Escherichia coli

J Biol Chem, 1998 Feb 6, 273(6), 3336 - 42
The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor; Sundaram M et al.; The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated . The experimental system consisted of the {3H}retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes . {3H}Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin . Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP . The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP . The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism . Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 659 - 64
Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica; Deng Z et al.; Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites . Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme . The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E . histolytica {Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim . Biophys . Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem . J . 316, 57-63} . The sequence identity between the two proteins was 17% . The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs . The cloned cDNA was expressed and the protein purified . The kinetic properties were identical with those of the enzyme isolated from the organism . Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E . histolytica PFK gene {Bruchhaus et al . (1996)} . The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E . histolytica trophozoites.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 631 - 5
Human insulin production from a novel mini-proinsulin which has high receptor-binding activity; Chang SG et al.; To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having the central C-peptide region replaced with a sequence forming a reverse turn . The mini-proinsulin was fused at the N-terminus to a 21-residue fusion partner containing a His10 tag for affinity purification . The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 25% of the total cell protein . The protein was sulphonated, cleaved by CNBr and the M2PI mini-proinsulin was purified using ion-exchange chromatography . The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide . Native human insulin was successfully generated by subsequent enzymic conversion of mini-proinsulin . The mini-proinsulin exhibited high receptor-binding activity, about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor-bound structure of insulin as well as the role of C-peptide in the folding and activity of proinsulin.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 589 - 96
Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli; Reche P et al.; Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain . A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E . coli . The biotinyl domain was readily released by cleavage with thrombin . Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes . No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro . The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated . It was also a poor substrate for lipoylation in vitro catalysed by the E . coli lipoyl protein ligase encoded by the lplA gene . The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes . The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E . coli but probably by a pathway different from that mediated by the lplA-dependent ligase . The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.

Structure, 1997 Nov 15, 5(11), 1485 - 99
Structural foundation for the design of receptor antagonists targeting Escherichia coli heat-labile enterotoxin; Merritt EA et al.; BACKGROUND: Escherichia coli heat-labile enterotoxin (LT) is the causative agent of traveller's diarrhoea, and it is also responsible for the deaths of hundreds of thousands of children per year in developing countries . LT is highly homologous in sequence, structure and function to cholera toxin (CT) . Both toxins attack intestinal epithelial cells via specific binding to the branched pentasaccharide of ganglioside GM1 at the cell surface . A receptor-binding antagonist which blocked this interaction would potentially constitute a prophylactic drug conferring protection both against the severe effects of cholera itself and against the milder but more common disease caused by LT . RESULTS: Four derivatives of the simple sugar galactose, members of a larger series of receptor antagonists identified by computer modeling and competitive binding studies, have been co-crystallized with either the full LT AB5 holotoxin or the LT B pentamer . These crystal structures have provided detailed views of the toxin in complex with each of the four antagonists: melibionic acid at 2.8 A resolution, lactulose at 2.65 A resolution, metanitrophenylgalactoside (MNPG) at 2.2 A resolution and thiodigalactoside (TDG) at 1.7 A resolution . The binding mode of each galactose derivative was observed 5-15 times, depending on the number of crystallographically independent toxin B pentamers per asymmetric unit . There is a remarkable consistency, with one important exception, in the location and hydrogen-bonding involvement of well-ordered water molecules at the receptor-binding site . CONCLUSIONS: The bound conformations of these receptor antagonist compounds preserve the toxin-galactose interactions previously observed for toxin-sugar complexes, but gain additional favorable interactions . The highest affinity compound, MNPG, is notable in that it displaces a water molecule that is observed to be well-ordered in all other previous and current crystal structures of toxin-sugar complexes . This could be a favorable entropic factor contributing to the increased affinity . The highest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 A2) of the binding-site surface covered by the full receptor GM1 pentasaccharide, despite being considerably smaller . This provides an encouraging basis for the creation of subsequent generations of derived compounds that can compete effectively with the natural receptor.

Infect Immun, 1998 Mar, 66(3), 980 - 6
A monoclonal antibody generated by antigen inoculation via tick bite is reactive to the Borrelia burgdorferi Rev protein, a member of the 2.9 gene family locus; Gilmore RD Jr et al.; Murine monoclonal antibodies directed against proteins of Borrelia burgdorferi B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks . This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae . One of the resultant antibodies reacted with a 17-kDa antigen from cultured B . burgdorferi, as seen by immunoblot analysis . This antibody was used to screen a B . burgdorferi genomic DNA lambda vector expression library, and an immunoreactive clone was isolated . DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames . These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of B . burgdorferi by Porcella et al . (S . F . Porcella, T . G . Popova, D . R . Akins, M . Li, J . D . Radolf, and M . V . Norgard, J . Bacteriol . 178:3293-3307, 1996) . By selectively subcloning genes found in this insert into an Escherichia coli plasmid expression vector, the observation was made that the rev gene product was the protein reactive with the 17-kDa-specific monoclonal antibody . The rev gene product was found to be expressed in low-passage, but not in high-passage, B . burgdorferi B31 . Correspondingly, the rev gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times . Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein . The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection.

Mol Cell Biol, 1998 Mar, 18(3), 1635 - 41
A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype; Nicolaides NC et al.; Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors . Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a "two-hit" inactivation of both alleles of a particular MMR gene . Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele . These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

Mol Cell Biol, 1998 Mar, 18(3), 1416 - 23
Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase; Cauthron RD et al.; Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site . Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules . In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A . Using {35S}methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197 . The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course . Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A . By both the gel shift assay and a {gamma-32P}ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197 . Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

Infect Immun, 1998 Mar, 66(3), 1270 - 2
Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs; Casey TA et al.; The role of Escherichia coli heat-stable enterotoxin B (STb) in neonatal porcine diarrhea caused by enterotoxigenic E . coli was examined by comparing adherent isogenic strains with or without STb . The cloned STb gene (in the plasmid pRAS1) was electroporated into a nonenterotoxigenic strain (226M) which expresses the F41 adhesin . Strain 226M pRAS1 adhered and expressed STb in vivo, causing fluid secretion in ligated ileal loops in neonatal pigs . Although strain 226M pRAS1 caused very mild diarrhea in some orally inoculated neonatal pigs, the weight loss in these pigs was similar to that caused by the parental strain without STb . We conclude that STb does not significantly contribute to diarrhea caused by enterotoxigenic E . coli in neonatal pigs.

Infect Immun, 1998 Mar, 66(3), 1037 - 44
Antigenicity of recombinant proteins derived from rhoptry-associated protein 1 of Plasmodium falciparum; Fonjungo PN et al.; Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine . We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice . Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting . These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia . Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1 . The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region . Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody . Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.

Infect Immun, 1998 Mar, 66(3), 944 - 9
Curli, fibrous surface proteins of Escherichia coli, interact with major histocompatibility complex class I molecules; Olsen A et al.; Curli are thin, coiled fibers expressed on the surface of Escherichia coli that bind several matrix and plasma proteins such as fibronectin, laminin, plasminogen, tissue plasminogen activator, and H-kininogen . In this work, we examined the interactions between curli-expressing E . coli and human major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules . Curliated E . coli was found to interact with an MHC-I-expressing lymphoma cell line as shown by scanning electron microscopy, whereas the binding to a mutant variant of this cell line expressing small amounts of MHC-I molecules was significantly lower . Moreover, curli-expressing E . coli bound purified radiolabeled MHC-I but not MHC-II molecules, whereas an isogenic curli-deficient mutant strain showed no affinity for either MHC-I or MHC-II . Purified insoluble curli could also bind 125I-labeled MHC-I molecules, and in Western blot experiments the 15-kDa curlin subunit protein bound intact MHC-I molecules as well as beta2-microglobulin, the light chain of MHC-I molecules . A direct interaction between monomeric MHC-I molecules and a bacterial surface protein has previously not been reported . The binding of curli to MHC-I molecules, which are present on virtually all cells in higher vertebrates, will provide curliated E . coli with ample opportunities to interact with a great variety of hosts and host cells . This should facilitate the adaptation of E . coli to different ecological niches, and in human infections the interaction between curli and MHC-I molecules could contribute to adherence and colonization.

Infect Immun, 1998 Mar, 66(3), 907 - 11
D-lactate production and {14C}succinic acid uptake by adherent and nonadherent Escherichia coli; McCabe K et al.; Escherichia coli isolates of different adherence phenotypes produced different amounts of D-lactate . Alterations of culture conditions did not influence the amount of D-lactate produced . The observed pH decreases in tissue culture medium corresponded with increases in D-lactate concentration . Very little {14C}succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E . coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed . The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E . coli.

Shock, 1998 Feb, 9(2), 95 - 100
Rapid and prolonged impairment of gut barrier function after thermal injury in mice; Eaves-Pyles T et al.; Loss of gut barrier function after burn injury can be important in the pathogenesis of systemic infections and organ dysfunction . The purpose of this study was to determine how rapidly impairment of gut barrier function occurs after burn injury and how long it persists . BALB/c mice were gavaged with 10(10) (111)In-oxine-labeled Escherichia coli 3 h before inflicting a 20% total body surface area burn . They were then killed at 5, 15, 30, 60, 120, or 240 min post-burn . Additional mice were given a 20% or 30% burn injury and were randomized into eight groups, which were killed at either 4 h or 1, 2, 4, 7, 14, or 21 days post-burn . Each mouse was gavaged with 10(10) (111)In-oxine-labeled E . coli 4 h before sacrifice to determine the magnitude of translocation . Gut barrier function was impaired as early as 5 min post-burn and was maximal by 4 h . Rapid improvement was observed by 24 h, followed by slow improvement, but with persistent abnormality through 21 days post-burn . Killing of translocated bacteria was impaired at 4 h and day 7 post-burn, according to the percentage of viable E . coli that remained alive in the tissues . The magnitude of gut dysfunction following burn injury is temporally related.

