literature are a result of different proteolytic processing and originate from the same gene.

J Bacteriol, 1998 Oct, 180(20), 5466 - 72
Demonstration that the TyrR protein and RNA polymerase complex formed at the divergent P3 promoter inhibits binding of RNA polymerase to the major promoter, P1, of the aroP gene of Escherichia coli; Wang P et al.; In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P . Wang, J . Yang, and A . J . Pittard, J . Bacteriol . 179:4206-4212, 1997) . Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1 . The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P . Wang, J . Yang, B . Lawley, and A . J . Pittard, J . Bacteriol . 179:4213-4218, 1997) . In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors . Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

J Bacteriol, 1998 Oct, 180(20), 5463 - 5
Autoregulation of the pTF-FC2 proteic poison-antidote plasmid addiction system (pas) is essential for plasmid stabilization; Smith AS et al.; The pasABC genes of the proteic plasmid addiction system of broad-host-range plasmid pTF-FC2 were autoregulated . The PasA antidote was able to repress the operon 25-fold on its own, and repression was increased to 100-fold when the PasB toxin was also present . Autoregulation appears to be an essential requirement for pas-mediated plasmid stabilization because when the pas genes were placed behind the isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated tac promoter, they were unable to stabilize a heterologous test plasmid.

J Bacteriol, 1998 Oct, 180(20), 5458 - 62
Efficiency of the pTF-FC2 pas poison-antidote stability system in Escherichia coli is affected by the host strain, and antidote degradation requires the lon protease; Smith AS et al.; The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (pas) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in Escherichia coli CSH50, a 2.5-fold increase in E . coli JM105, and no detectable stabilization in E . coli strains JM107 and JM109 . The lethality of the PasB toxin was far higher in the E . coli strains in which the pas was most effective . Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin . A decrease in host cell death following plasmid loss from an E . coli lon mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.

J Bacteriol, 1998 Oct, 180(20), 5421 - 5
Fumarate regulation of gene expression in Escherichia coli by the DcuSR (dcuSR genes) two-component regulatory system; Zientz E et al.; In Escherichia coli the genes encoding the anaerobic fumarate respiratory system are transcriptionally regulated by C4-dicarboxylates . The regulation is effected by a two-component regulatory system, DcuSR, consisting of a sensory histidine kinase (DcuS) and a response regulator (DcuR) . DcuS and DcuR are encoded by the dcuSR genes (previously yjdHG) at 93.7 min on the calculated E . coli map . Inactivation of the dcuR and dcuS genes caused the loss of C4-dicarboxylate-stimulated synthesis of fumarate reductase (frdABCD genes) and of the anaerobic fumarate-succinate antiporter DcuB (dcuB gene) . DcuS is predicted to contain a large periplasmic domain as the supposed site for C4-dicarboxylate sensing . Regulation by DcuR and DcuS responded to the presence of the C4-dicarboxylates fumarate, succinate, malate, aspartate, tartrate, and maleate . Since maleate is not taken up by the bacteria under these conditions, the carboxylates presumably act from without . Genes of the aerobic C4-dicarboxylate pathway encoding succinate dehydrogenase (sdhCDAB) and the aerobic succinate carrier (dctA) are only marginally or negatively regulated by the DcuSR system . The CitAB two-component regulatory system, which is highly similar to DcuSR, had no effect on C4-dicarboxylate regulation of any of the genes.

J Bacteriol, 1998 Oct, 180(20), 5413 - 20
Molecular cloning and expression analysis of the Rhodobacter capsulatus sodB gene, encoding an iron superoxide dismutase; Cortez N et al.; Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals . After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB) . The R . capsulatus sodB gene was expressed in E . coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species . Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism . The sodB gene was expressed at low levels in R . capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen . Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R . capsulatus in aerobic cultures.

J Bacteriol, 1998 Oct, 180(20), 5406 - 12
Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements; Rao HG et al.; The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity . FEN retained activity after preincubation at 95 degrees C+ for 15 min . A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides . FEN cleaved the strand with the free 5' end adjacent to the single-strand-duplex junction . Deletion of the free 3' end prevented cleavage . Hybridization of a complementary oligonucleotide to the free 3' end moved the cleavage site by 1 to 2 nucleotides . Hybridization of excess complementary oligonucleotide to the free 5' end failed to block cleavage, although this substrate was refractory to cleavage by the 5'-3' exonuclease activity of Taq DNA polymerase . For verification, the free 5' end was replaced by an internally labeled hairpin structure . This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin . A circular duplex substrate with a 5' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phiX174 . This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.

J Bacteriol, 1998 Oct, 180(20), 5369 - 74
Legionella pneumophila catalase-peroxidases: cloning of the katB gene and studies of KatB function; Bandyopadhyay P et al.; Legionella pneumophila, the causative organism of Legionnaires' pneumonia, is spread by aerosolization from man-made reservoirs, e.g . , water cooling towers and air conditioning ducts, whose nutrient-poor conditions are conducive to entrance into stationary phase . Exposure to starvation conditions is known to induce several virulence traits in L . pneumophila . Since catalase-peroxidases have been extremely useful markers of the stationary-phase response in many bacterial species and may be an avenue for identifying virulence genes in L . pneumophila, an investigation of these enzymes was initiated . L . pneumophila was shown to contain two bifunctional catalase-peroxidases and to lack monofunctional catalase and peroxidase . The gene encoding the KatB catalase-peroxidase was cloned and sequenced, and lacZ fusion and null mutant strains were constructed . Null mutants in katB are delayed in the infection and lysis of cultured macrophage-like cell lines . KatB is similar to the KatG catalase-peroxidase of Escherichia coli in its 20-fold induction during exponential growth and in playing a role in resistance to hydrogen peroxide . Analysis of the changes in katB expression and in the total catalase and peroxidase activity during growth indicates that the 8- to 10-fold induction of peroxidase activity that occurs in stationary phase is attributable to KatA, the second L . pneumophila catalase-peroxidase.

J Bacteriol, 1998 Oct, 180(20), 5313 - 8
Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI); Shibayama K et al.; Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive alpha-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide . By comparing the amino acid sequence of WaaO with those of 11 homologous alpha-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified . Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of alpha-glycosidic linkage from alpha-linked nucleotide diphospho-sugar donor . Hydrophobic cluster analysis revealed a conserved domain at the N termini of these alpha-glycosyltransferases . This domain was similar to that previously reported for beta-glycosyltransferases . Thus, this domain is likely to be involved in the formation of beta-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction . Site-directed mutagenesis analysis of E . coli K-12 WaaO revealed four critical amino acid residues.

Plant Physiol, 1998 Oct, 118(2), 451 - 9
Characterization of a granule-bound starch synthase isoform found in the pericarp of wheat; Nakamura T et al.; Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI) . The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine . However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose . Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules . A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm . This protein was designated GBSSII . The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli . GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI . This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.

J Virol, 1998 Nov, 72(11), 9374 - 9
RNA-binding activity of the E1B 55-kilodalton protein from human adenovirus type 5; Horridge JJ et al.; The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs . This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.

J Biol Chem, 1998 Oct 16, 273(42), 27055 - 7
A 17-amino acid insert changes UDP-N-acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc; Wang-Gillam A et al.; We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A . D . (1996) J . Biol . Chem . 271, 13147-13154) . Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P . Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa . Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm . An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus . We expressed the AGX1 and AGX2 genes in Escherichia coli . The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P . On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P . These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.

Genes Dev, 1998 Oct 1, 12(19), 3110 - 22
Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release; Artsimovitch I et al.; Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA . To distinguish these mechanisms we tested whether the effects of the his leader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript . The his pause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3' end . It also can terminate transcription when base changes extend its stem to </=8 nucleotides from the 3' end . All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3'-proximal RNA spacing or created a suitably positioned RNA loop . Oligonucleotides that paired </=8 nucleotides from the pause RNA 3' end could trigger transcript release, but only when added to an already paused complex . These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex . Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3' end from the active site.

Genes Dev, 1998 Oct 1, 12(19), 3032 - 43
Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein; Kitagawa R et al.; Replication of the Escherichia coli chromosome is initiated at a unique site, oriC . Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle . A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA . A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events . This mutant phenotype was suppressed by DnaA-titrating plasmids . Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype . Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle . Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.

Science, 1998 Oct 9, 282(5387), 296 - 8
Controlling gene expression in living cells through small molecule-RNA interactions; Werstuck G et al.; Short RNA aptamers that specifically bind to a wide variety of ligands in vitro can be isolated from randomized pools of RNA . Here it is shown that small molecule aptamers also bound their ligand in vivo, enabling development of a method for controlling gene expression in living cells . Insertion of a small molecule aptamer into the 5' untranslated region of a messenger RNA allowed its translation to be repressible by ligand addition in vitro as well as in mammalian cells . The ability of small molecules to control expression of specific genes could facilitate studies in many areas of biology and medicine.

J Invest Dermatol, 1998 Oct, 111(4), 662 - 7
Antibody responses to melanoma/melanocyte autoantigens in melanoma patients; Huang SK et al.; Melanogenesis-related proteins play important roles in melanin synthesis and antigenicity of melanomas . Identification of highly expressed melanoma-associated antigens (MAA) that are immunogenic in humans will provide potential targets for cancer vaccines . Melanogenesis-related proteins have been shown to be MAA . Autoantibody responses to these MAA have been shown to react with melanoma cells and melanocytes, and suggested to play a role in controlling melanoma progression . To assess antibody responses to potential melanoma/melanocyte autoantigens, the open-reading frame sequences of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and melanoma-associated glycoprotein antigen family (gp100/pmel17) genes were cloned and expressed as recombinant proteins in E . coli . Purified recombinant antigens were employed to detect antibodies in sera of melanoma patients and normal healthy donors . By affinity enzyme-linked immunosorbent assay and western blotting, all recombinant antigens were shown to be antigenic . The main subclass of antibody response to these antigens was IgG . Most importantly this study demonstrated anti-TRP-2 and anti-gp100/pmel17 IgG responses in melanoma patients . Only one of 23 normal donors had an antibody response to the antigens tested . MAA-specific IgG antibodies in sera were assessed in melanoma patients (n = 23) pre- and post-polyvalent melanoma cell vaccine treatment . Polyvalent melanoma cell vaccine treatment enhanced anti-MAA antibody responses; however, only anti-TRP-2 and anti-gp100/pmel17 antibody response was enhanced . These studies suggest that four melanogenesis-related proteins are autoimmunogenic and can be used as potential targets for active-specific immunotherapy.

Cell Stress Chaperones, 1998 Sep, 3(3), 200 - 7
Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes; Kawata Y et al.; Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes . The two proteins, one a GroEL homologue (cpn60: M . glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M . glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP . Both proteins were purified from crude extracts of M . glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography . The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpn10 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpn10 were 58 000 and 10 000, respectively . Both cpn60 and cpn10 possessed amino acid sequences which were highly homologous to other group I chaperonins . M . glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10 . The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES . The similarities between the Escherichia coli GroE proteins are discussed.

Vet Immunol Immunopathol, 1998 Aug 31, 64(4), 313 - 22
Interactions between lipopolysaccharides and blood factors on the stimulation of equine polymorphonuclear neutrophils; Benbarek H et al.; In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown . We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils . The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (> or = 99% neutrophils; viability > or = 98%) . Increasing concentrations of Escherichia coli (E . coli) LPS (from 0.01-10 microg ml(-1)) were used to activate the neutrophils . When LPS was used directly, without another stimulator, the respiratory burst of neutrophils was not activated (N=12 horses; n=5 assays per horse) . On the contrary, when LPS was added to whole blood, the neutrophils isolated from this blood were stimulated in a LPS dose-dependent manner, but polymyxin B added to whole blood suppressed this stimulation (N=2; n=6) . LPS dissolved in autologous equine plasma stimulated the isolated neutrophils in a dose-dependent manner from 0.1-10 microg ml(-1) (N=5; n=12) . Heat inactivation of the plasma abolished this CL increase (N=2; n=5) . LPS added to equine albumin did not stimulate the isolated neutrophils (N=2; n=5) . On the contrary, the addition of gamma-globulins (1 mg ml(-1)) to LPS (10 microg ml(-1)) led to the stimulation of neutrophils (N=2; n=5) . We concluded that LPS did not directly stimulate the isolated equine neutrophils, but that plasmatic factors are needed for the stimulation of these cells by LPS.

Analyst, 1998 Jun, 123(6), 1369 - 72
Determination of the pH difference across a cell membrane using a methylammonium-selective membrane electrode; Katsu T et al.; A method was developed for determining pH differences across cell membranes using a methylammonium-selective membrane electrode, based on monitoring of the pH gradient-induced uptake of methylammonium in situ . The methylammonium electrode was constructed using calix{6}arene-hexaacetic acid hexaethyl ester as a neutral carrier and bis(2-ethylhexyl) sebacate as a membrane solvent in a poly(vinyl chloride) membrane matrix . This electrode exhibited a near-Nernstian response to methylammonium in the concentration range 2 x 10(-5)-1 x 10(-2) M with a slope of 58 mV per concentration decade in a buffer solution of 150 mM choline chloride-10 mM TRIS-HCl (pH 7.5) . The limit of detection was 5 x 10(-6) M . In experiments using liposomes, the uptake of methylammonium into liposomes occurred effectively when the pH of the outside suspension medium was alkaline, and the determination of changes in methylammonium concentrations in the outer medium was quantitatively related to changes in the pH differences across the liposomal membrane . The transmembrane pH differences in Escherichia coli cells were also determined by this method.

Diagn Microbiol Infect Dis, 1998 Aug, 31(4), 503 - 9
Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction; Yavzori M et al.; A polymerase chain reaction (PCR) protocol for rapid (7 h) detection of enterotoxigenic Escherichia coli (ETEC) is described . This protocol has been validated on 57 stool samples from young children by comparing it with the colony hybridization technique . A good agreement was found between the two methods with Cohen's kappa statistics of 0.87 and 0.79 for the detection of the heat-stable toxin (ST) and heat-labile toxin (LT), respectively . Of 26 samples positive for LT and 15 samples positive for ST by colony hybridization, 21 (81%) and 15 (100%) were also found to be positive for LT and ST by PCR, respectively . Only one sample identified as LT-negative by colony hybridization was found to be positive by PCR . However, 3 of 42 samples of ST-negative by colony hybridization were detected as positive by PCR . A reconstruction experiment revealed that PCR could detect LT-producing and ST-producing ETEC at minimal concentrations of 2.5 x 10(3) cfu and 2.5 x 10(2) cfu per gram of feces, respectively . These data indicate the possible use of this method for rapid identification of ETEC-associated diarrhea in clinical and epidemiological settings.

Enferm Infecc Microbiol Clin, 1998 Jun-Jul, 16(6), 258 - 60
{Detection of pathogenicity factors in strains of classical enteropathogenic Escherichia coli}; Frias C et al.; OBJECTIVE: To determine the number of strains of classic enteropathogenic E . coli (EPEC) that have the eae gene, that is considered a pathogenicity factor . MATERIAL AND METHODS: The presence of the eae gene has been evaluated on 62 EPEC strains of ten different serogroups, isolated from children with gastroenteritis . RESULTS: Amplification of the eae gene was positive in 10 out of 62 EPEC strains analyzed (16%) corresponding to seven different serogroups . DISCUSSION: The low frequency of the detection of the eae gene on EPEC strains shows the limited correlation between the pathogenicity and the serogroup of the strains and would corroborate the need to reexamine this subject prospectively in our country.

Appl Microbiol Biotechnol, 1998 Aug, 50(2), 193 - 8
Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli; Saito K et al.; The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from alpha-D-glucose 1-phosphate and D-glucose were cloned from a basidiomycete, Grifola frondosa . Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns . Consistent with the novelty of TSase, there were no homologous proteins registered in the data-bases . Recombinant TSase with a histidine tag at the NH2-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G . frondosa strain . Incubation of alpha-D-glucose 1-phosphate and D-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 476 - 8
Expression, purification, crystallization and preliminary X-ray diffraction analysis of human uroporphyrinogen decarboxylase; Laterriere M et al.; A recombinant human uroporphyrinogen decarboxylase (E.C . 4.1.1.37, UROD) has been expressed in Escherichia coli and purified to homogeneity . Crystals grew by the hanging-drop vapor-diffusion technique from a starting solution containing 1.5 mg ml-1 protein . The crystals belong to the trigonal space group P3121 or its enantiomer P3221 and diffract to 3 A resolution . The unit-cell parameters are a = b = 103.4, c = 75.7 A and gamma = 120 degrees . The asymmetric unit contains one molecule . Preliminary structural predictions suggest for the protein a TIM-barrel type tertiary structure.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 473 - 5
Crystallographic studies of casein kinase I delta toward a structural understanding of auto-inhibition; Longenecker KL et al.; A recombinant form of mammalian casein kinase I delta (CKIdelta) containing the catalytic domain and an auto-inhibitory domain was expressed in Escherichia coli, purified and crystallized . X-ray data were collected to 2.4 A resolution, and the crystals belong to space group C2221 . Molecular replacement using the structure of the catalytic domain of CKIdelta yielded strong electron density for residues in the model, but no interpretable density was found for the inhibitory domain . A conserved intermolecular contact suggests the formation of dimers which would inhibit the activity of this protein kinase.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 467 - 9
Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli; Gallagher DT et al.; The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms . Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions . Diffraction data sets to 2.8 A resolution (native) and 2 . 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 461 - 6
Enhanced electron-density envelopes by extended solvent definition; Blom N et al.; Extended delineation of water molecules, monitored using Rfree values, afforded considerable improvement in quality of electron-density maps for structure determination of mammalian class I and E . coli class II aldolases . Augmented solvent definition results in an additional decrease in Rfree values of 3-4% and is reflected in significantly enhanced electron-density envelopes enabling tracing of amino-acid sequences through regions of otherwise discontinuous or weak electron density.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 458 - 60
Expression, crystallization and preliminary X-ray analysis of ligand-free human glutathione S-transferase M2-2; Patskovska LN et al.; Human glutathione-S-transferase M2-2 (hGSTM2-2) was expressed in Escherichia coli and purified by GSH-affinity chromatography . The recombinant enzyme and the protein isolated from human tissue were indistinguishable based on physicochemical, enzymatic and immunological criteria . The catalytically active dimeric hGSTM2-2 was crystallized without GSH or other active-site ligands in two crystal forms . Diffraction from form A crystals extends to 2.5 A and is consistent with the space group P21 (a = 53.9, b = 81.5, c = 55.6 A, beta = 109.26 A) with two monomers in the asymmetric unit . Diffraction from form B crystals extends to 3 A and is consistent with a space group P212121 (a = 57.2, b = 80.7, c = 225.9 A) with two dimers in the asymmetric unit . This is the first report of ligand-free mu-class GST crystals, and a comparison with liganded complexes will provide insight into the structural consequences of substrate binding which are thought to be important for catalysis.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 436 - 7
Crystallization and preliminary X-ray studies of hORF6, a novel human antioxidant enzyme; Choi HJ et al.; HORF6 is a member of the novel antioxidant enzyme family found in humans . A recombinant form of hORF6 expressed and purified from E . coli has been crystallized by the hanging-drop method using various PEG's as precipitating agents . HORF6 crystallizes in two different monoclinic space groups, P21 and C2 . The P21 crystals have unit-cell dimensions of a = 47.85, b = 75.17, c = 63.30 A and beta = 110.21 degrees and contain two monomers per asymmetric unit, while the C2 crystals have unit-cell dimensions of a = 165.27, b = 95.44, c = 166.44 A and beta = 128.97 degrees and contain more than six monomers per asymmetric unit . The P21 crystals with the smaller unit cell diffract X-rays better and behave well for the X-ray analysis . A native data set from a single crystal of the P21 space group gas been collected to 2.0 A resolution.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 433 - 5
Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2); Maenaka K et al.; Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells . These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell . The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified . The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method . Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A . A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 427 - 9
Crystallization of the NADP-dependent beta-keto acyl carrier protein reductase from Escherichia coli; Rafferty JB et al.; The NADP-dependent beta-keto acyl carrier protein reductase (BKR) from E . coli has been crystallized by the hanging-drop method of vapour diffusion using poly(ethylene glycol) of average molecular weight 1450 . The crystals belong to the hexagonal space group P6122 or P6522 with unit-cell dimensions a = b = 67.8, c = 355.8 A . Calculated values for Vm and consideration of the packing suggest that the asymmetric unit contains a dimer . BKR catalyses the first reductive step in the elongation cycle of fatty-acid biosynthesis . It shares extensive sequence homology with the enzyme which catalyzes the second reductive step in the cycle, enoyl acyl carrier protein reductase (ENR), and thus provides an opportunity to study the evolution of enzyme function in a metabolic pathway . The structure determination will permit the analysis of the molecular basis of its catalytic mechanism and substrate specificity.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 407 - 8
Crystallization of the alanine dehydrogenase from Phormidium lapideum; Sedelnikova S et al.; Amino-acid dehydrogenases catalyse the interconversion of their respective amino acids to the corresponding keto acid, with concomitant reduction of NAD or NADP . The enzymes phenylalanine, glutamate, leucine and valine dehydrogenase all share a similar three-dimensional subunit structure and a high degree of sequence similarity, indicating that they belong to an enzyme superfamily related by divergent evolution . In contrast, alanine dehydrogenase shows no sequence similarity with any of these enzymes despite catalysing a reaction with the same chemistry and thus it is predicted that it possesses a different three-dimensional structure . The alanine dehydrogenase from Phormidium lapideum has been crystallized in space group R32, cell dimensions a = b = 123.1 and c = 184.8 A, with a monomer in the asymmetric unit . The structure determination of this enzyme will shed light on how nature has evolved two different systems to carry out the same reaction.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 697 - 9
Crystallization and preliminary X-ray analysis of ferric enterobactin receptor FepA, an integral membrane protein from Escherichia coli; Smith BS et al.; Diffraction-quality crystals have been obtained of the integral membrane protein ferric enterobactin receptor (FepA) from the outer membrane of Escherichia coli . Crystals were grown using the zwitterionic detergent lauryldimethylamine oxide (LDAO), the precipitants polyethylene glycol (PEG) 1000 and sodium chloride, and the additive heptane-1,2,3-triol; they have the symmetry of the orthorhomic space group C2221 with a = 112.2, b = 137.2 and c = 135 . 4 A and diffract to 2.5 A resolution . The crystals were flash-cooled and a preliminary data set was collected at 103 K . The crystals are suitable for three-dimensional structure analysis.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 690 - 2
Subcloning, crystallization and preliminary X-ray analysis of the signal receiver domain of ETR1, an ethylene receptor from Arabidopsis thaliana; Grantz AA et al.; The signal receiver domain of ETR1, an ethylene receptor from Arabidopsis thaliana, has been subcloned and expressed in E . coli and purified by affinity chromatography . Crystals of both native and a selenomethionine-substituted form of the receiver domain have been obtained . Native crystals grew in 1.6 M Li2SO4 and 0.1 M HEPES pH 7 . 5 and once flash-frozen diffract to 2.1 A resolution . They belong to space group P41212 with unit-cell dimensions a = b = 48.4, c = 112.3 A.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 678 - 80
Crystallization of 5-keto-4-deoxyuronate isomerase from Escherichia coli; Dunten P et al.; 5-Keto-4-deoxyuronate isomerase from Escherichia coli has been crystallized after partial purification . The isomerase was found to be enriched in preparations of an unrelated recombinant protein . Crystals of the isomerase were obtained from two different precipitants despite the fact that the recombinant protein represented roughly 90% of the total protein present . The crystals diffract to 2.7 A resolution and are suitable for a structure determination . The role of the isomerase in E . coli is uncertain, as E . coli is not known to degrade the polysaccharides which are potential sources of 5-keto-4-deoxyuronate.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 647 - 9
Purification, crystallization and preliminary X-ray analysis of the Escherichia coli phytase; Jia Z et al.; A recombinant form of Escherichia coli phytase, which hydrolyzes phytic acid into phosphate and myo-inositol, has been expressed, purified and crystallized . Crystals have been obtained by the method of bulk crystallization in 10 mM sodium acetate buffer (pH 4.5) without using a conventional precipitant . The enzyme crystallized in space group P21, with unit-cell dimensions a = 74.9, b = 72.2, c = 82.4 A, and beta = 92.0 degrees . Crystals diffract to at least 2.2 A at a rotating-anode X-ray source and a 2.3 A resolution data set has been collected, giving completeness of 98.0% and an Rsym of 0.072 . Assuming there are two phytase molecules in the asymmetric unit, the solvent content is calculated to be 42.1% . A self-rotation function shows a clear twofold non-crystallographic symmetry relating two molecules of E . coli phytase in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 639 - 42
Purification, crystallization and preliminary X-ray diffraction studies of retinal dehydrogenase type II; Lamb AL et al.; One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (Ra1DH2) . Ra1DH2, expressed in the Escherichia coli BL21(DE3) strain, was purified and crystallized using ammonium sulfate as a precipitant . These crystals belong to the space group P212121 (a = 108, b = 150, c = 168 A, alpha = beta = gamma = 90 degrees).

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 143 - 5
Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays; Guerra B et al.; The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3) . The protein has been purified to homogeneity and crystallized . The crystals belong to the monoclinic space group C2, they have unit-cell parameters a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution . The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 140 - 2
Crystallization of a complex between a novel C-terminal transmitter, HPt domain, of the anaerobic sensor kinase ArcB and the chemotaxis response regulator CheY; Kato M et al.; The histidine-containing phosphotransfer (HPt) domain at the C-terminus of the anaerobic sensor kinase ArcB has been cocrystallized with the chemotaxis response regulator CheY by a hanging-drop vapor-diffusion method . Crystals belong to space group P212121 with unit-cell dimensions a = 55.32, b = 76.29 and c = 83.89 A, with one molecule in the crystallographic asymmetric unit . The crystals diffract to 2.7 A resolution . This is the first crystallization of a protein-protein complex formed by a transmitter domain of sensor kinase and a receiver domain of response regulator in the two-component signal-transduction system.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 135 - 6
The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis; Gruez A et al.; The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein subunit was crystallized using the hanging-drop technique, with PEG 4000 as precipitant . The crystals belong to space group P3112 or enantiomorph, with unit-cell parameters a = b = 171.0, c = 152.1 A . A solvent content of 75% was determined by a calibrated tetrachloromethane/toluene gradient which corresponds to three monomers per asymmetric unit . A 3 A resolution native data set was collected at beamline W32 of LURE, Orsay, France.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 132 - 4
Crystallization and preliminary X-ray analysis of the periplasmic receptor (PotF) of the putrescine transport system in Escherichia coli; Vassylyev DG et al.; The primary receptor (PotF) of the putrescine transport system in E . coli has been crystallized by the hanging-drop vapor-diffusion technique . The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A . The crystals diffract beyond 2.2 A with a rotating-anode X-ray source . A complete data set from the native crystals has been collected and processed at 2.3 A resolution . Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K . The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 111 - 3
Crystallization and preliminary X-ray studies of sialidase L from the leech Macrobdella decora; Luo Y et al.; Functional monomeric 83 kDa sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from Macrobdella leech, was expressed in Escherichia coli and readily crystallized by a macroseeding technique . The crystal belongs to space group P1 with unit-cell parameters a = 46.4, b = 69.3, c = 72.5 A, alpha = 113.5, beta = 95.4 and gamma = 107.3 degrees . There is one molecule per unit cell, giving a Vm = 2.4 A3 Da-1 and a solvent content of 40% . Native and mercury-derivative data sets were collected to 2.0 A resolution . Threading and molecular-replacement calculations confirmed the existence of a bacterial sialidase-like domain.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 102 - 4
Crystallization and preliminary crystallographic study of a component of the Escherichia coli tol system: TolB; Abergel C et al.; TolB from Escherichia coli is part of the Tol system used by the group A colicins to penetrate and kill cells . A TolB derivative tagged with six histidines was overexpressed, purified by chelation on a nickel affinity column and crystallized using the SAmBA software to define the optimal crystallization protocol . The crystals belong to the monoclinic system, space group P21 with unit-cell parameters a = 64.48, b = 41.06, c = 78.41 A, beta = 110.78 degrees . Frozen crystals diffract to 1.9 A resolution . Screening for heavy-atom derivatives both on the native TolB and various cysteine-substituted mutants is in progress . In addition, a selenomethionine-substituted protein is being produced in order to use the MAD method for structure determination.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 58 - 73
Accelerated X-ray structure elucidation of a 36 kDa muramidase/transglycosylase using wARP; Van Asselt EJ et al.; The X-ray structure of the 36 kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 A resolution . Subsequently, before any model building was carried out, phases were extended to 1.7 A resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases . The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened . The structure of the soluble lytic transglycosylase was refined at 1.7 A resolution with X-PLOR to a final crystallographic R factor of 18.9% . Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher . Furthermore, setting aside 5% of the data for an Rfree test set had a negative effect on the phase improvement . The mean WwARP, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the Rfree factor to judge different phase improvement protocols . The elongated Slt35 structure has three domains named the alpha, beta and core domains . The alpha domain contains mainly alpha-helices, while the beta domain consists of a five-stranded antiparallel beta-sheet flanked by a short alpha-helix . Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E . coli . The putative active site is at the bottom of a large deep groove in the core domain.