Med Hypotheses, 1998 Jan, 50(1), 61 - 5
The etiology of benign prostatic hypertrophy; Roper WG; Benign prostatic hypertrophy (BPH) is a hyperplasia and not a hypertrophy . No cogent total explanation for the etiology of this ubiquitous disease has ever been given . There is such a vast cascade of incongruities in respect of hormonal influences, on the one hand, and the degree, variation, timing and rate of development of the condition, on the other hand, that a 'missing link' has constantly been envisaged in its etiology . In the search for this, two simple facts stand out: (a) BPH starts at some time after involution commences; (b) it is the transitional zone that is affected in the major extent . This hypothesis proposes that, owing to dynamic embryological differences between the transitional and the other zones, its secretions are differently affected by involution, resulting in stimulatory factors affecting known hormonal influences upon prostatic growth, further resulting in the development of BPH . These stimulatory factors are considered to be the 'missing link' in the etiology of BPH . It is further suggested that one of these stimulatory factors may be E . coli endotoxin, repeatedly released within a contained prostatic environment . This is considered secondary to intermittent colonization of the transitional zone acini and/or ducts by E . coli, followed by the destruction of these E . coli . This colonization is considered mandatory, if the known 95% occurrence of invasive infection of BPH is to be explained.

Protein Eng, 1997 Oct, 10(10), 1205 - 11
Functional roles of the N-terminal amino acid residues in the Mn(II)-L-malate binding and subunit interactions of pigeon liver malic enzyme; Chou WY et al.; Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu- . In this work, various mutants of the enzyme with individual or combinational deletion (delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells . A major protein band corresponding to an Mr of approximately 65000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis . However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with Mr approximately 260000 or a mixture of tetramers and dimers with the exception of delta(K2K3G4) and delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed . K3A and K3E also dissociated substantially . K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers . All tetramers and dimers were enzymatically active in the gels . All mutants displayed a similar apparent Km value for NADP+ . The apparent Km for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the delta(K2/K3) and the delta(1-16) mutants . The small binding affinity of delta(K2/K3) with Mn(II)-L-malate was specific . With additional deletion at positions 3 and/or 4, the delta(K2K3), delta(K2G4/K3G4) or delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type . The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation . The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface . These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions.

Life Sci, 1998, 62(3), 247 - 55
Glomerular endothelial injury associated with free radical production induced by a fungal cell wall component, (1-->3) beta-D glucan; Iwamoto N et al.; Clinical evidence suggests that microangiopathy may be induced by fungal infection . The present study evaluated the toxic effect of (1-->3) beta-D glucan, a major component of fungal cell wall, on cultured transformed glomerular endothelial cells (TF-GEN) . When TF-GEN were exposed to increasing concentrations of (1-->3) beta-D glucan (beta-DG; 115 to 430 pg/ml) for 1 to 3 hours, concentration- and time-dependent increases in hydroxyl radical production were demonstrated by electron paramagnetic resonance spectrometry using 5, 5-dimethyl-1-pyrrolyne-N-oxide as a spin trap agent . The amount of radicals induced by 230 or 430 pg/ml beta-DG was comparable to that induced by E . coli LPS (1 or 10 microg/ml) . The beta-DG-induced free radical production was associated with a subsequent increase in LDH release from TF-GEN . When TF-GEN pretreated with U78517F (0.1 or 1.0 microM), a lipophilic antioxidant, were stimulated with LPS (1 or 10 microg/ml) or beta-DG (230 pg/ml) for 3 hours, free radical production by TF-GEN was significantly reduced in cells pretreated with the higher concentration of U78517F . Thus, fungal (1-->3) beta-D glucan induces glomerular endothelial injury by stimulating cellular free radical production . Such a mechanism may underlie microangiopathy in systemic fungal infections.

Photochem Photobiol, 1998 Feb, 67(2), 263 - 7
Fluorescence and photochemistry of recombinant phytochrome from the cyanobacterium Synechocystis; Sineshchekov V et al.; Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K . The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (P phi B), Syn(PCB)phy and Syn(P phi B)phy, respectively . Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(P phi B)phy . At room temperature, the spectra are blue shifted by 5-10 nm . The fluorescence intensity dropped down by approximately 15-20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol-1 in Syn(PCB)phy and Syn(P phi B)phy, respectively . Phototransformation of Pr upon red illumination was observed at temperatures above 160-170 K in Syn(PCB)phy and above 140-150 K in Syn(P phi B)phy with a 2-3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part . This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome . At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7-0.75 and 0.85-0.9 for Syn(PCB)phy and Syn(P phi B)phy, respectively . Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed . With respect to the photochemical parameters, Syn(PCB)phy and Syn(P phi B)phy are similar to each other and also to a small fraction of phyA (phyA") and to phyB . The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA'), which is distinguished by the high extent (ca 50%) of Pr phototransformation at 85 K . These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.

Photochem Photobiol, 1998 Feb, 67(2), 184 - 91
Photophysical properties and photobiological activity of the furanochromones visnagin and khellin; Borges ML et al.; The larger photobiological activity of visnagin (VI) versus khellin (KH) toward several living organisms, including fungi, viruses, yeasts and bacteria, induced a detailed investigation of the photophysical properties of these naturally occurring furanochromones, using laser-flash-photolysis, photoacoustic calorimetry and fluorescence (steady-state and time-resolved) techniques in solvents with different polarity and content of water, including micelles and vesicles . The results have shown that the magnitude of all the three rate constants out of S1 (radiative, kf; internal conversion, kic and intersystem crossing, kisc) for VI and KH strongly depend on the solvent, namely on its hydrogen bonding ability and polarity . The changes of kf and kisc are due to the solvent-assisted mixing and/or inversion of the two first singlet excited states (1n, pi and 1 pi, pi), while kic increases with a decrease of the S0-S1 energy gap . As a consequence, the quantum yield of triplet formation (phi T) strongly decreases from values of approximately 0.8 in dioxane to < 0.05 in water for both compounds . The magnitude of solvent polarity/hydrogen bonding ability required, at which the state order is inverted and phi T starts to decrease, is greater for VI than for KH and consequently phi T (VI) >> phi T (KH) over a broad range of water content including that appropriate to the environment of the compounds in a living system . These facts account for the larger photobiological activity of VI with respect to KH, regarding both the fungus Fusarium culmorum L . and the wild strain of Escherichia coli, studied by us.

Planta, 1998 Feb, 204(2), 242 - 51
Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit; Schroder R et al.; Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa {A . Chev.} C.F . Liang et A.R . Ferguson var . deliciosa cv . Hayward) was purified 3000-fold to homogeneity . The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8 . The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for {3H}XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan . Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of {3H}XXXG-ol by hydrolysis, and in the presence of {3H}XXXG-ol by hydrolysis and endotransglycosylation . Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA . The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes . Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6 . Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft . A full-length cDNA clone (AdXET5) was overexpressed in E . coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

Appl Microbiol Biotechnol, 1998 Jan, 49(1), 31 - 8
Purification and characterization of recombinant spider silk expressed in Escherichia coli; Arcidiacono S et al.; A partial cDNA clone, from the 3' end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands . This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a 43-kDa recombinant silk protein was expressed . Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk.

Pharm Res, 1998 Jan, 15(1), 116 - 21
Terplex DNA delivery system as a gene carrier; Kim JS et al.; PURPOSE: To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV-beta-gal plasmid efficiently in cultured smooth muscle cells (SMC) (1) . METHODS: Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used . RESULTS: AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation . The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay . Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC . GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added . Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation . Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in epsilon-methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used . CONCLUSIONS: Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity/charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation . This information is expected to be utilized for the development of much improved transfection vectors for in vivo gene therapy.

East Afr Med J, 1997 Aug, 74(8), 499 - 502
Individual and combined genotoxic response of boric acid and aflatoxin B1 in Escherichia coli PQ37; Odunola OA; The genotoxic potentials of boric acid in Escherichia coli PQ37 was assessed along in the presence of aflatoxin B1 using SOS chromotest . Boric acid induced beta-galactosidase synthesis on the tester bacteria both on the presence and absence of S9 activation mixture . Therefore, the inorganic acid may not require metabolic activation to be genotoxic for this bacteria . When present together with aflatoxin B1 in the assay medium, boric acid increased the degree of beta-galactosidase synthesis induced by the maximal inducing concentration of aflatoxin B1 before the toxin's activation . However, the degree of enzyme synthesis induced was significantly decreased when boric acid was present after aflatoxin activation . This suggests that boric acid may not interfere with nor block the expoxidation of aflatoxin B1 . It may however interact with the expoxide thereby inhibiting its activity . Boric acid may be a genotoxin and could possibly act as a syngenotoxic and/or a cogenotoxic agent.

Tumori, 1997 Jul-Aug, 83(4 Suppl 2), S3 - 15
{Erythropoietin: biochemical characteristics, biologic effects, indications and results of use in hematology}; Marmont AM; This review has two objects: a brief recapitulation of the biological background of erythropoietin (EPO), and a review of its clinical utilization in hematology . EPO, both in its naturally occurring and recombinant form (rH-EPO), is a single chain glycoprotein with an approximate molecular weight of 30.000 to 34.000 kD . Its heavy glycosilation is essential for its activity in vivo, since asialoEPO is readily cleared by the heptic asialoglycoprotein receptor . This impedes the recombinant molecule's synthesis in biologic cultures other than mammalian cells (Chinese hamster's ovary cells), and inevitably increases costs . If in vitro glycosilation of E . coli-derived rH-EPO could be achieved, the clinical utilization of the product would be considerably enhanced, most especially when very high doses are necessary, as discussed later . There is no antigenic diversity between natural and recombinant EPO, so that out of the enormous clinical experience only one single case of immunization has been recorded . Almost paradoxically there are however three published cases of pure red cell aplasia (PRCA) caused by immunization against autologous EPO . It is now established that in adults EPO is synthetized in renal peritubular interstitial cells, although some residual activity remains in the liver . Hypoxia results in a rapid induction of EPO expression, although the role of the oxygen sensor system is still debated . Cellular targets are notoriously erythroid progenitors and precursors (BFU-E, CFU-E, early and intermediate erythroblasts) . The global erythropoietic activity resulted in various effects (proliferation, differentiation, survival), but most probably each single effect is integrated with and complementary of the others . The utilization of rH-EPO in hematologic diseases came much later than its dramatic success in renal anemia . A variety of tools useful for assessing the possible beneficial effects of rH-EPO in clinical hematology has been proposed, among which a low level of endogenous EPO is a good predictor for therapeutic success . 'Hemopathic' anemia can be subdivided into three categories: patients with normal erythropoiesis due to inadequate EPO production (anemia of prematurity), patients with depressed but nonclonal erythropoiesis (chemotherapy, lymphoid malignancies such as multiple myeloma-MM and chronic lymphatic leukemia-CCL) and patients with at least partially clonal anemia, such as paroxysmal nocturnal hemoglobinuria (PNH), hemoglobinopaties, myelodysplastic syndromes (MDS) and others . Results in the first category of patients are, as expected, prompt and satisfactory with physiologic doses . Although therapeutic strategy for MM is moving fast to curative intents, the utilization of rH-EPO is indicated for the control of anemia in conservatively-treated patients . In the third category the most important and controversial area is MDS . Significant erythropoietic results are generally obtained in about 20% of patients; however, the association with G-CSF has considerably enhanced the response rate . In the field of bone marrow transplantation there is an inadequate production of endogenous EPO in the allogeneic setting, and randomized studies have shown the benefits of rH-EPO in this situation . However, the most important results have been obtained in post-major-ABO incompatible PRCA, when the removal of the recipient's isohemagglutinins does not resolve the anemia . High and very high doses of rH-EPO (even over 500 UI/kg/day for 2-4 weeks) may resolve this occasionally quite refractory condition . Although extremely expensive, this treatment may be life-saving when an otherwise successful allogeneic transplant is at the risk of failure because of this relatively uncommon but severe immunohematologic complication.