J Cell Biol, 1998 Oct 5, 143(1), 65 - 79
Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis; Gary JD et al.; The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kinase domain at its COOH terminus . Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase () . Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P2 . Consistent with this, we find that unlike wild-type cells, fab1Delta, fab1(tsf), and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P2, a phosphoinositide recently identified both in yeast and mammalian cells . PtdIns(4,5)P2 synthesis, on the other hand, is only moderately affected even in fab1Delta mutants . The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P2 synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P2 . Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P2 by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P2 has a distinct function . The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size . Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole . Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P2, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p . We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P2.

Drug Metab Dispos, 1998 Oct, 26(10), 1026 - 30
Catalytic properties of an expressed cytochrome P450 2B1 from a Wistar-Kyoto rat liver cDNA library; Kobayashi Y et al.; Cytochrome P450 2B1 clones were isolated from a phenobarbital-induced Wistar-Kyoto (WKY) hepatic cDNA library and were found to contain a Glu-322 --> Val substitution, compared with wild-type 2B1 from Sprague-Dawley rats . After heterologous expression in Escherichia coli and purification, activities of this 2B1 E322V variant were determined for ethoxycoumarin and androstenedione . The total activities and metabolite profiles did not differ between 2B1 E322V and wild-type 2B1 for these substrates . In addition, similar rate constants of inactivation were observed with the mechanism-based inactivators chloramphenicol, N-(2-p-nitrophenethyl)chlorofluoroacetamide, and 9-ethynylphenanthrene . These results suggest that the Glu-322 --> Val alteration in the 2B1 WKY variant does not significantly affect 2B1 activity . However, another clone obtained from the cDNA library contained two additional substitutions: Val-103 --> Ala and Glu-424 --> Lys . As residue 103 is within a predicted substrate recognition site (SRS-1), it was of interest to determine whether the Val --> Ala substitution conferred any unique catalytic activities on 2B1 . No differences in the metabolism of ethoxycoumarin or androstenedione were observed . However, the Val-103 --> Ala alteration caused an approximately threefold decrease in the rate constant of inactivation for 9-ethynylphenanthrene in comparison with either 2B1 E322V or wild-type 2B1 . Based on computer modeling, residue 103 is predicted to be near the active site but at a distance greater than 5A from 9-ethynylphenanthrene . Our results suggest that the Val-103 --> Ala alteration may have an indirect influence on the susceptibility of P450 2B1 to mechanism-based inactivators.

Drug Metab Dispos, 1998 Oct, 26(10), 1019 - 25
Uroporphyrinogen oxidation catalyzed by human cytochromes P450; Sinclair PR et al.; Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin . Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2 . Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX) . UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice . There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2 . With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2 . Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX . In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5 . Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX . Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study . From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source . These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue . The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2.

Mol Biochem Parasitol, 1998 Sep 1, 95(1), 53 - 68
Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase; Chaudhuri M et al.; The protozoal parasite Trypanosoma brucei depends on a mitochondrial non-cytochrome terminal oxidase known as the trypanosome alternative oxidase (TAO) in its mammalian host . We have recently cloned the cDNA from T . brucei bloodstream form and have characterized a 33 kDa mitochondrial protein as TAO . Here we report that the TAO is a single copy gene in T . brucei and its expression is down regulated at the level of transcript abundance during differentiation from the bloodstream to the procyclic trypanosomes . Like other alternative oxidases (AOXs) cloned from different plants and fungi, TAO possesses the conserved sequences at the centrally located predicted membrane spanning domains and the signature sequence at the C-terminal hydrophilic domain for a pair of putative iron binding motifs (E-X-X-H) . Phylogenetic analysis of the deduced protein sequences of eight different alternative oxidases cloned from different plants and fungi revealed that TAO is more closely related to the alternative oxidases of the fungi clade than that of plants . TAO has been functionally expressed in Escherichia coli . In the first of the two putative iron binding motifs, site-directed mutagenesis of E215 to A, L, N and Q resulted in the loss of the ability of the TAO gene to complement the heme deficiency of the E . coli mutants (SASX41B and GE1387) by conferring on them a CN-insensitive pathway of respiration . The conservative substitution of E215 by aspartate and histidine reduced the growth of the E . coli auxotrophs by approximately 80% . The mutations apparently did not have any effect on the stability of the expressed protein as revealed by the immunoblot analysis of the bacterial protein using TAO monoclonal antibody, which we have developed . Together, these points suggest that E215 plays an important role in the function of TAO . The steady state level of TAO mRNA is down-regulated in the procyclic stage presumably accounting for the low levels of TAO protein in these forms.

J Virol Methods, 1998 Sep, 74(1), 39 - 46
Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses; Burkala EJ et al.; Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia . The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV) . Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV . The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione . The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV . The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera . The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera . The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses . The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

FEBS Lett, 1998 Sep 18, 435(2-3), 211 - 4
The effects of SNAP/SNARE complexes on the ATPase of NSF; Matveeva E et al.; The ATPase of the N-ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle-target membrane fusion . Complexes containing different combinations of NSF, alpha-SNAP, Vamp-2 (synaptobrevin 2), syntaxin 1, and SNAP-25 were reconstituted and then tested for their effect on the ATPase of NSF . While NSF interacts with all alpha-SNAP-containing complexes, only the alpha-SNAP/t-SNARE complex significantly stimulated ATPase activity . This stimulation was dependent on increasing SNAP/t-SNARE complex and was saturable . The apparent stimulation of ATPase activity is due to a 10-fold increase in initial hydrolysis rate . Complex containing both v- and t-SNAREs bound significantly more alpha-SNAP but did not stimulate the ATPase of NSF.

Cell Adhes Commun, 1998 Jun, 5(4), 325 - 33
Expression profile of vascular cell adhesion molecule-1 (CD106) in inflammatory foci using rhenium-188 labelled monoclonal antibody in mice; Kairemo KJ et al.; Rhenium (Re)-188 is a generator (W-188/Re-188) produced high energy beta-emitter suitable for radionuclide therapy (T1/2 is 16.9 hrs and Emax 2.1 MeV (range 11 mm)) . We have labelled monoclonal antibody (MAb) raised against vascular cell adhesion molecule-1 (VCAM-1) with Re-188 using glucoheptonate chelation technique and SnCl2 as reducing agent . The labelling efficiency, free perrhenate and reduced Re were controlled with thin layer chromatography and the purification of Re-188-MoAbs was performed using gel filtration . Our results indicate that Re-188-labelled antibodies remain in vitro stable and the labelling purity is > 90% . We also have applied these Re-188-MoAbs for detection of inflammatory disease in a mouse . The effective half-lives of organs of interest after an injection of Re-188-anti-VCAM1 were as follows: blood 5.2 hr, kidney 4.7 hr, and liver 9.6 hr . Re-188-anti-VCAM-1 was found to accumulate mainly in kidney and liver . One hour after the injection, the kidney contained in average as high as 12.5% and the liver 2.8 ID/g tissue . After 6 hr, the kidney contained 5.5% ID/g and the liver 2.6% ID/g . At 24 hr, the kidney uptake was 0.5% ID/g and the liver uptake 0.8% ID/g, respectively . The inflamed foci, subcutaneous lesions in the footpad skin, were visualized using gamma camera . From the distribution data the uptakes in the inflamed foci as follows: at 1 hr 2.18 (inflammation) and 1.72% ID/g (control), at 6 hr 1.42 (inflammation) and 0.85% ID/g (control), and at 24 hr 0.17 (inflammation) and 0.084% ID/g (control), respectively . Anti-VCAM-1 MAb showed better targeting as compared to control MoAbs in inflammation (caused by E.coli lipoplysaccaride) . In conclusion, Re-188 is suitable for MAb labelling, and MAb against VCAM-1 may be used for detection of local inflammatory disease.

Biotechniques, 1998 Sep, 25(3), 426 - 33
Limitations for purification of murine interleukin-18 when expressed as a fusion protein containing the FLAG peptide; Elhofy A et al.; As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system . The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed . While significant amounts of recombinant IL-18 were present in E . coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody . Surprisingly, the majority of recombinant IL-18 present in E . coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody . However, we found that the BL21 strain of E . coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18 . Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.

Biochem Mol Biol Int, 1998 Sep, 45(6), 1149 - 54
RGD-containing trypsin with both platelet aggregation inhibitory activity and proteolytic activity; Nie X et al.; Arg-Gly-Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors . In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid turn region and a flexible loop of trypsin, respectively . Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E . coli and purified . Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined . 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis . 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5 microM, while the wild type trypsin shows no inhibitory activity.

Biochem Mol Biol Int, 1998 Sep, 45(6), 1113 - 9
A human B cell line AF10 expressing HIL-17; Zhou L et al.; AF10, a human B cell line, is a mycoplasma-free variant cloned from the human IgE myeloma cell line U266 . Total RNA isolated from the AF10 cells was used as template for RT-PCR, and a specific product of about 411bp corresponding to the coding region of hIL-17 lack of a leading sequence obtained . The sequence of the RT-PCR product is the same to that reported in the literature . The expressed rhIL-17 in E . coli can induce 10- to 15-fold increase of IL-6 secretion by mouse fibroblast 3T3 cells . It is for the first time until now that hIL-17 messager has been detected in B cell . There may exist some potential relationship between hIL-17 and myeloma cells.

Anal Biochem, 1978 Dec, 91(2), 600 - 12
Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation; McIntosh L et al.; Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described . It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA . Sauromatum but not Olisthodiscus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure . Nuclease levels were monitored by coincubating {3H}-labeled Escherichia coli ribosomal RNA with the experimental RNA samples . The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.

Plant Cell, 1998 Oct, 10(10), 1637 - 48
The plant U1 small nuclear ribonucleoprotein particle 70K protein interacts with two novel serine/arginine-rich proteins; Golovkin M et al.; The U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K protein (U1-70K), one of the three U1 snRNP-specific proteins, is implicated in basic and alternative splicing of nuclear pre-mRNAs . We have used the Arabidopsis U1-70K in the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with it . This screening has resulted in the isolation of two novel plant serine/arginine-rich (SR) proteins, SRZ-22 and SRZ-21 (SRZ proteins) . Neither the N-terminal region nor the arginine-rich C-terminal region of U1-70K alone interact with the SRZ proteins . The interaction of U1-70K with the SRZ proteins is confirmed further in vitro using a blot overlay assay . The plant SRZ proteins are highly similar to each other and contain conserved modular domains unique to different groups of splicing factors in the SR family of proteins . SRZ proteins are similar to human 9G8 splicing factor because they contain a zinc knuckle, precipitate with 65% ammonium sulfate, and cross-react with the 9G8 monoclonal antibody . However, unlike the 9G8 splicing factor, SRZ proteins contain a glycine hinge, a unique feature in other splicing factors (SC35 and ASF/SF2), located between the RNA binding domain and the zinc knuckle . SRZ-22 and SRZ-21 are encoded by two distinct genes and are expressed in all tissues tested with varied levels of expression . Our results suggest that the plant SRZ proteins represent a new group of SR proteins . The interaction of plant U1-70K with the SRZ proteins may account for some differences in pre-mRNA splicing between plants and animals.

FASEB J, 1998 Oct, 12(13), 1281 - 99
Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins; Frillingos S et al.; The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies . Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation . structure-function relationships in polytopic membrane proteins.

Biochem J, 1998 Oct 15, 335 ( Pt 2), 319 - 27
Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding; Macheroux P et al.; Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor . The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover . This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences . It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein . Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein . The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state . The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little . Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein . Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes . The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an alpha-beta-barrel using discrete state-space modelling.

Biochem J, 1998 Oct 15, 335 ( Pt 2), 247 - 57
Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes . Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation; Rahman S et al.; Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes . The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs . Wild-type Elegantin (ARGDNP) and several variants including Eleg . AM (ARGDMP), Eleg . PM (PRGDMP) and Eleg . PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli . The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin . Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg . AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg . PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity . The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg . AM and Eleg . PM had similar Kd (app) values . In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence . In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions . First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg . AM but inhibited poorly by Eleg . PM and Kistrin . In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the alpha5 beta1 complex . In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an alpha5 beta1-dependent manner . Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the alpha5 beta1 complex by the PRGD molecules . These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.

J Mol Biol, 1998, 283(1), 311 - 27
Structural and functional roles of heme binding module in globin proteins: identification of the segment regulating the heme binding structure; Inaba K et al.; To investigate structural and functional significance of a newly proposed structural unit in globins, the "heme binding module", we synthesized a "heme binding module"-substituted chimeric globin and characterized its function and structure . In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin alpha-subunit, plays a key role in constructing the heme proximal structure in globins . The replacement of the heme binding module in myoglobin with that of hemoglobin alpha-subunit converted the absorption spectra into that of the alpha-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement . The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the alpha-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site . The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins .

Protein Sci, 1998 Sep, 7(9), 1976 - 82
The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis; Pascarella S et al.; We describe a model for the three-dimensional structure of E . coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known . The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism . These proposals were supported by analysis of the properties of a number of site-directed mutants . New active site features are also proposed for further experimental testing.

Protein Sci, 1998 Sep, 7(9), 1875 - 83
Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala; Yang WZ et al.; The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases . Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution . Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure . Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix . The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix . This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion . The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments . The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.