Nature, 1998 Feb 19, 391(6669), 811 - 4
Role of the histone deacetylase complex in acute promyelocytic leukaemia; Lin RJ et al.; Non-liganded retinoic acid receptors (RARs) repress transcription of target genes by recruiting the histone deacetylase complex through a class of silencing mediators termed SMRT or N-CoR . Mutant forms of RARalpha, created by chromosomal translocations with either the PML (for promyelocytic leukaemia) or the PLZF (for promyelocytic leukaemia zinc finger) locus, are oncogenic and result in human acute promyelocytic leukaemia (APL) . PML-RARalpha APL patients achieve complete remission following treatments with pharmacological doses of retinoic acids (RA); in contrast, PLZF-RARalpha patients respond very poorly, if at all . Here we report that the association of these two chimaeric receptors with the histone deacetylase (HDAC) complex helps to determine both the development of APL and the ability of patients to respond to retinoids . Consistent with these observations, inhibitors of histone deacetylase dramatically potentiate retinoid-induced differentiation of RA-sensitive, and restore retinoid responses of RA-resistant, APL cell lines . Our findings suggest that oncogenic RARs mediate leukaemogenesis through aberrant chromatin acetylation, and that pharmacological manipulation of nuclear receptor co-factors may be a useful approach in the treatment of human disease.

Nature, 1998 Feb 19, 391(6669), 795 - 9
HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C; Braud VM et al.; The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability . Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules . Here we report the identification of ligands for HLA-E . We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin . This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood . On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR) . Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones . A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones . Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

Am J Physiol, 1998 Feb, 274(2 Pt 2), H580 - 90
Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness; Rigby SL et al.; Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte . We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression . Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes . Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia coli lipopolysaccharide (LPS) or saline (control; Ctl) . Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl{pCa value that resulted in half-maximal shortening (pCa50): 5.78 +/- 0.04} and LPS (pCa50: 5.72 +/- 0.02) . Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 +/- 0.02) and LPS (pCa50: 5.76 +/- 0.02) . Maximal tension generated by LPS myocytes (2.89 +/- 0.23 g/mm2) was significantly less (P < 0.05) than Ctl (3.75 +/- 0.34 g/mm2) . However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 +/- 0.98 g/mm2) was similar to Ctl (3.01 +/- 0.80 g/mm2) . We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes . Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased (P < 0.05) compared with Ctl myocytes.

Am J Physiol, 1998 Feb, 274(2 Pt 1), C319 - 32
pH-independent retrograde targeting of glycolipids to the Golgi complex; Schapiro FB et al.; A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood . In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells . The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy . This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex . Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane . The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB) . Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor . Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located . Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH . Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid "rafts" and that internalization is cytochalasin insensitive . We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.

FEMS Microbiol Lett, 1998 Feb 1, 159(1), 93 - 7
Effect of environmental factors on the dominant lethality caused by expression of a mutated DnaA protein with decreased intrinsic ATPase activity; Sakamoto K et al.; Induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells was previously shown to cause overinitiation of chromosomal DNA replication and a dominant lethal phenotype . Here it is shown that the dominant lethality required incubation at high temperatures; cells harboring the expression plasmid of DnaA E204Q showed very weak colony formation ability (less than 1/10(5) that of the wild-type DnaA) at 42 degrees C, whereas they showed a normal colony formation ability at 28 degrees C on LB agar plates . Overinitiation of chromosomal DNA replication caused by expression of DnaA E204Q also required incubation at high temperatures in LB medium . When the incubation was performed in synthetic (Tanaka) medium at 42 degrees C, neither the dominant lethality nor overinitiation caused by expressing DnaA E204Q was observed . These results suggest that the dominant lethality and the overinitiation caused by expressing DnaA E204Q require culture conditions that provide a high potential for cell growth.

Proteins, 1997, Suppl 1, 38 - 42
Protein modeling by multiple sequence threading and distance geometry; Aszodi A et al.; The application of homology modeling is often limited by the lack of known structures with sufficiently high sequence similarity to the target protein . The recent development of threading methods now enable the identification of likely folding patterns in a number of cases where the structural relatedness between target and template(s) is not detectable at the sequence level . We devised a hybrid method in which fold recognition was performed using the Multiple Sequence Threading (MST) method . The structural equivalences deduced from the threading output were used to guide the distance geometry program DRAGON in the construction of low-resolution C alpha/C beta models . The initial structures were converted to full-atom representation and refined using the general-purpose molecular modeling package QUANTA . The performance of the approach is illustrated on the CASP2 target T0004 (polyribonucleotide nucleotidyl-transferase S1 motif (PNS1) from Escherichia coli, PDB code: 1SRO) for which no obvious homologues with known structure were available . The correct fold of PNS1 was successfully identified, and the model was found to be more similar to the experimental PNS1 structure than the scaffold (C alpha RMSD of 6.2 A compared with 6.4 A) . Our results indicate that a sensitive fold recognition algorithm coupled with a distance geometry program capable of rapidly generating initial structures can successfully complement high-resolution homology modeling methods in cases where sequential similarity is low.

Biochemistry, 1998 Feb 17, 37(7), 2004 - 16
Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism; Tsaprailis G et al.; Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase {CCP(MI)} and horseradish peroxidase (HRP) in their ferric forms . CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl . CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl . The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic . The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl . Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data . The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding . Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture . The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI) . Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.

Biochemistry, 1998 Feb 17, 37(7), 1979 - 88
Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer's paired helical filament formation in vitro; Yanagawa H et al.; Tau is a microtubule-associated protein in mammalian brain . In Alzheimer's disease, this protein is present in the somatodendritic compartment of certain nerve cells, where it forms a portion of paired helical filament, the major constituent of the neurofibrillary tangle . For clarification of the mechanism of this formation, recombinant human tau and its fragments (N-terminal half, C-terminal half, and 4-repeats) expressed in Escherichia coli were prepared, eight peptide fragments (C-tails 1-8) of the C-tail region were synthesized, and the conformation and capacity for aggregation essential for filamentous structure formation in vitro were examined . Recombinant full-length tau, the N-terminal half, 4-repeats, and the C-terminal half did not form filamentous structures in aqueous solution after standing at 20 degrees C . Peptides corresponding to the C-tail region of tau, C-tail 5, C-tail 7, and C-tail 8, produced the paired filament or single straight filament in acidic solution . The rate of filament formation by each peptide was followed by circular dichroism, which showed the C-tails to have predominantly random coil structures immediately following dissolution in aqueous solution and be gradually converted to the beta-sheet structure . The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, whose negative peak intensity at 218 nm in circular dichroism increased due to filamentous structure formation . This delay was eliminated by seeding supersaturated solution of preformed filaments . C-tails interacted with recombinant full-length tau to form definite single straight filament . The C-tail region of tau is thus shown indispensable to the formation of paired helical filament and nucleation to reduce the rate of paired helical filament formation in amyloidogenesis in vitro . These findings may provide some clarification of the pathogenesis of Alzheimer's disease.

Biochemistry, 1998 Feb 17, 37(7), 1917 - 25
Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases; Misra HS et al.; A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases . The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E . coli DNA polymerase I . The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases . The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone . Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer . PNA sequences were also recognized as template when annealed with a DNA primer . These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases . The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases.

Biochemistry, 1998 Feb 17, 37(7), 1905 - 9
The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner; Fu GK et al.; LON proteases, which are ATP-dependent and exhibit ATPase activity, are found in bacteria, yeast, and humans . In Escherichia coli, LON is known to regulate gene expression by targeting specific regulatory proteins for degradation . The yeast and human LON proteins are encoded in the nucleus but localize to the mitochondrial matrix . In yeast, LON has been shown to be essential for the maintenance of the integrity of the mitochondrial genome . E . coli Lon has long been known to bind DNA, but we have only recently demonstrated that it binds preferentially to a specific TG-rich double-stranded sequence . We now show that human LON recognizes a very similar site in both the light and heavy chain promoters of the mitochondrial genome, in a region which is involved in regulating both DNA replication and transcription . Unlike E . coli Lon, however, human LON specifically binds to the TG-rich element only when it is presented in the context of a single DNA strand . These findings suggest that the human LON protease might regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters.

Biochemistry, 1998 Feb 17, 37(7), 1861 - 7
Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli; Braun M et al.; The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups . By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment . The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G . The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy . Here, we report that the fully assembled fragment can be overproduced in E . coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D . Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium . The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps . The preparation contains one noncovalently bound FMN per molecule . Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I . The preparation fulfills all prerequisites for crystallization of the fragment.