Electrophoresis, 1998 Sep, 19(12), 2169 - 74
Subnanomolar detection limit for sodium dodecyl sulfate-capillary gel electrophoresis using a fluorogenic, noncovalent dye; Harvey MD et al.; Picomolar limits of detection are obtained using the noncovalent, fluorogenic dye, Sypro Red . The size separation of four commonly used sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) molecular weight markers with 8% linear polyacrylamide (PAA) as the sieving matrix is used to construct a calibration curve for molecular weight determinations . SDS-CGE purity and molecular weight determination of purified chorismate mutase-prephenate dehydrogenase (CMPD) from Escherichia coli is shown to be comparable in accuracy with slab gel SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . A migration time precision study indicates excellent reproducibility . Sypro red labeling of SDS-bovine serum albumin (SDS-BSA) complexes at nanomolar protein concentrations suggests assay detection limits surpassing those of silver staining . This detectability exceeds that achieved in previous SDS-CGE laser-induced fluorescence studies . This approach is expected to be easily adapted for use with commercial polymer formulations and automated instrumentation.

Microbiol Res, 1998 Aug, 153(2), 173 - 8
The low level expression of chloramphenicol acetyltransferase (CAT) mRNA in Escherichia coli is not dependent on either Shine-Dalgarno or the downstream boxes in the CAT gene; Odjakova M et al.; Recent studies have shown that the canonical Shine-Dalgarno (SD)-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli . Alternative non-SD sequences (located upstream from the initiation codon) and also downstream sequences ("downstream boxes") complementary to 16S rRNA were found to be involved in the initiation of translation of mRNAs devoid of either SD or any leader sequences . In this study the chloramphenicol acetyltransferase (CAT) gene was modified to remove the 5' terminal non-translated region and/or the two potential downstream boxes in the CAT gene . Thus a series of ten CAT gene constructs was created and expressed in E . coli under a strong constitutive promoter . The results showed that CAT mRNAs devoid of both leader sequence nucleotides and the two downstream boxes in the CAT gene remained active in vivo and produced CAT protein in sufficient amounts for survival of the transformed cells at chloramphenicol concentrations up to 20-30 micrograms/ml.

Nutrition, 1998 Sep, 14(9), 667 - 71
Total parenteral nutrition supplemented with medium-chain triacylglycerols prevents atrophy of the intestinal mucosa in septic rats; Iba T et al.; Total parenteral nutrition (TPN) is associated with an increased incidence of bacterial translocation (BT) compared with enteral nutrition because of the disuse atrophy of the intestine . In this study, we assessed the effect of adding medium-chain triacylglycerols (MCT) to TPN for the prevention of mucosal atrophy in the intestine . Rats were subjected to either fat-free TPN, TPN with long-chain triacylglycerols (LCT), or TPN with MCT for 5 d and nutrition parameters were evaluated . In another set of rats receiving the same TPN regimen, 0.8 or 0.05 mg/kg endotoxin was administered on day 4 . Survival was evaluated and BT to the mesenteric lymph nodes, liver, and systemic blood was measured 24 h later . The mucosal heights of the jejunum and ileum were evaluated concurrently . The survival rate was significantly improved in the MCT group (P < 0.05) at the endotoxin dose of 0.8 mg/kg . The nutrition condition presented by phospholipid, total cholesterol, and total ketone body levels was the best in the MCT group compared to the other groups . The intestinal villous height in the ileum was significantly greater in the MCT group . However, the improvement of BT in MCT group was not statistically significant . In this endotoxin-challenged rat model, survival rate was improved by the supplementation of MCT . This effect may be presented in some part by the improvement in nutrition condition and by the prevention of mucosal atrophy in the intestine.

J Appl Physiol, 1998 Oct, 85(4), 1533 - 43
Dynamics of lung collapse and recruitment during prolonged breathing in porcine lung injury; Neumann P et al.; Oleic acid (OA) injection, lung lavage, and endotoxin infusion are three commonly used methods to induce experimental lung injury . The dynamics of lung collapse and recruitment in these models have not been studied, although knowledge of this is desirable to establish ventilatory techniques that keep the lungs open . We measured lung density by computed tomography during breath-holding procedures . Lung injury was induced with OA, lung lavage, or endotoxin in groups of six mechanically ventilated pigs . After a stabilization period, repetitive computed tomography scans of the same slice were obtained during prolonged expirations with and without positive end-expiratory pressure and during prolonged inspirations after 5 and 30 s of expiration . Lung collapse and recruitment occurred mainly within the first 4 s of breath-holding procedures in all three lung injury models, and some collapse and recruitment occurred even within 0.6 s . OA-injured lungs were significantly more unstable than lungs injured by bronchoalveolar lavage or endotoxin infusion . In this experimental setting, expiration times <0.6 s are required to avoid cyclic alveolar collapse during mechanical ventilation without extrinsic positive end-expiratory pressure.

J Appl Physiol, 1998 Oct, 85(4), 1421 - 8
Effect of urokinase on disseminated intravascular coagulation; Vasquez Y et al.; Our study evaluated the possible therapeutic effect of urokinase in treating the microthrombiotic effects of disseminated intravascular coagulation by assisting the activation of endogenous plasminogen . Twenty-six pigs were anesthetized, intubated, mechanically ventilated, and surgically catheterized . Septic shock was induced in all 26 pigs by an intravenous infusion of heat-killed Escherichia coli . The pigs were divided into two sets of experiments: in experiment 2 (n = 14), one-half received an intravenous dose of urokinase 1 h after heat-killed E . coli infusion and in experiment 3 (n = 12) one-half received an intravenous bolus dose and a continuous drip of urokinase 2 h after heat-killed E . coli infusion . The untreated pigs served as controls . Hemodynamic parameters, blood chemistries, and blood gases were analyzed . Urokinase given 1 h after bacterial toxin infusion significantly restored blood flow, resulting in an increase in cardiovascular and pulmonary function and improved survival rate (43% control vs . 100% treated, 24-h experimental period) . Treatment given after 2 h showed some significant effect on pulmonary function; however, within 10 h of E . coli infusion, mortality rates in control and treated groups were 100 and 83%, respectively . Early administration of urokinase after onset of disseminated intravascular coagulation restored blood flow and helped resolve organ damage.

Biochemistry, 1998 Oct 6, 37(40), 14230 - 6
The carboxyl-terminal tripeptide of the manganese-stabilizing protein is required for quantitative assembly into photosystem II and for high rates of oxygen evolution activity; Betts SD et al.; The extrinsic manganese stabilizing protein of photosystem II is required for Mn retention by the O2-evolving complex, accelerates the rate of O2 evolution, and protects photosytem II against photoinhibition . We report results from studies of the in vitro reconstitution of spinach photosytem II with recombinant manganese stabilizing protein with C-terminal deletions of two, three, and four amino acids . The deletions were the result of amber mutations introduced by site-directed mutagenesis . Removal of the C-terminal dipeptide (Glu-Gln) did not diminish the ability of the manganese stabilizing protein either to rebind to or to restore high rates of O2 evolution to photosystem II preparations depleted of the native protein . Deletion of the C-terminal tripeptide (Leu-Glu-Gln) resulted in weakened but specific binding of manganese stabilizing protein to photosystem II and minimal recovery of O2 evolution activity . Removal of the C-terminal tetrapeptide (Gln-Leu-Glu-Gln) eliminated the ability of the subunit to interact stably with all of its available binding sites on photosystem II, as evidenced by the fact that this mutant was totally inactive in restoring O2 evolution activity . Evidence is presented to indicate that these mutational effects on the binding and function of the manganese stabilizing protein may be due to major changes in tertiary structure . The truncation mutations lacking either the C-terminal tri- or tetrapeptide exhibit apparent size increases of 25 and 40%, respectively, when compared either to a mutant lacking the C-terminal dipeptide or to the wild-type protein.

Biochemistry, 1998 Oct 6, 37(40), 14030 - 7
Identification of the binding surface on Cdc42Hs for p21-activated kinase; Guo W et al.; The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization . The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes . Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs) . We have determined previously the solution structure of Cdc42Hs {Feltham et al . (1997) Biochemistry 36, 8755-8766} using NMR spectroscopy . A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis . The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46 . A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I) . A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum . Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis . These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 {Nassar et al . (1995) Nature 375, 554-560)}.

Biochemistry, 1998 Oct 6, 37(40), 13958 - 67
Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure; Abdulaev NG et al.; The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated . The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2 . Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme . Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides . Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides . Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm . Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B) . Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase . The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s) . Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures . The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.

Nature, 1998 Sep 24, 395(6700), 347 - 53
Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution; Sutton RB et al.; The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown . Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B . The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins . Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex . Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices . These residues are highly conserved across the entire SNARE family . The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins . The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions . These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Eur J Biochem, 1998 Sep 1, 256(2), 453 - 60
Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli; Nardone E et al.; The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form . After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained . ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins . In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced . The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity . The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase . Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity . Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution . The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.

Eur J Biochem, 1998 Sep 1, 256(2), 447 - 52
Fusion of two subunits does not impair the function of a {NiFeSe}-hydrogenase in the archaeon Methanococcus voltae; Pfeiffer M et al.; {NiFe}-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit . The {NiFeSe}-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition . Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU . It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom . We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance . We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M . voltae by gene replacement . The enzyme was purified and shown to be as active as the wild-type enzyme . M . voltae carries the genetic information for four different {NiFe}-hydrogenases . In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified . Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively . Their gene groups, vhc and frc are transcribed only upon selenium depletion . The selenium-containing subunit VhuU has been implicated in their negative regulation . However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc andfrc promoter activities as tested in reporter gene constructs . This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.

Neurochem Int, 1998 Sep, 33(3), 271 - 5
Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins; Simonovic M et al.; A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed . For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E . coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST) . The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose . G alpha-subunits were expressed and purified as His-tagged proteins . For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein . G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay . It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins . Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein . The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.

J Immunol, 1998 Oct 1, 161(7), 3438 - 43
C4d DNA sequences of two infrequent human allotypes (C4A13 and C4B12) and the presence of signal sequences enhancing recombination; Martinez-Quiles N et al.; The DNA sequences of the polymorphic region (C4d) that belong to the infrequent complement C4 allotypes C4A13 and C4B12 have been obtained . In addition, C4A4 and C4B2 C4d sequences have been completed . C4A13 shows a new combination of amino acids at the following polymorphic positions: Asp1054, Pro1101 Cys1102, Leu1105, Asp1106, Asn1157, Ala1188, and Arg1191 . These amino acids conform to the antigenic determinants Chido 1 and Rodgers 3; thus C4A13 is the only allele described thus far that carries both Ags . C4A13 and C4A4 carry the motif "ggctc*" (* means "deletion") at positions 14 to 19 in their intron 28; this motif had previously been reported only in C4B alleles . The C4B12 nucleotide sequence is analogous to C4B1b and C4B3 sequences, except for codon 1076, which is GCC in C4B1b and C4B3 and GGA in C4B12, which is coding for glycine in both cases . A recombination model for the generation of C4 alleles is formulated based on the analysis of these new sequences . One recombination would take place between positions 1157 and 1186 and would give rise to C4A13 and C4B5 or C4A3 (or C4A6) and C4B2; another one would occur between positions 1054 and 1076 and would generate C4A3 (or C4A6) and C4B12 or C4A2 and C4Bnew . Analysis of 1157 to 1186 and 1054 to 1076 fragments reveals the presence of putative sequence signals for recombination (similar to Escherichia coli chi recombination signal); the accumulation of such signals in fragments 1054 to 1076 supports the notion that a recombination hot spot for the C4 gene may exist and it also enhances new allele generation and intraspecies C4 gene homogenization.

Thromb Haemost, 1998 Sep, 80(3), 423 - 7
Proteolysis of tissue factor pathway inhibitor (TFPI) by plasmin: effect on TFPI activity; Li A et al.; An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI) . TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway . Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plasminogen activators . In this study, the interaction of plasmin with recombinant E . coli-derived TFPI (rTFPI) was examined . Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities . Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257 . Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity . These changes were abolished by addition of aprotinin, an inhibitor of plasmin . These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated . The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.

Chin J Biotechnol, 1998, 14(1), 1 - 8
Construction of Escherichia coli-Streptomyces shuttle expression vectors for gene expression in Streptomyces; Yang R et al.; pIJ4123 and pIJ6021 are high-copy-number vectors for gene expression in Streptomyces . They contain a strong, thiostrepton-inducible promoter, PtipA . Two E . coli-Streptomyces shuttle vectors, pHZ1271 and pHZ1272, were constructed by inserting a replicon and bla gene from E . coli into pIJ4123 and pIJ6021, respectively . Both vectors were structurally stable either in E . coli or in Streptomyces lividans . To demonstrate the utility of pHZ1272, the hemoglobin gene of Vitreoscillia (vhb) was cloned into pHZ1272 and expressed in Streptomyces lividans . The expression product (VHb) was detected by Western blotting and carbon monoxide binding activity analysis.

Annu Rev Biochem, 1998, 67, 71 - 98
Ribonucleotide reductases; Jordan A et al.; Ribonucleotide reductases provide the building blocks for DNA replication in all living cells . Three different classes of enzymes use protein free radicals to activate the substrate . Aerobic class I enzymes generate a tyrosyl radical with an iron-oxygen center and dioxygen, class II enzymes employ adenosylcobalamin, and the anaerobic class III enzymes generate a glycyl radical from S-adenosylmethionine and an iron-sulfur cluster . The X-ray structure of the class I Escherichia coli enzyme, including forms that bind substrate and allosteric effectors, confirms previous models of catalytic and allosteric mechanisms . This structure suggests considerable mobility of the protein during catalysis and, together with experiments involving site-directed mutants, suggests a mechanism for radical transfer from one subunit to the other . Despite large differences between the classes, common catalytic and allosteric mechanisms, as well as retention of critical residues in the protein sequence, suggest a similar tertiary structure and a common origin during evolution . One puzzling aspect is that some organisms contain the genes for several different reductases.

Appl Environ Microbiol, 1998 Oct, 64(10), 4093 - 4
Substrate selectivity and biochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli; Pollard JR et al.; 4-Hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli was identified as a class I aldolase . The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept alpha-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.

Appl Environ Microbiol, 1998 Oct, 64(10), 4028 - 34
Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus; Penas MM et al.; Hydrophobins are small (length, about 100 +/- 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium . Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae . We identified and isolated a hydrophobin (fruit body hydrophobin 1 {Fbh1}) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus . In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1 . We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments . Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.