Biochemistry, 1998 Feb 17, 37(7), 1828 - 38
The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties; Vanoni MA et al.; As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli . The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation . We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form . This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine . Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate . The glutamate synthase alpha subunit contains the {3Fe-4S} cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments . The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present . The FMN moiety but not the {3Fe-4S} cluster of the subunit appears to participate in this reaction . Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme . These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor . The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide . However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the {3Fe-4S} center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Biochemistry, 1998 Feb 17, 37(7), 1789 - 99
Modeling the carbohydrate-binding specificity of pig edema toxin; Cummings MD et al.; The wild-type binding pentamer of Shiga-like toxin IIe (SLT-IIe) binds both the globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) cell surface glycolipids, whereas the double mutant GT3 (Q65E/K67Q) exhibits a marked preference for Gb3 {Tyrrell, G . J., et al . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 524-528} . We modeled three unique sites (sites 1-3) for binding of the carbohydrate moiety of Gb3 to GT3 and SLT-IIe, on the basis of the three sites observed for the SLT-I pentamer {Ling, H., et al . (1998) Biochemistry 37, 1777-1788} . Examination of the three sites in light of various mutation and binding data strongly suggested that one of the binding sites plays a role in the change of specificity observed for the GT3 mutant . We applied several modeling techniques, and developed a model for binding of the carbohydrate moiety of Gb4 to this site of the SLT-IIe binding pentamer . This model is consistent with a wide variety of mutation and binding data and clearly shows the importance of the terminal GalNAc residue of Gb4, as well as that of the two mutated residues of GT3, to the intermolecular interaction.

Biochemistry, 1998 Feb 17, 37(7), 1777 - 88
Structure of the shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3; Ling H et al.; Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans . Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer) . The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin . Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide . The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues . All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane . The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.

Mol Microbiol, 1998 Jan, 27(2), 493 - 505
Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors; Wu H et al.; Expression from the Escherichia coli nir promoter is co-dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions) . Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70 . A novel mechanism to account for co-dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered . DNA sequence elements located upstream of the NarL/NarP-binding site are the targets for two or more proteins that act to repress Fnr-dependent activation of the nir promoter . This inhibition is counteracted by NarL or NarP . The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins . One of these repressors has been identified as the Fis protein, that binds to a site located 135-149bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position -146 . The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position -99.

Mol Microbiol, 1998 Jan, 27(2), 477 - 92
Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate; Hesslinger C et al.; An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme . Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase . Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl . On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase . Expression of the tdcABCDEFG operon was strongly catabolite repressed . Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase . By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate . These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA . These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction . Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.

Mol Microbiol, 1998 Jan, 27(2), 469 - 76
Correlation between requirement for SecA during export and folding properties of precursor polypeptides; de Cock H et al.; The structural complexity of a ligand in association with the molecular chaperones SecB and SecA was investigated using three species of precursor maltose-binding protein, which differ in their stability as a result of an amino acid substitution in each that affects the rate of folding of the polypeptide . In the presence of high concentrations of both SecB and SecA, the precursors were translocated in vitro with indistinguishable kinetics . However, when SecA was limiting, the translocation was more rapid for precursor species, which had lower stability in the native state relative to the stability of the wild-type precursor . We propose that, when in complex with SecB, precursors can form an element of tertiary structure and that these tertiary contacts are blocked when SecA is bound.

Mol Microbiol, 1998 Jan, 27(2), 455 - 68
MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond; Mayer C et al.; The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria . In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail . As in Escherichia coil, regulation takes place at the level of translation . MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E . coli, was shown to be an autoregulator of the MvaL1 operon . The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome . Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1 . Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies . In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron . A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co-regulatory function of MvaL10, the homologue of the regulatory protein L10 of the beta-operon of E . coil . However, we show that MvaL10 does not have a regulatory function.

Mol Microbiol, 1998 Jan, 27(2), 415 - 24
Genetic analysis of the stationary phase-induced mcb operon promoter in Escherichia coli; Mao W et al.; A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase-induced promoter of the mcb operon . Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter . Sequences between -344 and -164 upstream of the transcriptional start site were required for wild-type levels of mcb transcription in stationary phase . More dramatic reductions in both exponential and stationary phase expression were observed when sequences from -164 to -54 were deleted . Point mutations located between -105 and -138 decreased both exponential and stationary phase expression . All but one of these mutations decreased OmpR-dependent activation of Pmcb transcription . EmrR, also known as MprA, acts directly or indirectly at sequences downstream of -54 to repress Pmcb . A minimal promoter containing sequences from -34 to +79 was still induced > or = 10-fold in stationary phase . Point mutations within this region identified sequences at -8, -11, -30, -31 and -32 as important for Pmcb activity . These bases are in the gearbox sequence, present in Pmcb and several other stationary phase-induced Escherichia coli promoters.

Mol Microbiol, 1998 Jan, 27(2), 381 - 90
Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc; Decker K et al.; The maltose regulon consists of 10 genes encoding a multicomponent and binding protein-dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars . MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes . MalK, the energy-transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced . We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK . The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein . The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression . By DNasel protection assay, we found that Mlc protected a DNA region comprising positions +1 to +23 of the malT transcriptional start point . Using a mlc-lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression . As Mlc also regulates another operon (manXYZ, see pages 369-379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization.

Mol Microbiol, 1998 Jan, 27(2), 369 - 80
Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS; Plumbridge J; The manXYZ operon of Escherichia coli encodes a sugar transporter of the phosphoenol pyruvate (PEP)-dependent phosphotransferase system, which is capable of transporting many sugars, including glucose, mannose and the aminosugars, glucosamine and N-acetylglucosamine . Transcription of manX is strongly dependent on cyclic AMP (cAMP)/cAMP receptor protein (CAP) . A cAMP/CAP binding site is located at -40.5, and activation by cAMP/CAP is shown to be typical of a class II promoter . The 5' end of a transcript, potentially encoding two proteins, is expressed divergently from the manXYZ operon . Previously, two binding sites for the NagC repressor were detected upstream of manX, but a mutation in nagC has very little effect on manX expression . However, a mutation in the mlc gene, encoding a homologue of nagC, results in a threefold derepression of manX expression, suggesting that this protein is a more important regulator of manX expression than NagC . The Mlc protein binds to the NagC operators, binding preferentially to the promoter-proximal operator . Plasmids overproducing either the NagC protein or the Mlc protein repress the expression of manX, but the effect of the Mlc protein is stronger . The mlc gene is shown to be allelic with the previously characterized dgsA mutation affecting the mannose phosphoenolpyruvate-dependent phosphotransferase system (PTS).

Mol Microbiol, 1998 Jan, 27(2), 257 - 68
Cell division is required for resolution of dimer chromosomes at the dif locus of Escherichia coli; Steiner WW et al.; The dif locus is a RecA-independent resolvase site in the terminus region of the chromosome of Escherichia coli . The locus reduces dimer chromosomes, which result from sister chromatid exchange, to monomers . A density label assay demonstrates that recombination occurs at dif, and that it requires XerC and XerD . The frequency of this recombination is approximately 14% per site per generation, which is doubled in polA12 mutants . We have determined that recombination occurs late in the cell cycle, and that resolution is blocked if cell division is inhibited with cephalexin or by a ftsZts mutation . Fluorescence microscopy has demonstrated that abnormal nucleoids are present in cells incubated in cephalexin, and this is increased in polA12 mutants.

Br J Pharmacol, 1998 Jan, 123(1), 5 - 12
Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide-like pathway; Hoang LM et al.; 1 . In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+-activated K+ channels (BK channels) by internally applied E . coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells . 2 . Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4 degrees C for 2-4 days before recording with standard patch-clamp techniques . 3 . Acute application of LPS (100 microg ml(-1)) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3+/-0.30 fold . 4 . Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 microM) to inside-out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8+/-0.2 fold over control . 5 . Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels . 6 . Neither LPS nor SNP altered the reversal potential or conductance of BK channels . 7 . The NOS substrate L-arginine (1 microM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D-arginine (1 microM) inhibited the action of LPS on BK channels . 8 . The acute activation of BK channels by LPS was suppressed by pre-incubation of cells with N(omega)-nitro-L-arginine (50 microM) or N(omega)-nitro-L-arginine methyl ester (1 mM), two competitive antagonists of nitric oxide synthases . N(omega)-nitro-D-arginine (50 microM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS . 9 . These results indicate that excised, inside-out patches of CVSMC membrane exhibit a NOS-like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface . Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.

Br J Cancer, 1998 Feb, 77(4), 537 - 46
Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins; Deonarain MP et al.; Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties . We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold . This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase . As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery . We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin . All these molecules are produced in Escherichia coli (E . coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity . Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

Biochem Pharmacol, 1998 Feb 1, 55(3), 313 - 7
Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis; Kakuta Y et al.; Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones . The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively . In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester . These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol . Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol . The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A.

Oncogene, 1998 Feb 19, 16(7), 883 - 90
Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein; Bessard AC et al.; Recombinant human p53 isolated either from E . coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA . We report here that Xenopus p53 has a very similar behavior . Using a new set of monoclonal antibodies directed either to the amino- or the carboxy-terminus of Xenopus p53, we demonstrate that the frog protein can be activated by specific carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element . In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxy-terminus of both p53 . Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent . This is confirmed by the finding that small poly-lysine peptides can activate both human and Xenopus p53 . In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 32 degrees C since activity at 37 degrees C, a temperature well above the natural temperature of Xenopus, is lost . Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53.

Plant Mol Biol, 1998 Feb, 36(3), 393 - 405
Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin; Bak S et al.; A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated . A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied . Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained . Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis . In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile . In vivo administration of oxime to E . coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E . coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction . CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin . Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e . the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin . Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.

Plant Mol Biol, 1998 Jan, 36(1), 43 - 54
Stress responses in alfalfa (Medicago sativa L) . XXII . cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase; He XZ et al.; Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism . The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4' position (B-ring) of daidzein . Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo . Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones . A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources . The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum . The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE) . The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain . Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo . Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation . The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed.