Appl Environ Microbiol, 1998 Oct, 64(10), 3724 - 30
Bias in template-to-product ratios in multitemplate PCR; Polz MF et al.; Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood . To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers . Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates . Variability between replicates also contributed to the observed bias but in a comparatively minor way . Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis . When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification . In contrast, gene copy number was found to be an unlikely cause of the observed bias . Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios . Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

Protein Expr Purif, 1998 Oct, 14(1), 146 - 52
Overexpression of functional hydrolase domain of rat liver 10-formyltetrahydrofolate dehydrogenase in Escherichia coli; Krupenko SA et al.; Rat liver 10-formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers . We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector . We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences . The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity . The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore) . The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture . Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E . coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells . The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC . These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E . coli cells and purified in one step by a simple chromatographic procedure .

Protein Expr Purif, 1998 Oct, 14(1), 139 - 45
Purification of active chloroplast sedoheptulose-1,7-bisphosphatase expressed in Escherichia coli; Dunford RP et al.; Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere . This makes SBPase an appropriate target for intensive study . We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag . The identity of the recombinant SBPases was confirmed by SDS-PAGE analysis and immunological detection with a specific antibody . Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography . Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity . Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts . The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography .

Protein Expr Purif, 1998 Oct, 14(1), 131 - 8
Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei; Dabrowski S et al.; Complete PCR-derived DNA fragments containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector . The clones expressing thermostable His-tagged DNA polymerases were selected . The cloned fragments were sequenced . The DNA sequences were verified to be authentic by sequencing several clones . The nucleotide (nt) sequence revealed that DNA polymerase of P . woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566 . It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P . furiosus (Pfu DNA polymerase) . The results confirm that nucleotide sequences of both archaeons (P . furiosus and P . woesei) are highly similar . The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-affinity chromatography . We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG . However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition . The proteins were purified by heat treatment (to denature E . coli proteins), followed by metal-affinity chromatography on Ni2+-Sepharose columns . The enzymes were characterized and displayed high DNA polymerase activity and thermostability . This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases .

Protein Expr Purif, 1998 Oct, 14(1), 104 - 12
Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nepsilon-methyltransferase; Zheng Q et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco . A pea (Pisum sativum L . cv Laxton's Progress No . 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body . Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity . However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37 . Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity . Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity . Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E . coli cell culture . The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts . However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification . The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco .

Protein Expr Purif, 1998 Oct, 14(1), 87 - 96
The chloroplast small heat shock protein--purification and characterization of pea recombinant protein; Harndahl U et al.; We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using an Escherichia coli expression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography . After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step . The purified recombinant protein had an isoelectric point of 5 . 0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment . This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens alpha-crystallin . The crystallins are known from both crystallographic and spectroscopic data to be all-beta proteins . In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% alpha-helical secondary structure .

Protein Expr Purif, 1998 Oct, 14(1), 79 - 86
A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein; Ruscetti T et al.; Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks . During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains . To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli . We modified expression to facilitate protein folding by including zinc and reducing the induction temperature . Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner . To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein . In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column . This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation . This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies .

Protein Expr Purif, 1998 Oct, 14(1), 65 - 70
Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose; Caldas TD et al.; The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively . Both possess cysteine residues that are important for activity . We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin . Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose . Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column . Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins . The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity . The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols .

Protein Expr Purif, 1998 Oct, 14(1), 54 - 64
Expression and purification of HIV-I p15NC protein in Escherichia coli; Ozturk DH et al.; An efficient method for the expression and purification of nucleocapsid precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed and used to obtain large quantities of this viral protein for structural studies, protein biochemistry, and high-throughput screening efforts . We have engineered an existing p15NC clone into a new vector developed at the University of Heidelberg, Germany . Using PCR, we introduced new restriction sites and a strong ribosome-binding site in the p15NC gene and expressed authentic p15NC protein . Our protocol enabled us to rapidly obtain soluble and highly stable p15NC expressed in Escherichia coli and to purify several milligrams of p15NC to homogeneity . In the current purification scheme, lysis of cell paste followed by a simple three-step FPLC procedure yields about 0.4-0.5 mg of purified p15NC per gram of E . coli cell paste expressing the protein with an overall yield of 45% . The purified p15NC retained its ability to bind full-length HIV-I p15NC mRNA in solution- or solid-phase-based assays . A specific stem-loop forming RNA fragment (24-mer) and its antisense DNA oligomer (21-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC . In addition, antisense DNA oligos with bulky 5-iodouracil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete with the full-length p15NC mRNA in filter-binding competition assays . In addition, RNA-dependent cleavage of the purified p15NC in vitro by HIV-I protease occurred at rates similar to those reported previously .

Protein Expr Purif, 1998 Oct, 14(1), 31 - 7
Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization; Xie Y et al.; Human retinol-binding protein (RBP) is a monomeric 21-kDa protein that is currently the subject of numerous studies owing to its role in the cellular uptake and utilization of retinol . When the RBP gene is overexpressed in Escherichia coli, inclusion bodies of aggregated RBP are found in the cells . These inclusion bodies are solubilized in 5.0 M GdmCl containing 10 mM DTT . Refolding of RBP is carried out in the presence of vitamin A by diluting denatured and reduced RBP into a redox refolding buffer consisting of 3 mM cysteine/0.3 mM cystine at 4 degreesC . Ion exchange chromatography (HPLC) is utilized to purify refolded RBP to homogeneity as demonstrated by SDS-PAGE and electrospray MS . The native structure of refolded RBP was established by its ability to bind to vitamin A and the plasma protein transthyretin . The reconstitution of RBP outlined within affords a 50-60% overall yield, i.e., 73 mg of pure RBP/L of E . coli culture .

Protein Expr Purif, 1998 Oct, 14(1), 1 - 7
High-level expression of branching enzyme II from maize endosperm in Escherichia coli; Libessart N et al.; The gene that encodes the mature branching enzyme II (BEII) protein from maize (Zea mays L.) endosperm was amplified by means of a polymerase chain reaction technique and inserted into a T7-based expression vector . Although this has been an efficient expression system of maize BEII in Escherichia coli, an example is presented in this report which allows a greater expression of mBEII protein from the bacterial system by changing only one codon . The key to the level of expression appears to be related to the conversion of the third thymine base in the 285 position codon of the mBEII cDNA to cytosine without altering the encoded mBEII protein product . The crude cell extracts of enzyme prepared from E.coli exhibited seven-fold higher expression of branching enzyme activity compared to expression of the native enzyme . The enzymes from wild-type and the silent mutation genes were purified . The proteins were indistinguishable kinetically and immunologically . Thus, we obtained a significantly improved expression of mBEII protein in the bacterial system .

J Surg Res, 1998 Oct, 79(2), 179 - 84
Interleukin 10 inhibits alveolar macrophage production of inflammatory mediators involved in adult respiratory distress syndrome; Lo CJ et al.; BACKGROUND: Adult respiratory distress syndrome (ARDS) causes severe morbidity and mortality in trauma patients . One potential method to attenuate the lung injury is to inhibit alveolar macrophage production of proinflammatory mediators . The purpose of this study was to investigate the cellular mechanism of interleukin 10 (IL-10) inhibition on LPS-stimulated macrophage (Mphi) . We hypothesized that IL-10 inhibited phospholipase C signal pathways in Mphi . IL-10 inhibition would be restored by calcium ionophores and protein kinase C (PKC) activation . METHODS: Rabbit alveolar Mphi were obtained by bronchoalveolar lavage . Mphi were treated with Escherichia coli LPS (10 ng/ml) in the presence of various concentrations of human IL-10 . Cell lysates and supernatant were analyzed for proagulants (PCA) and tumor necrosis factor (TNF), respectively . TNF mRNA expression of alveolar Mphi was also measured by Northern Blot assay . Macrophage PGE2 production was measured by ELISA . RESULTS: IL-10 inhibited the production of both TNF and PCA by LPS-stimulated Mphi . In addition, IL-10 also reduced TNF mRNA expression . Similarly, PGE2 production by LPS-stimulated Mphi was also attenuated by IL-10 . An increase in the intracellular {Ca2+} induced by A23187 failed to reverse this IL-10-mediated inhibition . In comparison, phorbol myristate acetate, a protein kinase C (PKC) activator, restored TNF and PCA production despite the presence of IL-10 . CONCLUSIONS: IL-10 inhibits Mphi production of inflammatory mediators . This inhibition is, at least in part, mediated by modulating the PKC activity .

Am J Hum Genet, 1998 Oct, 63(4), 1049 - 59
Adenosine deaminase deficiency: genotype-phenotype correlations based on expressed activity of 29 mutant alleles; Arredondo-Vega FX et al.; Adenosine deaminase (ADA) deficiency causes lymphopenia and immunodeficiency due to toxic effects of its substrates . Most patients are infants with severe combined immunodeficiency disease (SCID), but others are diagnosed later in childhood (delayed onset) or as adults (late onset); healthy individuals with "partial" ADA deficiency have been identified . More than 50 ADA mutations are known; most patients are heteroallelic, and most alleles are rare . To analyze the relationship of genotype to phenotype, we quantitated the expression of 29 amino acid sequence-altering alleles in the ADA-deleted Escherichia coli strain SO3834 . Expressed ADA activity of wild-type and mutant alleles ranged over five orders of magnitude . The 26 disease-associated alleles expressed 0.001%-0.6% of wild-type activity, versus 5%-28% for 3 alleles from "partials." We related these data to the clinical phenotypes and erythrocyte deoxyadenosine nucleotide (dAXP) levels of 52 patients (49 immunodeficient and 3 with partial deficiency) who had 43 genotypes derived from 42 different mutations, including 28 of the expressed alleles . We reduced this complexity to 13 "genotype categories," ranked according to the potential of their constituent alleles to provide ADA activity . Of 31 SCID patients, 28 fell into 3 genotype categories that could express <=0.05% of wild-type ADA activity . Only 2 of 21 patients with delayed, late-onset, or partial phenotypes had one of these "severe" genotypes . Among 37 patients for whom pretreatment metabolic data were available, we found a strong inverse correlation between red-cell dAXP level and total ADA activity expressed by each patient's alleles in SO3834 . Our system provides a quantitative framework and ranking system for relating genotype to phenotype.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1046 - 8
Expression, purification, crystallization and preliminary X-ray analysis of cyclophilin A from the bovine parasite Trypanosoma brucei brucei; Dao-Thi MH et al.; Cyclophilin A from the bovine parasite Trypanosoma brucei brucei has been cloned, expressed in Escherichia coli, purified and crystallized in the presence of cyclosporin A using ammonium sulfate as a precipitant . The crystals belong to the orthorhombic crystal system with unit-cell dimensions of a = 118.61, b = 210.15 and c = 153.21 A . A data set complete to 2.7 A has been collected using rotating-anode radiation, however the crystals diffract to at least 2.1 A resolution using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1020 - 2
Crystallization and preliminary X-ray analysis of the beta-isoform of glutamate decarboxylase from Escherichia coli; Malashkevich VN et al.; Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the alpha-decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA) . Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADalpha and GADbeta . Well diffracting crystals of GADbeta were obtained by taking advantage of the possibility of expressing each isoform separately . They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 A and contain one GAD hexamer in the asymmetric unit . High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1017 - 9
Crystallization and preliminary X-ray diffraction studies on the water soluble form of rat heme oxygenase-1 in complex with heme; Omata Y et al.; The water-soluble portion of rat heme oxygenase-1 which lacks 22 hydrophobic amino-acid residues at the C-terminus was expressed in E . coli and crystallized in the form of a complex with heme by the vapor-diffusion method using polyethylene glycol 4000 as the precipitant . The crystals belong to the tetragonal space group P41212 or P43212, with unit-cell dimensions of a = b = 56.7, c = 186 . 7 A . The crystal contains one heme-heme oxygenase-1 complex in an asymmetric unit and diffracts X-rays beyond 3.0 A resolution with a conventional X-ray source.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 996 - 8
Crystallization and preliminary X-ray analysis of Escherichia coli GlnK; MacPherson KH et al.; The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized . The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit . The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A . These crystals have a single subunit in the asymmetric unit . High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 986 - 8
Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase; Goldman S et al.; DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme . The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method . The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees . The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 975 - 81
Crystallization of ccdB; Dao-Thi MH et al.; CcdB is a small dimeric protein that poisons DNA-topoisomerase II complexes . Its crystallization properties in terms of precipitant type, precipitant concentration, pH and protein concentration have been investigated leading to a novel crystal form which, in contrast to previously reported crystals, is suitable for structure determination using the multiple isomorphous replacement (MIR) method . The space group of this new form is C2, with unit-cell parameters a = 74.94, b = 36.24, c = 35.77 A, beta = 115.27 degrees . The asymmetric unit contains a single monomer . Flash-frozen crystals diffract to at least 1.5 A resolution, while room-temperature diffraction can be observed up to 1.6 A . The double mutant S74C/G77Q, which acts as a super-killer, crystallizes in space group I222 (or I212121) with unit-cell dimensions a = 105.58, b = 105.80, c = 91.90 A . These crystals diffract to 2.5 A resolution.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 735 - 49
The refined structure of dUTPase from Escherichia coli; Dauter Z et al.; Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C . 3.6 . 1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis . A crystal of the recombinant E . coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A . Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution . The resulting 2.2 A MIRAS phases differed from the final set by 40 degrees on average and produced an excellent map which was easy to interpret . The model contains 132 water molecules and refined to an R value of 13.7% . 136 residues have clear electron density out of 152 expected from the gene sequence . The 16 C-terminal residues are presumably disordered in the crystal lattice . The monomer is a 'jelly-roll' type, containing mostly beta-sheet and only one short helix . The molecule is a tight trimer . A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its beta-sheet . Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the beta-sheet and in loop regions . A similar molecular architecture has recently been found in two other trimeric dUTPases.