Plant Mol Biol, 1998 Mar, 36(4), 553 - 63
RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen; Beecher B et al.; Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives . For example, SI Nicotiana alata rejects pollen from SC N . plumbaginifolia, but the reciprocal pollination is compatible . This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection . However, the two systems differ in degree of specificity . In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype . Rejection of N . plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase . Here, we have tested whether a non-S-RNase can cause rejection of N . plumbaginifolia pollen . The Escherichia coli rna gene encoding RNAseI was engineered for expression in transgenic (N . plumbaginifolia x SC N . alata) hybrids . Expression levels and pollination behavior of hybrids expressing E . coli RNaseI were compared to controls expressing SA2-RNase from N . alata . Immunoblot analysis and RNase activity assays showed that RNaseI and SA2-RNase were expressed at comparable levels . However, expression of SA2-RNase caused rejection of N . plumbaginifolia pollen, whereas expression of RNaseI did not . Thus, in this system, RNase activity alone is not sufficient for rejection of N . plumbaginifolia pollen . The results suggest that S-RNases may be specially adapted to function in pollen rejection.

Plant Mol Biol, 1998 Mar, 36(4), 521 - 8
Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants; Davis SJ et al.; Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems . However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility . It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein . Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP) . The excitation and emission spectra for this protein are nearly identical to wild-type GFP . When introduced into A . thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form . Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP) . The increased fluorescence output of smGFP will further the use of this reporter in higher plants . In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

Plant Mol Biol, 1998 Mar, 36(4), 509 - 20
Expression of the tobacco anionic peroxidase gene is tissue-specific and developmentally regulated; Klotz KL et al.; Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation . Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv . Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli beta-glucuronidase (GUS) coding region and the nopaline synthase terminator . Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ . In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages . In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues . Activity in roots was limited to cortical cells and vascular-associated parenchyma cells . In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis . Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N . sylvestris . These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development.

Plant Mol Biol, 1998 Jan, 36(2), 307 - 14
The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability; Rott R et al.; A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure . These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators . Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products . Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract . To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E . coli thrA by biolistic transformation of Chlamydomonas chloroplasts . Each 3' UTR was inserted in both the sense and antisense orientations . The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted . However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation . These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

Plant Mol Biol, 1998 Jan, 36(2), 219 - 27
A flavonoid 7-O-methyltransferase is expressed in barley leaves in response to pathogen attack; Christensen AB et al.; We have shown previously that transcripts corresponding to the cDNA clone pBH72-F1, with similarities to O-methyltransferases (OMT), accumulated in barley leaves in response to attack by the pathogenic fungus Blumeria graminis (Plant Mol Biol 26 (1994) 1797) . To investigate the accumulation pattern in the defence response and the organ localization of the pBH72-F1-encoded polypeptide (F1-OMT), an antiserum was raised against Escherichia coli expressed F1-OMT . The 43 kDa protein was absent in normal leaves but accumulated strongly in response to pathogen attack . The F1-OMT protein accumulated faster in barley lines inoculated with an avirulent B . graminis isolates compared to a virulent isolate . Additionally, F1-OMT related proteins were detected in developing kernels . F1-OMT was expressed as a functional enzyme in E . coli and the substrate specificity was investigated . The enzyme exhibited OMT activity towards flavonoid aglycones with the highest activity against apigenin (4',5,7-trihydroxyflavone) . In contrast, caffeic acid did not serve as substrate for F1-OMT . The product of F1-OMT was analyzed by HPLC and GC-MS and found to be genkwanin (4',5-dihydroxy-7-methoxyflavone) . Initial velocity data were best represented by a sequential bi-bi mechanism, and kinetic parameters of KSAM = 10.9 microM, Kapigenin = 4.6 microM and a specific activity of 0.45 mukat/g were obtained . Barley F1-OMT, apigenin 7-O-methyltransferase, is suggested to be involved in the production of a methylated flavonoid phytoalexin.

Plant Mol Biol, 1998 Jan, 36(2), 195 - 204
The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression; Schwechheimer C et al.; The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression . The contribution of different components in a transcription factor and target gene system was assayed by measuring transcriptional activation . Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays . The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18 . Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency . Evidence is presented that these modifications can result in even more active transcription factors when they are combined . Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels.

Biochemistry, 1998 Feb 10, 37(6), 1722 - 30
Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli; Bhattacharyya D et al.; The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains . The two domains are of nearly equal size and are connected by a hinge region . The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue . This protein functions in DNA repair by direct dealkylation of mutagenic O6-alkylguanine . The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own . Each of the two domains functions independently as an alkyl acceptor . The purified homogeneous protein is unstable at 37 degrees C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity . However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation . Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein . This temperature-dependent inactivation of the Ada protein is inhibited by DNA . In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure . The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea . The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant . The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine . These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.

Biochemistry, 1998 Feb 10, 37(6), 1697 - 705
Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction; Peterson KL et al.; The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376 . By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties . The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA . There was no selective solvent deuterium isotope effect on enzyme catalysis . These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376 . The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction {Kumar, N . R., and Srivastava, D . K . (1994) Biochemistry 33, 8833-8841} . Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.

Biochemistry, 1998 Feb 10, 37(6), 1632 - 9
Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase; Mogi T et al.; Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump . Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines . This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I . Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp . Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity . Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center . On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.

Neuroscience, 1998 Apr, 83(3), 691 - 700
Transgenic mice with cerebral expression of human immunodeficiency virus type-1 coat protein gp120 show divergent changes in short- and long-term potentiation in CA1 hippocampus; Krucker T et al.; The human immunodeficiency virus type-1 envelope glycoprotein gp120 is shed from the virus and from infected cells and thus can diffuse and interact with a variety of central nervous system cells . Transgenic mice constitutively expressing glial fibrillary acidic protein-driven gp120 from brain astrocytes display neuronal and glial changes resembling abnormalities in human immunodeficiency virus type-1-infected human brains . To assess the neurophysiology of these transgenic mice and determine whether gp120 expression impairs synaptic plasticity, we examined CA1 population excitatory postsynaptic potentials in hippocampal slices from transgenic mice and from non-transgenic controls, using a double-blind protocol . Compared with slices from non-transgenic littermate controls, slices from gp120 transgenic mice showed four significant alterations: (i) increased mean slopes of normalized population excitatory postsynaptic potentials; (ii) larger paired-pulse facilitation after induction of long-term potentiation at 50 ms interpulse intervals; (iii) markedly elevated short-term potentiation after 10 and 20 shocks at 100 Hz; and (iv) a significant reduction in the magnitude of CA1 long-term potentiation . In slices from transgenic mice expressing Escherichia coli beta-galactosidase from the same promoter, paired-pulse facilitation and long-term potentiation were normal . These results indicate that brain slice preparations from gp120 transgenic mice can be used to assess pathophysiological effects of gp120 on neuronal networks . Because short-term potentiation involves presynaptic mechanisms, our results suggest that gp120 expression in these mice enhances either presynaptic glutamate release or postsynaptic glutamate receptor function, or both . These changes could lead to increased Ca2+ influx, thereby contributing to neuronal dysfunction and injury . As long-term potentiation is a cellular model of learning and memory, our results may be relevant to memory (cognitive) impairments seen in patients with AIDS.

Mech Ageing Dev, 1997 Dec 30, 99(3), 257 - 71
Transgenic mouse models for studying mutations in vivo: applications in aging research; Vijg J et al.; To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed . The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations . Procedures have been worked out to efficiently recovery the plasmids into E . coli host cells . A positive selection system, permitting only E . coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered . Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain . Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Int Arch Allergy Immunol, 1998 Feb, 115(2), 150 - 6
Involvement of the N-terminus of Der p 2 in IgE and monoclonal antibody binding; Hakkaart GA et al.; The major house dust mite allergen Der p 2 was expressed as a recombinant fusion protein in Escherichia coli either with glutathione-S-transferase as fusion partner or with a poly-histidine tag . Both recombinant fusion proteins failed to react with 3/14 Der p 2-specific monoclonal antibodies (mAbs) . When Der p 2 was expressed in yeast with one alanine linked N-terminally to the allergen, no reactivity was observed . When expressed without any fusion partner, all 14 mAbs showed reactivity . The addition of a single N-terminal alanine also disrupted an important epitope for IgE . In RAST inhibitions, an average decrease in inhibitory potency of 72+/-32% was observed (n = 16) with a maximum decrease of 91% . These observations suggest that the N-terminus of Der p 2 is involved in an important epitope for IgE that is disrupted by the addition of one single amino-acid . Recombinant Der p 2 molecules should therefore preferably lack any fusion peptide.

Nucl Med Commun, 1997 Dec, 18(12), 1189 - 93
The value of 67Ga-citrate scanning in psoas abscess; Chiu NT et al.; Psoas abscess is a serious health problem which presents with non-specific symptoms and signs . To reduce morbidity and mortality, it is important to diagnose the presence and extent of a psoas abscess accurately using imaging studies . Because the 67Ga scan may facilitate the early diagnosis of insidious infection and assist CT-guided percutaneous drainage of the abscess, we examined the value of 67Ga scans in 18 patients with psoas abscess . The imaging results of 67Ga scans (18 patients), computed tomography (CT) (16 patients) and bone scans (13 patients) were analyzed . In this series, concomitant infections were very common (94%) in patients with psoas abscess . For detecting psoas abscess, the sensitivity of 67Ga scanning (92%) and CT (91%) was similar . However, 67Ga scanning is superior to CT in demonstrating concomitant infectious foci at other sites . Bone scanning is a sensitive tool for depicting osteomyelitis, which was common in this series of patients . We also found that increased vascularity in the psoas area was demonstrated by three-phase bone scanning in 60% of patients.