Am J Obstet Gynecol, 1998 Sep, 179(3 Pt 1), 750 - 5
Evidence for mechanisms of the acute-phase response to endotoxin in late-gestation fetal goats; Yoneyama Y et al.; OBJECTIVE: The aim of this study was to evaluate the relationship between febrile response to fetal endotoxin administration and fetal plasma endogenous pyrogen, tumor necrosis factor-alpha, the possible putative pyrogen mediator prostaglandin E2, and the endogenous antipyretic arginine vasopressin in late-gestation pregnant goats . STUDY DESIGN: Changes in fetal core temperature, plasma tumor necrosis factor-alpha, prostaglandin E2, and arginine vasopressin levels were measured after administration of Escherichia coli endotoxin (70 microg/kg of fetal weight) to 10 fetal goats in late gestation . RESULTS: Fetal body temperature did not rise after endotoxin administration . Fetal plasma tumor necrosis factor-alpha and arginine vasopressin increased to 87.5 +/- 15.2 pg/mL and 25.1 +/- 4.8 pg/mL, respectively, after 1 to 2 hours (P < .05) . Fetal plasma prostaglandin E2 levels did not change significantly throughout the study . CONCLUSION: The absence of a febrile response to endotoxin in late-gestation fetal goats is accompanied by a deficient responses in prostaglandin generation in the periphery and increased activity of the antipyrogen arginine vasopressin.

Trends Biochem Sci, 1998 Aug, 23(8), 277 - 81
A unified DNA- and dNTP-binding mode for DNA polymerases; Singh K et al.; Crystal structures of various DNA polymerases show a common structural topology that resembles a right hand and has distinct finger, palm and thumb subdomains . Early models of the klenow fragment (KF) of Escherichia coli polymerase I showed DNA entering a large cleft that faces the palm subdomain where the catalytic site is situated1,2 . However, subsequent resolution of the structures of HIV-1 reverse transcriptase, KF and polymerase beta (pol beta) bound to DNA3-5 yielded conflicting data that suggested a different orientation for DNA bound to pol beta compared with DNA bound to other polymerases . The debate, on the correct orientation of the template-primer DNA, that followed failed to reach a consensus . Using an alternative superposition scheme, we now provide convincing evidence for a common DNA-binding mode that is applicable to all polymerases, including pol beta.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1594 - 6
Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis; Noguchi T et al.; The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)) . It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs) . We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides . It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis . This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1528 - 34
Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other derivatives; Yamasaki M et al.; Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter . A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo . The purification involved a sequential renaturation process and three-step chromatography . Refolding succeeded in very high yield using a urea system . The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis . According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations . This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF . KW-2228 ({Ala1, Thr3, Tyr4, Arg5, and Ser17}-hG-CSF) and derivative A ({Ala1, Thr3, Tyr4, Arg5}-hG-CSF) are easily crystallized and they show similar in vitro activity . On the other hand, neither rhG-CSF nor derivative B ({Ser17}-hG-CSF) are crystallized under the same conditions . Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization . The change of Cys17 to Ser may not influence the stability and activity of hG-CSF.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1515 - 21
Construction of a BAC library of the rice blast fungus Magnaporthe grisea and finding specific genome regions in which its transposons tend to cluster; Nishimura M et al.; We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones . The insert size ranged from 35 to 175 kbp, with an average of 120 kbp . The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome . This library is the first to be constructed using a rice pathogenic wild type isolate . Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency . Total library clones were arranged onto two nylon membranes for efficient screening . Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions . As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed . Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively . The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number . The results show that these transposons were distributed in clusters in the M . grisea genome.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1498 - 503
Lipoxygenase-1 from soybean seed inhibiting the activity of pancreatic lipase; Satouchi K et al.; There are some similar characteristics in protein nature between the lipase inhibitor from soybean seed and soybean lipoxygenase-1 (LOX-1) . Thus, the inhibiting protein for pancreatic lipase was prepared from defatted soybean meal by the procedure for the isolation of LOX-1 {Axelrod et al., Methods in Enzymology, 71, 441-451 (1981)} . The LOX-1 from soybean seed dose-dependently inhibited the release of fatty acid from a soybean oil emulsion, and the concentration of LOX-1 to cause half inhibition of the lipase activity was 3.2 x 10(-7) M . The LOX-1 obtained from E . coli transfected with a plasmid carrying the soybean LOX-1 gene also inhibited the lipase activity . However, the lipase-inhibiting activity by the LOX-1 was not affected by the presence of nordihydroguaiaretic acid, an inhibitor for LOX, in the reaction mixture . These results show that the soybean LOX-1 inhibits lipase activity regardless of its lipoxygenase activity.

No To Shinkei, 1998 Aug, 50(8), 731 - 8
{Gene transduction for experimental brain tumors using recombinant adenovirus vector}; Ichikawa T et al.; Recent advances in molecular biology have permitted significant progress toward the treatment of malignant brain tumors using gene transduction methods . Adenovirus vectors have recently been shown to transduce genes successfully into brain tumor cells both in vitro and in vivo . We have investigated the feasibility of gene transduction for brain tumors using adenovirus vectors . To evaluate in vitro transduction rate by adenovirus vectors, rat 9L gliosarcoma cells or human glioblastoma cells were infected with recombinant replication-deficient adenovirus vectors containing the E . coli beta-galactosidase gene (Adex-CALacZ) and stained with X-Gal . We observed a multiplicity of infection (MOI)-dependent rate . Approximately 100% transgene expression was achieved at a MOI of 5 after seven days of incubation . To evaluate transgene expression in a rat brain tumor model, AdexCALacZ was stereotactically injected into established rat 9L brain tumors . Intratumoral injection of AdexCALacZ resulted in high transgene expression in tumor cells . Although injection of AdexCALacZ in the normal basal ganglia resulted in broad and diffuse transduction into endogenous neural cells, direct intratumoral injection resulted in transduction that was relatively restricted to the tumor cells as well as some neighboring normal cells . Transduction rates were relatively elevated at the margin of the tumor . Our results suggest that adenovirus vectors might be a feasible method to transfer therapeutic genes into malignant brain tumors.

Mutat Res, 1998 Oct 12, 418(2-3), 121 - 9
Genotoxicity tests for new chemicals in Germany: routine in vitro test systems; Broschinski L et al.; According to regulations in the European Union, new chemical substances must be notified before they can be introduced onto the market . One of the prerequisites for notification is that toxicological properties, including mutagenicity, are examined . In this paper, a report on routine in vitro mutagenicity testing is given for 776 new substances notified in Germany between 1982 and 1997 . In general, the methodological quality of testing was in line with internationally accepted guidelines . Bacterial gene mutation tests (Bact) were conducted for nearly all of the substances, 13.4% were positive . Of the Bact-positive substances, 36 were also tested in the in vitro chromosomal aberration test (CAbvit) and the mammalian cell gene mutation test (MCGM) . Twenty-six of these (72 . 2%) were negative in both mammalian cell tests indicating that the genotoxic potentials of the substances are not relevant for man . Of all new substances, 333 were tested in CAbvit, here the percentage of positive findings was 25.2% . More than 80% of the in vitro clastogens were negative in the Bact . With respect to a sensitive detection of genotoxic potentials of substances, the combination 'Bact+CAbvit' is appropriate for basic testing . In our database CHL cells were more sensitive to clastogenic effects than other cell types . Only very few clastogens were identified as 'high toxicity clastogens' . MCGM tests were performed for 118 substances, quite often as follow-up in case of positive Bact tests . In total, 12.7% of the substances were positive in the MCGM . However, there was a clear difference in the frequencies of positive findings in HPRT tests (5.5%) and mouse lymphoma assays (MLA; 37.0%) . None of the MCGM-positive substances was a 'unique positive', i.e., negative in Bact and CAbvit .

J Biol Chem, 1998 Oct 9, 273(41), 26705 - 13
A pathway where polyprenyl diphosphate elongates in prenyltransferase . Insight into a common mechanism of chain length determination of prenyltransferases; Ohnuma S et al.; Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates . Each enzyme forms the final product with a specific chain length . The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme . To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase . The side chains of these amino acids are situated on the same side of an alpha-helix . In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T . (1996) J . Biol . Chem . 271, 18831-18837) . A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40 . A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40 compound with C35 and C45 . Mutated farnesyl-diphosphate synthases also show similar patterns . These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases . Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.

J Biol Chem, 1998 Oct 9, 273(41), 26670 - 4
Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells; Ajouz B et al.; Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with the highest conductance, which is activated at the highest membrane tension, has been cloned . We investigated the putative involvement of MscL in the effluxes caused by osmotic downshock . Osmotic shock caused the release of potassium glutamate, trehalose, and glycine betaine from wild type cells and cells lacking MscL . There was no difference between the two strains, but the extreme rapidity of the efflux process, as shown herein for glycine betaine, suggests that it is channel-mediated . Osmotic downshock also induces the release of some cytosolic proteins from EDTA-treated cells . We investigated the release of thioredoxin . This protein was totally released from wild type cells but was retained by MscL- cells . Release was restored by expression of the gene coding for MscL . Thus MscL is not necessary for the excretion of osmoprotectants, but it does open in vivo during shock and catalyzes the efflux of thioredoxin and possibly other small cytosolic proteins . It follows that the other mechanosensitive channels, which are known to be activated at lower tension, must also open during osmotic shock . Their opening and that of MscL could account for the rapid release of osmolytes.

J Biol Chem, 1998 Oct 9, 273(41), 26645 - 51
Solution structure of the epsilon subunit of the F1-ATPase from Escherichia coli and interactions of this subunit with beta subunits in the complex; Wilkens S et al.; The solution structure of the epsilon subunit of the Escherichia coli F1-ATPase has been determined by NMR spectroscopy . This subunit has a two-domain structure with an N-terminal 10-stranded beta sandwich and a C-terminal antiparallel two alpha-helix hairpin, as described previously (Wilkens, S., Dahlquist, F . W., McIntosh, L . P., Donaldson, L . W., and Capaldi, R . A . (1995) Nat . Struct . Biol . 2, 961-967) . New data show that the two domains interact in solution in an interface formed by beta strand 7 and the very C-terminal alpha-helix . This interface involves only hydrophobic interactions . The dynamics of the epsilon subunit in solution were examined . The two domains are relatively tightly associated with little or no flexibility relative to one another . The epsilon subunit can exist in two states in the ECF1F0 complex depending on whether ATP or ADP occupies catalytic sites . Proteolysis of the epsilon subunit in solution and when bound to the core F1 complex indicates that the conformation of the polypeptide in solution closely resembles the conformation of epsilon when bound to the F1 in the ADP state . Chemical and photo-cross-linking show that the epsilon subunit spans and interacts with two beta subunits in the ADP state . These interactions are disrupted on binding of ATP + Mg2+, as is the interaction between the N- and C-terminal domains of the epsilon subunit.

J Biol Chem, 1998 Oct 9, 273(41), 26618 - 23
Molecular analysis of two pyruvate dehydrogenase kinases from maize; Thelen JJ et al.; Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases . Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases . Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases . In addition, specific Trp and Cys residues are also invariant in these sequences . The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity . Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms . PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification . The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis . Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa . Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from {gamma-32P}ATP into the alpha subunit of pyruvate dehydrogenase.

J Biol Chem, 1998 Oct 9, 273(41), 26543 - 8
Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 domain; Pacheco-Rodriguez G et al.; ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins that require specific guanine nucleotide-exchange proteins (GEPs) to accelerate the conversion of inactive ARF-GDP to active ARF-GTP . Cytohesin-1, a 46-kDa ARF GEP, contains a central Sec7 domain of 188 amino acids similar in sequence to a region of the yeast Sec7 protein . Cytohesin-1 and its 22-kDa Sec7 domain (C-1 Sec7), synthesized in Escherichia coli, were assayed with recombinant non-myristoylated ARFs and related proteins to compare their GEP activities . Both were effective with native mammalian ARFs 1 and 3 . Cytohesin-1 accelerated GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) binding to recombinant human ARF1 (rARF1), yeast ARF3, and ARD1 (a 64-kDa guanine nucleotide-binding protein containing a C-terminal ARF domain) . In contrast, C-1 Sec7 enhanced GTPgammaS binding to recombinant human ARFs 1, 5, and 6; yeast ARFs 1, 2, and 3; ARD1; two ARD1 mutants that contain the ARF domain; and Delta13ARF1, which lacks the N-terminal alpha-helix . Neither C-1 Sec7 nor cytohesin-1 increased GTPgammaS binding to human ARF-like ARL proteins 1, 2, and 3 . Thus, ARLs, initially differentiated from ARFs because of their inability to activate cholera toxin, differ also in their failure to interact functionally with C-1 Sec7 or cytohesin-1 . As C-1 Sec7 was much less substrate-specific than cytohesin-1, it appears that structure outside of the Sec7 domain is important for ARF specificity . Data obtained with mutant ARF constructs are all consistent with the conclusion that the ARF N terminus is an important determinant of cytohesin-1 specificity.

J Biol Chem, 1998 Oct 9, 273(41), 26528 - 33
Constitutive and adaptive detoxification of nitric oxide in Escherichia coli . Role of nitric-oxide dioxygenase in the protection of aconitase; Gardner PR et al.; Nitric oxide (NO.) is a naturally occurring toxin that some organisms adaptively resist . In aerobic or anaerobic Escherichia coli, low levels of NO . exposure inactivated the NO.-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO.-defensive proteins was blocked with chloramphenicol . Protection of aconitase in aerobically grown E . coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO . consumption . Constitutive and adaptive cellular NO . consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide . Exposure of aerobic cells to NO . accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO . dioxygenase (NOD) . Anaerobic E . coli exposed to NO . or nitrate induced a modest O2-independent and cyanide-resistant NO.-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity . The latter activity was attributed to NOD . The results support a role for NOD in the aerobic detoxification of NO . and suggest functions for NOD and a cyanide-resistant NO . scavenging activity in anaerobic cells.

J Biol Chem, 1998 Oct 9, 273(41), 26477 - 86
Sequential hydrolysis of ATP molecules bound in interacting catalytic sites of Escherichia coli transcription termination protein Rho; Stitt BL et al.; Escherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle . Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites . In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous . A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s-1, followed by steady-state hydrolysis at 30 s-1 per hexamer . The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis . The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis . Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating . These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features.

J Biol Chem, 1998 Oct 9, 273(41), 26470 - 6
A point mutation (G338S) and its suppressor mutations affect both the pH response of the NhaA-Na+/H+ antiporter as well as the growth phenotype of Escherichia coli; Rimon A et al.; pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli . In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter . Monitoring Na+-dependent collapse of DeltapH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active . Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22Na from proteoliposomes as well as DeltapH-driven influx . Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5 . DeltanhaA DeltanhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in the presence of both ions at neutral pH . Several possibilities that could explain the Na+ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity . The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only Deltapsi exists in the cells . The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type . Therefore an increase in intracellular Na+ due to a reduced antiporter affinity could not explain the results . It is suggested that the loss of growth at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA . Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA . Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.