Comp Immunol Microbiol Infect Dis, 1997 Sep, 20(4), 299 - 307
Role of iron and iron chelation therapy in oxygen free radical mediated tissue injury in an ascending mouse model of chronic pyelonephritis; Gupta R et al.; The contribution of iron towards the free radical generation leading to renal tissue damage was assessed using a non-obstructive ascending mouse model for chronic pyelonephritis . The parameters studied include luminol dependent chemiluminescence (LDCL), histopathology and some biochemical investigations . We found that iron enhanced the renal tissue damage and led to renal scarring, and end point in chronic renal inflammation, irrespective of the bacterial strain studied . In addition a role of iron chelation therapy as a treatment for chronic renal inflammation is also suggested.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 538 - 44
Molecular cloning of human D-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation; Nishihira J et al.; D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5,6-quinone (D-dopachrome) into 5,6-dihydroxyindole . This protein has an amino acid sequence that is highly homologous with that of macrophage migration inhibitory factor (MIF), which has the potential to catalyze D-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and is an important cytokine for T-lymphocyte activation . We isolated and sequenced a 566 bp-long cDNA encoding human D-dopachrome tautomerase . The cDNA contains an open reading frame encoding 118 amino acids, including the initiator methionine . The amino acid sequence of the protein shares 80% homology with that of the rat enzyme . Northern blot analysis demonstrated that mRNA of D-dopachrome tautomerase is expressed in a large amount in the liver, and to lesser extent in other organs, including the heart, lung and pancreas . After purification of D-dopachrome tautomerase expressed in E . coli, we confirmed that the recombinant protein catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole . Its catalytic mechanism is not well understood . We found that the protein completely lost the enzyme activity when the N-terminal proline residue was replaced with alanine by site-directed mutagenesis . This fact suggests that the N-terminal proline is essential for the catalytic mechanism . Although the precise pathophysiological function of D-dopachrome tautomerase remains to be elucidated, the present results could contribute to further understanding of isomerase activity in relation to the immune response.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 438 - 43
Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber; Chen X et al.; We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs . Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs . We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column . Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate . The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX . When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 372 - 6
Role of glycosylation in the lipid-binding activity of the exchangeable apolipoprotein, apolipophorin-III; Soulages JL et al.; Non-glycosylated recombinant Locusta migratoria apolipophorin-III, apoLp-III, was expressed in E . coli and its physical-chemical properties were compared to those of the glycosylated native apoLp-III . Fluorescence quantum yield and acrylamide quenching studies indicated a slightly higher accessibility of the Trp residues in the recombinant apoLp-III . Far-UV CD spectroscopy indicated that the recombinant apoLp-III has a lower alpha-helical content than the glycosylated apoLp-III . Both proteins spontaneously formed discoidal recombinant lipoprotein particles when incubated with dimyristoylphosphatidylcholine (DMPC) . Interaction with lipid promotes an increase in alpha-helical content . CD and fluorescence studies indicate that both proteins adopt the same conformation in the lipid-bound state . However, the kinetics of association of the recombinant protein with DMPC is 5-fold faster than that of the native protein . The results suggest that glycosylation inhibits the lipid binding activity by preventing the exposure of hydrophobic domains and/or decreasing the conformational flexibility of the protein.

Schweiz Arch Tierheilkd, 1997, 139(11), 495 - 9
{Current resistance status of Escherichia coli strains from bovine mastitis milk samples}; Stephan R et al.; Between December 1996 and March 1997, 95 E.coli strains were isolated from mastitis milk samples from 95 different animals . In 29.5% resistance could be observed against one or several of the examined antibiotics . However, Cefoperazone, Polymyxin B, Colistin and Gentamycin proved effective against the majority of these strains . Between 0 and 23% of E.coli were resistant against the single tested antibiotics . As opposed to earlier investigations a smaller number of Chloramphenicol-resistant strains could be found . One strain with multiple resistance was also resistant against Gentamycin . However, in the treatment of mastitis the resistance of a particular agent is only one among several contributing factors.

Genetika, 1997 Nov, 33(11), 1487 - 93
{Genetic study of the mechanism of tandem duplication formation in conjugational crosses in Escherichia coli K-12}; Sukhodolets VV; The formation of heterozygous tandem duplications of the deo operon was studied in conjugational matings HfrH deoA deoB::Tn5 thyA x HfrH deoC deoD thyA . When the HfrH deoC deoD thr::Tn9 car::Tn10 thyA donor strain was used, the thr::Tn9 and car::Tn10 transposon insertions linked to the deo operon were integrated into some duplications . Duplications carrying the recipient thr+ and car+ alleles in the duplicated state were not found among duplications that did not include the thr::Tn9 and car::Tn10 donor markers . These data indicate that heterozygous deo operon duplications are primarily formed directly during the recombinational interaction between chromosomes in the merozygote, but not on the basis of preexisting sister duplications in the recipient strain . Thus, genetic recombination occurring during the process of conjugation in E . coli induces both symmetrical and unequal crossing over . Nevertheless, when the HfrH deoA deoB::Tn5 thr::Tn9 car::Tn10 thyA strain was used as a recipient, a deo operon duplication containing thr::Tn9 and car::Tn10 markers was found in the homozygous state . Consequently, some heterozygous duplications can also be formed on the basis of preexisting sister duplications.

Science, 1998 Feb 6, 279(5352), 873 - 6
Conjugative transfer by the virulence system of Legionella pneumophila; Vogel JP et al.; Legionella pneumophila, the causative agent of Legionnaires' pneumonia, replicates within alveolar macrophages by preventing phagosome-lysosome fusion . Here, a large number of mutants called dot (defective for organelle trafficking) that were unable to replicate intracellularly because of an inability of the bacteria to alter the endocytic pathway of macrophages were isolated . The dot virulence genes encoded a large putative membrane complex that functioned as a secretion system that was able to transfer plasmid DNA from one cell to another.

J Biol Chem, 1998 Jan 30, 273(5), 3076 - 81
Human small intestinal maltase-glucoamylase cDNA cloning . Homology to sucrase-isomaltase; Nichols BL et al.; It has been hypothesized that human mucosal glucoamylase (EC 3.2.1 . 20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal alpha-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing . As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments . Human maltase-glucoamylase was purified by immunoisolation and partially sequenced . Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction . The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da) . Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous . Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities . Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.

J Biol Chem, 1998 Jan 30, 273(5), 3045 - 50
Kinetic analysis of the catalytic mechanism of serotonin N-acetyltransferase (EC 2.3.1.87); De Angelis J et al.; Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin biosynthesis . This enzyme is of special biological interest because large changes in its activity drive the large night/day rhythm in circulating melatonin in vertebrates . In this study the kinetic mechanism of AANAT action was studied using bacterially expressed glutathione S-transferase (GST)-AANAT fusion protein . The enzymologic behavior of GST-AANAT and cleaved AANAT was essentially identical . Two-substrate kinetic analysis generated an intersecting line pattern characteristic of a ternary complex mechanism . The dead end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus tryptamine . Tryptophol was not an alternative substrate but was a dead end competitive inhibitor versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acetyl-CoA first . N-Acetyltryptamine, a reaction product, was a noncompetitive inhibitor versus tryptamine and uncompetitive with respect to acetyl-CoA . Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framework for inhibitor design.

J Biol Chem, 1998 Jan 30, 273(5), 3033 - 8
A novel alternatively spliced form of murine vascular endothelial growth factor, VEGF 115; Sugihara T et al.; Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115 . It differs from VEGF 120 by 37 amino acids at the carboxyl terminus . VEGF 115-specific sequence reacted to a single transcript in mouse tissues . Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes . The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter . The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody . A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells . Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones . We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115 . The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

J Biol Chem, 1998 Jan 30, 273(5), 2799 - 807
Enzymatic synthesis of lipopolysaccharide in Escherichia coli . Purification and properties of heptosyltransferase i; Kadrmas JL et al.; Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core . Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose . The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available . In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA . Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity . NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product . The subunit molecular mass is 36 kDa . Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5 . The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA . Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation . The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA . The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose . Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs . Pure RfaC can also be used together with Kdo2-{4'-32P}lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.

J Biol Chem, 1998 Jan 30, 273(5), 2639 - 44
Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom; Koschak A et al.; Five novel peptidyl inhibitors of Shaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus . The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli . HgTX1 inhibits 125I-margatoxin binding to rat brain membranes as well as depolarization-induced 86Rb+ flux through homotetrameric Kv1.1, Kv1 . 2, and Kv1.3 channels stably transfected in HEK-293 cells, but it displays much lower affinity for Kv1.6 channels . A HgTX1 double mutant (HgTX1-A19Y/Y37F) was constructed to allow high specific activity iodination of the peptide . HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting 125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values approximately 0.3 pM) . 125I-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels and to rat brain membranes (Kd values 0.1-0.25 pM, respectively) but with lower affinity to Kv1.6 channels (Kd 9.6 pM), and it does not interact with either Kv1.4 or Kv1.5 channels . Several subpopulations of native Kv1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using antibodies specific for Kv1 subunits . HgTX1 represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and Kv1 subunit assembly in different tissues.

J Biol Chem, 1998 Jan 30, 273(5), 2610 - 6
Molecular cloning of the cDNA encoding human deoxyribonuclease II; Yasuda T et al.; A rapid amplification of cDNA ends method, using degenerate oligonucleotides based upon the N-terminal amino acid sequence of human hepatic deoxyribonuclease II (DNase II), allowed a novel cDNA encoding DNase II to be constructed from thyroid gland RNA . The composite nucleotide sequence (1593 bases) included an open reading frame of 1080 bases, which encoded a single polypeptide of 360 amino acids (signal peptide, 16; propeptide, 91; mature protein, 253) . Although the sequence of DNase II showed no significant homology to other mammalian proteins, its cDNA structural organization resembled those of the lysosomal cathepsin families . The two parts of the cDNA corresponding to the propeptide and the mature protein were expressed in Escherichia coli, and the recombinant polypeptides thus obtained were strongly stained with an anti-DNase II antibody on Western blotting . DNase II is ubiquitously expressed in human tissues, and the DNase II gene (DNASE2) was assigned to chromosome 19.

J Biol Chem, 1998 Jan 30, 273(5), 2553 - 60
Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1); Vitale N et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation . Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently . A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis . ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension . We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins . The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1 . Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis . The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs . Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension . The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.

J Biol Chem, 1998 Jan 30, 273(5), 2532 - 42
Identification and partial purification of human double strand RNase activity . A novel terminating mechanism for oligoribonucleotide antisense drugs; Wu H et al.; We have identified a double strand RNase (dsRNase) activity that can serve as a novel mechanism for chimeric antisense oligonucleotides comprised of 2'-methoxy 5' and 3' "wings" on either side of an oligoribonucleotide gap . Antisense molecules targeted to the point mutation in codon 12 of Harvey Ras (Ha-Ras) mRNA resulted in a dose-dependent reduction in Ha-Ras RNA . Reduction in Ha-Ras RNA was dependent on the oligoribonucleotide gap size with the minimum gap size being four nucleotides . An antisense oligonucleotide of the same composition, but containing four mismatches, was inactive . When chimeric antisense oligonucleotides were prehybridized with 17-mer oligoribonucleotides, extracts prepared from T24 cells, cytosol, and nuclei resulted in cleavage in the oligoribonucleotide gap . Both strands were cleaved . Neither mammalian nor Escherichia coli RNase HI cleaved the duplex, nor did single strand nucleases . The dsRNase activity resulted in cleavage products with 5'-phosphate and 3'-hydroxyl termini . Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected . The cytosolic enzyme was purified approximately 165-fold . It has an approximate molecular weight of 50,000-65,000, a pH optimum of approximately 7.0, requires divalent cations, and is inactivated by approximately 300 mM NaCl . It is inactivated by heat, proteinase K, and also by a number of detergents and several organic solvents.

J Biol Chem, 1998 Jan 30, 273(5), 2501 - 4
Modeling charge interactions and redox properties in DsbA; Warwicker J; Accurate prediction of charge interactions in macromolecules presents a significant challenge for computational biology . A model for the low Cys30 pKa and oxidizing power of DsbA (Gane, P . J., Freedman, R . B., and Warwicker, J . (1995) J . Mol . Biol . 249, 376-387) has been investigated experimentally (Hennecke, J., Spleiss, C., and Glockshuber, R . (1997) J . Biol . Chem . 272, 189-195), with substitutions for Glu37 and Glu38 and with residues 38-40 removed . Measured changes in Cys30 pKa and redox potential were relatively small and reported to be in contrast to model predictions . It is now shown, particularly with calculations of wild-type:mutant differences for a range of salt concentrations, that the data are consistent with the model and support the key finding that a number of different factors contribute to the oxidizing power of DsbA, so that any particular one need not necessarily be large . A feature of the model is a low protein dielectric, and higher values (which are becoming popular in predictions of pH dependence) are inconsistent with both the difference data and the wild-type Cys30 pKa.

EMBO J, 1998 Jan 15, 17(2), 626 - 33
A specific 3' exonuclease activity of UvrABC; Gordienko I et al.; Specific cutting of undamaged DNA by UvrABC nuclease is observed . It occurs seven nucleotides (nt) from the 3' terminus of oligonucleotides annealed to single-stranded M13 DNA circles . Although the location of the UvrABC cut on undamaged DNA is similar to that of the cut on the 5' side of a damaged DNA site during the dual incision reaction, the cut of undamaged DNA is not an intermediate in the dual incision step . On DNA duplexes with a single AAF adduct, the anticipated cut at the eighth phosphodiester bond 5' of the lesion is present, but extra cuts at 7-nt increments are observed at the 15th and 22nd phosphodiester bonds . We suggest that these additional cuts are made by the UvrABC activity observed on undamaged DNA; such activity is referred to as ABC 3' exonuclease and may play a significant role by providing a suitable gap for RecA-mediated recombinational exchanges during repair of interstrand crosslinks and closely opposed lesions . This ABC 3' exonuclease activity depends on higher concentrations of Uvr proteins as compared with dual incision and may be relevant to reactions that occur when UvrA and UvrB are increased during SOS induction.

EMBO J, 1998 Jan 15, 17(2), 363 - 7
Release of normal bases from intact DNA by a native DNA repair enzyme; Berdal KG et al.; Base excision repair is initiated by DNA glycosylases removing inappropriate bases from DNA . One group of these enzymes, comprising 3-methyladenine DNA glycosylase II (AlkA) from Escherichia coli and related enzymes from other organisms, has been found to have an unusual broad specificity towards quite different base structures . We tested whether such enzymes might also be capable of removing normal base residues from DNA . The native enzymes from E.coli, Saccharomyces cerevisiae and human cells promoted release of intact guanines with significant frequencies, and further analysis of AlkA showed that all the normal bases can be removed . Transformation of E . coli with plasmids expressing different levels of AlkA produced an increased spontaneous mutation frequency correlated with the expression levels, indicating that excision of normal bases occurs at biologically significant rates . We propose that the broad specificity 3-methyladenine DNA glycosylases represent a general type of repair enzyme 'pulling' bases in DNA largely at random, without much preference for a specific structure . The specificity for release of damaged bases occurs because base structure alterations cause instability of the base-sugar bonds . Damaged bases are therefore released more readily than normal bases once the bond activation energy is reduced further by the enzyme . Qualitatively, the model correlates quite well with the relative rate of excision observed for most, if not all, of the substrates described for AlkA and analogues.

EMBO J, 1998 Jan 2, 17(1), 297 - 305
Genetic code in evolution: switching species-specific aminoacylation with a peptide transplant; Wakasugi K et al.; The genetic code is established in aminoacylation reactions whereby amino acids are joined to tRNAs bearing the anticodons of the genetic code . Paradoxically, while the code is universal there are many examples of species-specific aminoacylations, where a tRNA from one taxonomic domain cannot be acylated by a synthetase from another . Here we consider an example where a human, but not a bacterial, tRNA synthetase charges its cognate eukaryotic tRNA and where the bacterial, but not the human, enzyme charges the cognate bacterial tRNA . While the bacterial enzyme has less than 10% sequence identity with the human enzyme, transplantation of a 39 amino acid peptide from the human into the bacterial enzyme enabled the latter to charge its eukaryotic tRNA counterpart in vitro and in vivo . Conversely, substitution of the corresponding peptide of the bacterial enzyme for that of the human enabled the human enzyme to charge bacterial tRNA . This peptide element discriminates a base pair difference in the respective tRNA acceptor stems . Thus, functionally important co-adaptations of a synthetase to its tRNA act as small modular units that can be moved across taxonomic domains and thereby preserve the universality of the code.

EMBO J, 1998 Jan 2, 17(1), 101 - 12
A novel sec-independent periplasmic protein translocation pathway in Escherichia coli; Santini CL et al.; The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme . The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif . By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase . The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation . The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export . The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins . Depletion of Ffh, a core component of the signal recognition particle of E . coli, appears to have a slight effect on the export of the TMAO reductase . These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.

Biochem J, 1998 Jan 15, 329 ( Pt 2), 275 - 82
Dipeptidyl peptidase III is a zinc metallo-exopeptidase . Molecular cloning and expression; Fukasawa K et al.; We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta . It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-beta-naphthylamide . Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-beta-naphthylamides were not hydrolysed at all . The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme . The zinc dissociation constant was 250 fM at pH 7 . 4 as determined by the zinc binding study . We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme . The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da . Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-beta-naphthylamide . The recombinant protein was purified and the amino acid sequence of the protein was confirmed . We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme . It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

Biochem J, 1998 Jan 15, 329 ( Pt 2), 243 - 7
Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain; Qiu Y et al.; Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits . Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E . coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein . The Vmax for the Asp-90-->Ala and Arg-269-->Ala substitutions is decreased to 1/21 and 1/50 respectively, the {S}0.5 for aspartate is increased 540-fold and 826-fold respectively, and the {S}0.5 for carbamoyl phosphate is increased 60-fold for both . These substitutions decrease the oligomeric size of the protein . Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer . The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme . Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase.

Nucleic Acids Res, 1998 Jan 15, 26(2), 662 - 8
Homogenous repair of singlet oxygen-induced DNA damage in differentially transcribed regions and strands of human mitochondrial DNA; Anson RM et al.; Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture . The primary product of this reaction is the DNA lesion 7-hydro-8-oxo-deoxyguanosine (8-oxo-dG) . The DNA damage was quantitated using Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) in a gene-specific damage and repair assay . Assay conditions were refined to give incision at all enzyme-sensitive sites with minimal non-specific cutting . Cultured fibroblasts were exposed to photoactivated methylene blue under conditions that would produce an average of three oxidative lesions per double-stranded mitochondrial genome . Within 9 h, 47% of this damage had been removed by the cells . This removal was due to repair rather than to replication, cell loss or degradation of damaged genomes . The rate of repair was measured in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region . Fpg-sensitive alkali-resistant oxidative base damage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional activity.

Nucleic Acids Res, 1998 Jan 15, 26(2), 655 - 61
Three-dimensional structure of the yeast ribosome; Verschoor A et al.; The 80S ribosome from Saccharomyces cerevisiae has been reconstructed from cryo electron micrographs to a resolution of 35 A . It is strikingly similar to the 70S ribosome from Escherichia coli, while displaying the characteristic eukaryotic features familiar from reconstructions of ribosomes from higher eukaryotes . Aside from the elaboration of a number of peripherally located features on the two subunits and greater overall size, the largest difference between the yeast and E.coli ribosomes is in a mass increase on one side of the large (60S) subunit . It thus appears more elliptical than the characteristically globular 50S subunit from E.coli . The interior of the 60S subunit reveals a variable diameter tunnel spanning the subunit between the interface canyon and a site on the lower back of the subunit, presumably the exit site through which the nascent polypeptide chain emerges from the ribosome.

Nucleic Acids Res, 1998 Jan 15, 26(2), 650 - 4
Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay; Zaitsev EN et al.; We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein . This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding . The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence . DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not . Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP . These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein . Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.

Nucleic Acids Res, 1998 Jan 15, 26(2), 638 - 44
Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana; Nakajima S et al.; UV radiation induces two major classes of pyrimidine dimers: the pyrimidine {6-4} pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD) . Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage . These photolyases are specific for either CPDs or 6-4 products . A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis . We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana . This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli . We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog . We have therefore termed this gene UVR3 . Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo.

Nucleic Acids Res, 1998 Jan 15, 26(2), 631 - 7
Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus; Zhang D et al.; RPA is the replicative single-strand DNA (ssDNA) binding protein of eukaryotic chromosomes . This report shows that human RPA interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus (EBV) . RPA binds to EBNA1 both in solution, and when EBNA1 is bound to the EBV origin . RPA is a heterotrimer, and the main contact with EBNA1 is formed through the 70 kDa subunit of RPA, the subunit which binds to ssDNA . We propose that this interaction between RPA and EBNA1 is an early step in activation of the latent origin of EBV.