J Biol Chem, 1998 Oct 9, 273(41), 26415 - 20
Individual substitutions of clustered arginine residues of the sensor kinase KdpD of Escherichia coli modulate the ratio of kinase to phosphatase activity; Jung K et al.; Escherichia coli responds to K+ limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase . KdpD, the sensor kinase of this system, is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE . Here we demonstrate that individual replacements of clustered arginine residues located close to transmembrane domain TM4 modulate the ratio of kinase to phosphatase activity . Thus KdpD-Arg511 --> Gln is characterized by an increase in the kinase activity and a loss of the phosphatase activity . However, when Arg at position 511 is replaced with Lys, activities of the corresponding protein are comparable with wild-type KdpD . In contrast, replacement of arginine residues at positions 503, 506, or 508 with glutamine or lysine causes a decrease of the kinase and an increase of the phosphatase activities . Changes of the activities of these KdpD proteins correspond with alterations in kdpFABC expression . Thus KdpD-Arg511 --> Gln causes constitutive expression of kdpFABC . KdpD proteins with Arg replacements at positions 503, 506, or 508 are unable to respond to osmolarity, whereas the sensing of K+ limitation is not influenced . Simultaneous replacement of arginine residues 508 and 511 or 506, 508, and 511 with glutamine leads to a decrease of the phosphatase activity . However, kdpFABC expression is dependent on K+ and osmolarity . Finally, when Arg513 is replaced with glutamine the amount of KdpD detected in the membrane is drastically reduced . These results imply that there is an equilibrium between the kinase and phosphatase activities of KdpD, which can be shifted by the replacement of one arginine residue . An electrostatic switch mechanism within the protein is proposed through which the ratio of kinase to phosphatase is regulated . Finally, these results lend support to the notion that KdpD can be activated by two distinct stimuli, K+ limitation and osmolarity.

J Biol Chem, 1998 Oct 9, 273(41), 26400 - 7
Topology of the Na+/proline transporter of Escherichia coli; Jung H et al.; Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y . (1987) Mol . Gen . Genet . 208, 70-75) . We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis . Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model . Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results . As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated . Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed . Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells . In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells . These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm . In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus . Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9) . These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2 . Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm . The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.

J Biol Chem, 1998 Oct 9, 273(41), 26394 - 9
Potential of Escherichia coli GTP cyclohydrolase II for hydrolyzing 8-oxo-dGTP, a mutagenic substrate for DNA synthesis; Kobayashi M et al.; MutT protein of Escherichia coli prevents the occurrence of A:T --> C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), which is produced by active oxygen species . In a search for mutT-related genes, we found that the ribA gene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced . Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively . dGTP was not hydrolyzed by these preparations . GTP cyclohydrolase II catalyzes conversion of GTP to 2, 5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis . The Km values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same . In the mutT- background, ribA- cells showed higher spontaneous mutation frequencies as compared with that of ribA+ cells . Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.

J Biol Chem, 1998 Oct 9, 273(41), 26349 - 60
Structural analysis of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha; Oh JI et al.; The fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase of Ralstonia eutropha H16 was cloned and sequenced . Sequence comparisons indicated a high resemblance of FdsA (alpha-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor . The NH2-terminal region (residues 1-240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia coli as well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases . FdsB (beta-subunit) and FdsG (gamma-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA . It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases . No significant similarity to any known protein was observed for FdsD (delta-subunit) . The predicted product of fdsC showed the highest resemblance to FdhD from E . coli, a protein required for the formation of active formate dehydrogenases in this organism . Transcription of the fds operon is subject to formate induction . A promoter structure resembling the consensus sequence of sigma70-dependent promoters from E . coli was identified upstream of the transcriptional start site determined by primer extension analysis.

Antimicrob Agents Chemother, 1998 Oct, 42(10), 2620 - 5
An Escherichia coli system expressing human deoxyribonucleoside salvage enzymes for evaluation of potential antiproliferative nucleoside analogs; Wang J et al.; Deoxyribonucleoside salvage in animal cells is mainly dependent on two cytosolic enzymes, thymidine kinase (TK1) and deoxycytidine kinase (dCK), while Escherichia coli expresses only one type of deoxynucleoside kinase, i.e., TK . A bacterial whole-cell system based on genetically modified E . coli was developed in which the relevant bacterial deoxypyrimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the lac promoter was introduced . The TK level in extract from induced bacteria with cDNA for human TK1 was found to be 20,000-fold higher than that in the parental strain, and for the strain with human dCK, the enzyme activity was 160-fold higher . The in vivo incorporation of deoxythymidine (Thd) and deoxycytidine (dCyd) into bacterial DNA by the two recombinant strains was 20 and 40 times higher, respectively, than that of the parental cells . A number of nucleoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluoro-dCyd, and several 5-halogenated deoxyuridine analogs, were tested with the bacterial system, as well as with human T-lymphoblast CEM cells . The results showed a close correlation between the inhibitory effects of several important cytostatic and antiviral analogs on the recombinant bacteria and the cellular system . Thus, E . coli expressing human salvage kinases is a rapid and convenient model system which may complement other screening methods in drug discovery projects.

Fungal Genet Biol, 1998 Aug, 24(3), 335 - 44
In vivo addition of telomeric repeats to foreign DNA generates extrachromosomal DNAs in the taxol-producing fungus Pestalotiopsis microspora; Long DM et al.; Transformation of the taxol-producing filamentous fungus Pestalotiopsis microspora with a plasmid containing the bacterial hygromycin resistance gene fused to Aspergillus regulatory sequences resulted in the in vivo formation of extrachromosomal DNAs with telomeric repeats in the majority of transformants . Repeats of the telomeric sequence 5'-TTAGGG-3' were appended to nontelomeric transforming DNA termini . No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs . Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs . The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection . Transformation of wild-type P . microspora with a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector . The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications .

J Biochem (Tokyo), 1998 Oct, 124(4), 842 - 7
Single-step purification and characterization of MBP (maltose binding protein)-DnaJ fusion protein and its utilization for structure-function analysis; Ishii Y et al.; DnaJ is a molecular chaperone, which contains a zinc finger-like motif and cooperates with DnaK to mediate the folding of newly synthesized and denatured proteins . DnaJ was overproduced and purified using the maltose binding protein (MBP) fusion vector . The fusion protein (MBP-DnaJ) was expressed in a soluble form in Escherichia coli and purified to homogeneity using amylose resin in a single step . The UV-visible absorption spectrum of MBP-DnaJ showed peaks at 355 and 475 nm . Moreover, these absorption peaks disappeared upon treatment with ethylenediaminetetraacetic acid (EDTA) or p-hydroxymercuriphenylsulfonic acid (PMPS) . Inductively coupled plasma (ICP) spectrometry demonstrated that MBP-DnaJ contains Fe ions as well as Zn ions . MBP-DnaJ mediated the replication of the lambda phage in vivo, stimulated the ATPase activity of DnaK and prevented the aggregation of denatured rhodanase, indicating that fusion of MBP to the N-terminal of DnaJ does not affect the functions of DnaJ . To study the roles of bound metal ions, metal-free MBP-DnaJ, and MBP-DnaJ containing 2 Zn ions were prepared . MBP-DnaJ containing Fe and Zn ions, and MBP-DnaJ containing 2 Zn ions stimulated the ATPase activity of DnaK, prevented the aggregation of denatured rhodanase and bound to DNA to similar extents . On the other hand, metal-free MBP-DnaJ showed much lower DNA-binding ability and lower ability to prevent rhodanese aggregation . Therefore, the bound metal species do not affect the function of the zinc finger-like motif of DnaJ, whereas removal of the metal ions from DnaJ diminishes its binding ability as to DNA and denatured proteins.

J Biochem (Tokyo), 1998 Oct, 124(4), 835 - 41
Protection of mammalian cells from the toxicity of bleomycin by expression of a bleomycin-binding protein gene from Streptomyces verticillus; Kumagai T et al.; A gene, blmA, encodes a bleomycin (Bm)-binding protein, designated BLMA, from Bm-producing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli cells . In the present study, by transfection of the gene into COS-1 cells with a plasmid designated pEF-BOS/blmA, which contains a strong promoter from the human polypeptide chain elongation factor 1alpha, we transiently overproduced BLMA at a high level of approximately 4% of the whole cell protein . Although NIH/3T3 cells transfected with pEF-BOS/blmA, designated NIH/3T3-BR cells, stably expressed BLMA, its expression level was about 0.1% of the total protein . Using an anti-BLMA monoclonal antibody reported previously {Sugiyama et al . (1995) FEBS Lett . 362, 80-84}, we revealed that BLMA is localized in the nucleus of pEF-BOS/blmA-transfected COS-1 and NIH/3T3-BR cells . Semi-permeabilized nuclear transport experiments showed that BLMA penetrates the nuclear envelope by energy- and transporter-independent passive diffusion, suggesting that the karyophilic nature of BLMA may be due to the acidic nature of the protein . NIH/3T3-BR cells were 130-fold more resistant to Bm than the host cells . NIH/3T3 cells exhibited a swollen nuclear envelope and a malformed spindle body and overexpressed at least 4 kinds of stress proteins including calreticulin and mitochondrial matrix protein P1 when exposed to 25 microg/ml of Bm, whereas NIH/3T3-BR cells grew without morphological alteration and expressed no stress proteins under the same conditions . Furthermore, reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression of interleukin-6, an inflammatory cytokine, is activated by addition of Bm in NIH/3T3 cells, but not in the NIH/3T3-BR cells . These results suggest that BLMA contributes to protection of mammalian cells from the inflammatory effect of Bm.

J Biochem (Tokyo), 1998 Oct, 124(4), 816 - 23
Activities of mutant Sar1 proteins in guanine nucleotide binding, GTP hydrolysis, and cell-free transport from the endoplasmic reticulum to the Golgi apparatus; Saito Y et al.; Sar1p belongs to a unique subfamily of the small GTPase superfamily and is essential for the formation of vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus . We have obtained mutants of the yeast SAR1 gene, which show several different phenotypes in cell growth and protein transport {Nakano, A . , Otsuka, H., Yamagishi, M., Yamamoto, E., Kimura, K., Nishikawa, S., and Oka, T . (1994) J . Biochem . 116, 243-247; Yamanushi, T., Hirata, A., Oka, T., and Nakano, A . (1996) ibid . 120, 452-458} . In this study, we have purified five mutant Sar1 proteins using an Escherichia coli expression system and characterized their biochemical properties in detail . Three of them prefer GDP binding to GTP binding and are thus regarded as GDP-form mutants, and one is insensitive to the GTPase-activating protein and is almost fixed in the GTP-bound state . The GDP mutants are defective in vesicle formation in vitro, whereas the GTP mutant can drive vesicle formation but not the overall transport to the Golgi . These mutants will be useful for further understanding of the regulation of the GTPase cycle of Sar1p.

J Biochem (Tokyo), 1998 Oct, 124(4), 769 - 77
Topological mutation of Escherichia coli dihydrofolate reductase; Iwakura M et al.; Proteins appear to contain structural elements which determine the folded structure . If such elements are present, the order of structural elements in the primary structure, i.e . the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers . To experimentally examine this, "topological" mutants (designated as GHF33 and GHF34) of Escherichia coli dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the betaF strand and betaG-betaH strands in the primary sequence . In this way, the chain topology of wild-type DHFR, betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaF-->betaG-->betaH, was changed to betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaG-->betaH-->betaF . Such topological mutant proteins were stably expressed and accumulated in E . coli cells, and highly purified . Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence . In terms of kcat, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with Km values of 3.3 microM (GHF33) and 2.1 microM (GHF34), respectively . The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with DeltaGH2O values of 4.0 and 4.8 kcal/mol . Whereas, the Km and DeltaGH2O values for wild-type DHFR were 0.9 microM and 6.1 kcal/mol, respectively . The significance of the topological mutations was discussed with respect to protein folding and protein evolution.

J Biochem (Tokyo), 1998 Oct, 124(4), 694 - 6
CYP51-like gene of Mycobacterium tuberculosis actually encodes a P450 similar to eukaryotic CYP51; Aoyama Y et al.; A CYP51-like gene (Z80226) of Mycobacterium tuberculosis was expressed in Escherichia coli . The product exhibited absorption spectra characteristic of P450 . The expressed P450 formed a stoichiometric complex with ketoconazole, one of the specific ligands of CYP51 . These findings indicate that the CYP51-like gene of M . tuberculosis actually encodes a P450 having active-site environments similar to those of CYP51, confirming the predicted orthologous nature of this gene to eukaryotic CYP51 . Although eukaryotic CYP51s are membrane-binding proteins, the expressed product was accumulated only in the soluble fraction of the host cells.

Am J Physiol, 1998 Oct, 275(4 Pt 1), G862 - 7
A choline-rich diet improves survival in a rat model of endotoxin shock; Rivera CA et al.; This study investigated whether dietary choline can prevent endotoxin shock . Female Sprague-Dawley rats fed chow or chow plus choline chloride (0.025-0.4%) for 3 days were given lipopolysaccharide (LPS) via the tail vein . Eighty-three percent and 56% of chow-fed rats survived after 2.5 or 5.0 mg/kg LPS, respectively . Choline increased survival in a dose-dependent manner, with maximal effects observed at 0.4%; this dose of choline prevented mortality completely after 2.5 or 5 mg/kg LPS . Choline also improved the microscopic appearance of the lungs and blunted increases in serum aspartate aminotransferase levels . Intracellular Ca2+ was monitored in liver and lung macrophages during LPS exposure . Ca2+ increases in macrophages from choline-fed rats were blunted by 40-60% compared with chow-fed controls . Feeding choline also blunted tumor necrosis factor-alpha production . Feeding glycine, which prevents macrophage activation via a chloride channel, in addition to choline was even more effective than feeding choline alone, suggesting that glycine and choline act via distinct sites . These data are consistent with the hypothesis that choline diminishes endotoxin shock by preventing macrophage activation.

Biotechnol Appl Biochem, 1998 Oct, 28 ( Pt 2), 163 - 7
Expression of recombinant Lym-1 single-chain Fv in Escherichia coli; Bin Song K et al.; Lym-1 single-chain Fv (sFv) can be used for targeted radiodiagnosis and therapy of B-lymphocytic malignancies . Lym-1 sFv was constructed and expressed as a glutathione S-transferase fusion protein, using a (G4S)3 linker connecting the C-terminus of the VH domain and the N-terminus of the VL domain of Lym-1 . Six histidine residues and an E Tag epitope were introduced at the C-terminus of the sFv . Lym-1 sFv was purified with glutathione-Sepharose 4B affinity chromatography followed by digestion with thrombin . Lym-1 sFv of 28 kDa was confirmed by Western blotting with anti-(E Tag) monoclonal antibody . An antigen binding assay of Lym-1 and a CD study indicated that it is functionally active.

Pharm Res, 1998 Sep, 15(9), 1348 - 55
Beta cyclodextrins enhance adenoviral-mediated gene delivery to the intestine; Croyle MA et al.; PURPOSE: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer . In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum . METHODS: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E . coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection . In vivo experiments were performed using an intestinal loop ligation technique . RESULTS: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models . The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization . Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus . Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes . This enhances internalization of viral particles and results in increased levels of gene expression . CONCLUSIONS: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors . They are simple to prepare and do not affect the ability of the virus to transduce target cells.

Korean J Parasitol, 1998 Sep, 36(3), 183 - 90
Immunodiagnosis of clonorchiasis using a recombinant antigen; Yong TS et al.; A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically . One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum . Recombinant C . sinensis antigen with 28 kDa as a beta-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis . The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases . The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG . On RNA slot blot analysis . C . sinensis adult worm RNA showed a positive reaction with the cloned gene . Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.

Insect Biochem Mol Biol, 1998 Sep, 28(9), 651 - 8
Molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the diamondback moth, Plutella xylostella; Huang HS et al.; Four glutathione S-transferase (GST, EC 2.5.1.18) isozymes have been characterized in the larvae of the diamondback moth (DBM), Plutella xylostella L., a cosmopolitan insect pest of crucifiers . This work aimed at cloning and heterologously expressing the cDNA of DBM GST-3, an isozyme involved in this insect resistance to some organophosphorus insecticides, and studying the molecular basis for its increased expression in the resistant strains . Reverse-transcription polymerase chain reaction (RT-PCR) using midgut mRNA from a methyl parathion resistant MPA strain and degenerate primers complimentary to the N-terminal and internal amino acid sequences of GST-3 generated a 128 bp DNA product . A clone of 809 bp, obtained by screening a midgut cDNA library of MPA strain using this PCR product as probe, encoded a protein of 216 amino acids (calculated Mr 24,083 and pI 8.50) . This GST of DBM, PxGST3, shared the highest (46.3%) amino acid sequence identity, among insects, to MsGST1 of Manduca sexta . PxGST3 mRNA level was considerably higher in MPA than in susceptible strains, and Southern blots suggested that gene amplification was probably not involved in the increased expression of this GST isozyme . Enzymatically active PxGST3 expressed heterologously in E . coli exhibited similar biochemical and toxicological properties as GST-3 purified from DBM larvae . It is the first cloned GST with a well-defined role in insecticide resistance.

Genetics, 1998 Oct, 150(2), 543 - 51
Reassigning cysteine in the genetic code of Escherichia coli; Doring V et al.; We investigated directed deviations from the universal genetic code . Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12 . Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids . Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs . A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it . Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E . coli . Propagating the amidase missense mutation in E . coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys . The toxicity of cysteine miscoding was low in all our strains . The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.

EMBO J, 1998 Oct 1, 17(19), 5822 - 31
Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein; Sharpe PL et al.; The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7 . Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD . The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein . TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7 . In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7 . The combination of L29 and ACP also stimulates Tn7 transposition in vitro . Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC+D reactions.

EMBO J, 1998 Oct 1, 17(19), 5811 - 21
Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F; Piron M et al.; Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation . Rotavirus mRNAs are capped, but lack poly(A) sequences . During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end . We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast . Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI) . The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli . In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected . PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli . These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation . These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.

EMBO J, 1998 Oct 1, 17(19), 5543 - 50
Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins; Stewart EJ et al.; Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles . The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway . However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation . This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm . Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively . Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role . In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes . Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth . Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4688 - 95
Mapping of a protein-RNA kissing hairpin interface: Rom and Tar-Tar*; Comolli LR et al.; An RNA 'kissing' complex is formed by the association of two hairpins via base pairing of their complementary loops . This sense-antisense RNA motif is used in the regulation of many cellular processes, including Escherichia coli ColE1 plasmid copy number . The RNA one modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to the kissing hairpins and stabilizing their interaction . We have used heteronuclear two-dimensional NMR spectroscopy to map the interface between Rom and a kissing complex formed by the loop of the trans -activation response (Tar) element of immunodeficiency virus 1 (HIV-1) and its complement . The protein binding interface was obtained from changes in amide proton signals of uniformly 15N-labeled Rom with increasing concentrations of unlabeled Tar-Tar* . Similarly, the RNA-binding interface was obtained from changes in imino proton signals of uniformly 15N-labeled Tar with increasing concentrations of unlabeled Rom . Our results are in agreement with previous mutagenesis studies and provide additional information on Rom residues involved in RNA binding . The kissing hairpin interface with Rom leads to a model in which the protein contacts the minor groove of the loop-loop helix and, to a lesser extent, the major groove of the stems.

Am J Physiol, 1998 Oct, 275(4 Pt 1), L687 - 93
Alveolar type II-like cells release G-CSF as neutrophil chemotactic activity; Koyama S et al.; We evaluated the potential of A549 cells, an alveolar type II epithelial cell line, to release granulocyte colony-stimulating factor (G-CSF), in addition to interleukin (IL)-8 and leukotriene B4, as neutrophil chemotactic activity (NCA) . Human recombinant IL-1beta stimulated A549 cells to release NCA in a time- and dose-dependent fashion . The released NCA was blocked by mouse anti-human G-CSF polyclonal antibody . Molecular-sieve column chromatography revealed that IL-1beta induced the release of a 19- to 20-kDa chemotactic mass that was inhibited by anti-human G-CSF antibody . IL-1beta stimulated the release of G-CSF in a dose-dependent fashion, but the time-dependent profile of G-CSF showed that the concentration of G-CSF declined after 48 h . Tumor necrosis factor (TNF)-alpha, Escherichia coli lipopolysaccharide (LPS), and bradykinin (BK) stimulated A549 cells to release NCA that was inhibited by anti-G-CSF antibody . The release of G-CSF in response to TNF-alpha, LPS, and BK was significantly increased . The similar concentrations of human recombinant G-CSF (10-1,000 pg/ml) as in the supernatant fluid induced neutrophil chemotaxis . G-CSF mRNA was expressed time and dose dependently at 4 h and declined after 4 h in response to IL-1beta as evaluated by RT-PCR . The expression of G-CSF mRNA was also observed by TNF-alpha, LPS, and BK stimulation . These data suggest that type II alveolar epithelial cells may produce G-CSF as NCA and may participate in the regulation of leukocyte extravasation.

Am J Physiol, 1998 Oct, 275(4 Pt 1), C1048 - 57
Cl- transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon; Sahi J et al.; Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach approximately 90% confluence on plastic and develop transepithelial resistances of 120-250 Omega . cm2 on porous substrates . Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl- transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts . Basal Cl- influx was increased (P < 0.01) by PGE1 (1 microM), forskolin (1 microM), 8-bromoadenosine 3'5'-cyclic monophosphate (100 microM), heat-stable Escherichia coli enterotoxin (STa; 1 microM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 microM), histamine (1 microM), and phorbol 12,13-dibutyrate (1 microM) in both cell types . The Cl- channel blocker diphenylamine 2-carboxylic acid (50 microM) and the Na+-K+-2Cl- cotransport inhibitor furosemide (1 microM), but not the K+ channel blocker Ba2+ (3 mM), inhibited these Cl- permeabilities . These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II . Thus cAMP-, cGMP-, and Ca2+-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl- transport . This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.

Indian J Biochem Biophys, 1998 Apr, 35(2), 63 - 6
Rapid and reliable site directed mutagenesis using Kunkel's approach; Handa P et al.; Oligonucleotide based site directed mutagenesis (SDM) is an invaluable technique in molecular biology . Among the various methods developed for SDM, the PCR-based approach, Kunkel's and the Eckstein's procedures are widely used . The Kunkel's method, on account of its cost effectiveness and simplicity, is preferred by many a scientist . However, a general drawback of this method is the high background due to persistence of the parent template resulting in low efficiency of mutagenesis . In this report, we describe a modification of the Kunkel's method to increase the efficiency of selecting against the wild type strand . We have used Sequenase for the extension reaction, and introduced an in vitro UDG step to enhance the biological selection against the parent strand . Consequently, the efficiency of the modified method is enhanced to allow screening of the mutants directly by DNA sequencing . A step by step single tube protocol which is over in less than three hours makes it a method of choice for efficient and cost-effective site directed mutagenesis.

Plant J, 1998 Aug, 15(4), 563 - 8
A gene encoding phosphatidylinositol-4-phosphate 5-kinase is induced by water stress and abscisic acid in Arabidopsis thaliana; Mikami K et al.; Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol-4-phosphate to produce phosphatidylinositol-4,5-bisphosphate as a precursor of two second messengers, inositol-1,4,5-triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization . Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants . Searching the Arabidopsis expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same Arabidopsis PIP5K and isolated a corresponding full-length cDNA encoding a protein that we designated AtPIP5K1 . Recombinant AtPIP5K1 expressed in Escherichia coli possessed a PIP5K activity in vitro . Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K . The expression of the AtPIP5K1 mRNA was induced rapidly by treating Arabidopsis plants with drought, salt and abscisic acid, which suggests that AtPIP5K11 is involved in water-stress signal transduction . These data give evidence for a close link between phosphoinositide signaling cascades and water-stress responses in plants.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4778 - 82
Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo; Deana A et al.; In order to test the effect of silent mutations on the regulation of gene expression, we monitored several steps of transcription and translation of the ompA gene in vivo , in which some or all codons between codons 6 and 14, frequently used in Escherichia coli , had been exchanged for infrequent synonymous codons . Northern blot analysis revealed an up to 4-fold reduction in the half-life of the mutated messengers and a >10-fold reduction in their steady-state amounts . Western blot analysis showed a 10-fold reduction in the amount of OmpA protein . Use of a system expressing a Rho-specific anti-terminator allowed us to detect a strong transcription polarity effect in the silent mutants . These results demonstrate that silent mutations can severely inhibit several steps of gene expression in E . coli and that code degeneracy is efficiently exploited in this species for setting signals for gene control and regulation.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4669 - 75
Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions; Zhang QM et al.; Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA . Escherichia coli must have several pathways to repair such mismatches and DNA modifications . In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli . The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis . In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose . Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal . We screened approximately 40 000 colonies and selected several mutator strains . The strain GC39 showed the highest mutation rate to Lac+ . The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome . The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli . The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains . Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A . Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair . These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4618 - 25
Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI; Sriskanda V et al.; A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft . Motif V links the two structural domains . Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure . We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate . This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI . The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated . We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick . These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4582 - 7
The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli; Fujikawa K et al.; The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay . 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay . The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage . 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4557 - 65
The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule; Grimm M et al.; Deviations from the universal genetic code have evolved independently several times in ciliated protozoa . Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine . We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells . Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon . This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate . It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract . Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons . Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position . We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.

Structure, 1998 Sep 15, 6(9), 1207 - 14
Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution; Peat TS et al.; BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively . IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function . The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently . RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution . The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A . Unmodified P . aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores . CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible . The C-terminal domain is found to be homologous to the cold-shock protein CspA of E . coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 466 - 8
An essential methionine residue involved in substrate binding by phosphofructokinases; Wang X et al.; An alignment of all PPi-dependent phosphofructokinases and all allosteric ATP-dependent PFKs shows relatively few residues that are fully conserved . One residue that is conserved is a methionine residue that appears from the crystal structure of Escherichia coli PFK to be interacting with fructose 6-P . Very conservative substitutions for this methionine with leucine or isoleucine by site-directed mutagenesis of E . coli ATP-PFK and Entamoeba histolytica PPi-PFK produced profound decreases either in the apparent affinity for fructose 6-P or in maximal velocity, or both . Methionine provides a highly specific interaction with fructose 6-P for binding and for transition state stabilization.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 409 - 13
Gene synthesis, expression, and mutagenesis of zucchini mavicyanin: the fourth ligand of blue copper center is Gln; Kataoka K et al.; The gene coding for the 109-amino-acid, non-glycosylated form of mavicyanin was synthesized and expressed in Escherichia coli . The recombinant protein refolded from E . coli inclusion bodies was purified and characterized . Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety . The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins . To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis . The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin . The midpoint potential of Gln95-->Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins . The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 381 - 4
Autophosphorylation of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system requires dimerization; Seok YJ et al.; Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition . In vitro autophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein . Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity . The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer . These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 206 - 11
Uridine diphosphoglucose dehydrogenase regulates proteoglycan expression: cDNA cloning and antisense study; Wegrowski Y et al.; Using a reverse-transcriptase-polymerase chain reaction approach human and murine UDPG-dehydrogenase (GDH) was cloned from fibroblast mRNAs . Human enzyme is 97% and 27% identical with its murine and E . coli orthologs . Murine mRNA of 3.1 kb size is expressed in all the tissue studied at a level independent of glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA . In human fibroblast in vitro, 2 GDH transcripts were observed . They were expressed proportionally to GAPDH . The simple pattern of human GDH Southern blotting suggests a single copy gene . An antisense oligonucleotide directed to the ATG region of the human enzyme inhibited 35S-sulphate incorporation into extracellular macromolecules, especially proteoglycans . These data indicate that GDH expression may regulate proteoglycan synthesis in the cells.

J Mol Biol, 1998 Oct 9, 282(5), 1083 - 91
Folding and association of beta-Galactosidase; Nichtl A et al.; beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present . Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme . Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates . The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC . The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC . Tetramers form from these dimers in a fast reaction . By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association . Copyright 1998 Academic Press.

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