Nucleic Acids Res, 1998 Jan 15, 26(2), 602 - 7
Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases; Lavrik OI et al.; Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes . It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits . To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique . The photoreactive oligonucleotide was synthesized in situ by DNA polymerases . 5-{N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1}deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF) . The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA . The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE . The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA . The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer . This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Nucleic Acids Res, 1998 Jan 15, 26(2), 549 - 53
Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning; Poot RA et al.; Helix 2 of the central pseudoknot structure in Escherichia coli 16S rRNA is formed by a long-distance interaction between nt 17-19 and 918-916, resulting in three base pairs: U17-A918, C18-G917and A19-U916 . Previous work has shown that disruption of the central base pair abolishes ribosomal activity . We have mutated the first and last base pairs and tested the mutants for their translational activity in vivo , using a specialized ribosome system . Mutations that disrupt Watson-Crick base pairing result in strongly impaired translational activity . An exception is the mutation U916-->G, creating an A.G pair, which shows almost no decrease in activity . Mutations that maintain base complementarity have little or no impact on translational efficiency . Some of the introduced base pair substitutions substantially alter the stability of helix 2, but this does not influence ribosome functioning, neither at 42 nor at 28 degrees C . Therefore, our results do not support models in which the pseudoknot is periodically disrupted . Rather, the central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.

Nucleic Acids Res, 1998 Jan 15, 26(2), 537 - 43
Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern; Brule H et al.; Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level . This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes . Here we describe the sequence of human mt-tRNAPropurified from placenta . It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications . These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs . Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase . This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation . Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.

Nucleic Acids Res, 1998 Jan 15, 26(2), 462 - 8
The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision; Moolenaar GF et al.; The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC . The exact requirements for the two incision events, however, are different . It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC . This interaction, however, is dispensable for the 5'-incision . Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision . The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair . Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions . Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding . In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA . Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place . We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.

Nucleic Acids Res, 1998 Jan 15, 26(2), 420 - 6
In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome; Ashoub A et al.; The genetic information of potato leafroll virus (PLRV), a typical member of the subgroup 2 luteoviruses, is contained in a single-stranded (+) sense RNA of approximately 5.9 kb . A single subgenomic RNA (sgRNA1) of approximately 2.3 kb has been characterized as the mRNA for the 3' clustered viral open reading frames ORF3, ORF3/5 and ORF4 . Here we demonstrate by Northern blot analyses of polysomal RNAs from PLRV-infected Solanum tuberosum and Physalis floridana plants that, as with luteoviruses belonging to subgroup 1, in planta synthesis of a second 0.8 kb subgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteoviral genomes significantly . PLRV-specific hybridization probes as well as primer extension experiments map sgRNA2 to the 3'-end of the PLRV RNA genome (positions 5190-5987) . Similarly, for the closely related cucurbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size and position (positions 4888-5669) was identified . PLRV sgRNA2 may code for two viral proteins of 7.1 (ORF6) and 14 kDa (ORF7) respectively, while the CABYV proteins are 8.7 (ORF6) and 8.3 kDa (ORF7) in size, with PLRV ORF7 displaying nucleic acid binding activity . In vivo experiments by transient expression of chimeric GUS fusions in potato protoplasts demonstrated that sgRNA2 functions as a bicistronic mRNA with high expression of ORF6 and low translational efficiency for synthesis of ORF7.

Nucleic Acids Res, 1997 Dec 15, 25(24), 5010 - 6
A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures; Matveeva O et al.; A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis . The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding . In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once . Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension . The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting . New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified . Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.

Nucleic Acids Res, 1997 Dec 15, 25(24), 4946 - 53
A novel nucleic acid-binding protein that interacts with human rad51 recombinase; Kovalenko OV et al.; Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase . Analysis in vitro confirmed the interaction between Rad51 and Pir51 . Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine . The Pir51 gene locus was mapped to chromosome 12p13.1-13 . 2 by fluorescence in situ hybridization . The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity . Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA . The protein also binds RNA . The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.

Nucleic Acids Res, 1997 Dec 15, 25(24), 4899 - 906
Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase; Sissler M et al.; Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArgand non-cognate tRNAAspare described . This work was motivated by the fact that the in vitro transcript of tRNAAspis of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated . The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAspis recognized by its variable region side . Here we show by footprinting with enzymatic and chemical probes that transcribed tRNAAspis contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns . Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs . These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArgand tRNAAsp . The mirror image alternative interaction patterns of unmodified tRNAAspwith either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases . This study indicates that complex formation between unmodified tRNAAspand either ArgRS and AspRS is solely governed by the proteins.

Nucleic Acids Res, 1997 Dec 15, 25(24), 4883 - 90
Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase; Liu M et al.; Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition . Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency . The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors . This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs . Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition . Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m . A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix . The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal . This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.

Vet Immunol Immunopathol, 1997 Nov, 59(3-4), 253 - 70
Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE; Marti E et al.; An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a {His}6-tag and purified over a Ni-NTA column . The recombinant protein was used to immunise hens . Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf) . The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the {His}6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins . In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa . A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions . These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction . Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot . Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE . The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE . It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG . The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner . In addition, preincubation with horse IgG, a nonrelevant {His}6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic . This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

Am J Respir Crit Care Med, 1998 Feb, 157(2), 498 - 504
Nitric oxide biosynthesis in an exotoxin-induced septic lung model: role of cNOS and impact on pulmonary hemodynamics; Schutte H et al.; Nitric oxide (NO) is an important vasodilator that is produced by constitutive (cNOS) as well as inducible (iNOS) isoforms of nitric oxide synthase . The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E . coli infections, was found to be a potent stimulator of NO liberation in isolated endothelial cells, and that it also causes thromboxane generation and related vasoconstriction in rabbit lungs . We investigated the effect of different concentrations of HlyA on pulmonary NO synthesis in buffer-perfused rabbit lungs . NO release into the alveolar as well as the intravascular compartment was monitored on-line by chemiluminescence detection of expired NO and by measurement of (peroxy-)nitrite/nitrate release into the perfusate . HlyA induced a pressor response and an immediate dose-dependent increase of exhalative and intravascular NO liberation, further enhanced by the addition of the NOS substrate L-arginine . The nonspecific NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), but not the iNOS selective inhibitors aminoguanidine and 2-(2-aminoethyl)-2-thiopseudourea-dihydrobromide, blocked the HlyA-evoked NO liberation into both the alveolar and the intravascular compartments . Enhancement of NO formation (L-arginine) slightly reduced, and inhibition of NO synthesis (L-NMMA) amplified greatly, the HlyA-elicited vasoconstrictor response . Inhibition of the pressor response by a thromboxane receptor antagonist did not interfere with the exotoxin-elicited NO formation . We conclude (1) that marked NO biosynthesis occurs in this model of the septic lung, (2) that the signal transduction in response to HlyA proceeds via activation of cNOS directly related to exotoxin activity and not to secondary changes in shear stress, and (3) that this vasodilator release mitigates the HlyA-induced pulmonary vasoconstriction . These findings may have important implications for therapeutic approaches using NOS inhibitors in sepsis.

FEBS Lett, 1998 Jan 23, 422(1), 79 - 84
C-terminal end of v-src protein interacts with peptide coded by gadd7/adapt15-like RNA in two-hybrid system; Mizenina O et al.; The significant differences in the metastatic properties of hamster fibroblasts transformed by the Rous sarcoma virus (RSV) were associated with mutations in the v-src carboxy-terminal region . To identify the capacity of this region for protein-protein interaction the two-hybrid system was used . The cDNA clone (vseap1), producing the protein specifically bound with the v-src C-terminal part in yeast cells in vivo and in GST-fusion system in vitro was isolated . Vseap1 shared 68% of homology with stressful agents induced RNA-gadd7/adapt15 . Two vseap1 specific messenger RNAs were identified: 0.9-kbp RNA expressed in all transformed cells and three times less in embryo fibroblasts; 3.1-kbp transcript was deleted in the cells with suppressed v-src activity and H2O2 resistance.

FEBS Lett, 1998 Jan 23, 422(1), 19 - 22
A reappraisal of the mechanism of the photoenzyme protochlorophyllide reductase based on studies with the heterologously expressed protein; Townley HE et al.; It is widely believed that protochlorophyllide reductase is a flavoenzyme effecting catalysis by a radical mechanism . Here the cyanobacterial reductase has been isolated from Escherichia coli overexpressing the Synechocystis gene . The purified enzyme, while retaining full activity, has no detectable flavine . No radical derived ESR signal was observed during catalysis or on photoexcitation under non-catalytic conditions . Mechanistic implications of the findings are discussed.

FEBS Lett, 1998 Jan 23, 422(1), 5 - 9
A novel centromere monospecific serum to a human autoepitope on the histone H3-like protein CENP-A; Valdivia MM et al.; Centromere autoantibodies are commonly found in the serum of patients with some systemic autoimmune diseases . Previous studies have shown that a major human centromere autoantigen is the histone H3-like protein CENP-A . Although the human cDNA has been cloned, native CENP-A has been neither isolated nor expressed in Escherichia coli, and specific antibodies to this chromatin-associated centromere protein are not available yet . In this report, a highly charged peptide on CENP-A (residues 3-17) was used to generate a monospecific antibody that reacts by immunoblots with the 17 kDa centromeric protein . Immunofluorescence analysis showed reactivity of this anti-CENP-A serum in several but not all mammalian culture cells analyzed, suggesting that the sequence of this histone-like centromere protein could be more variable throughout evolution than originally thought . Selective extractions of human placenta nuclear proteins and immunoblot analysis indicated that CENP-A behaves in a similar way to the core histone polypeptides after nuclease digestion of chromatin . Also, immunoblot analysis demonstrated that the CENP-A peptide used as immunogen is a target region on the CENP-A molecule in several but not all CREST patients analyzed with high titers of autoantibodies to the centromere . Lastly, we found that in Jurkat cells induced to apoptosis, CENP-A remains associated with the centromere, in contrast to other human autoantigens studied during apoptosis.

Indian J Exp Biol, 1997 Oct, 35(10), 1076 - 9
Optimisation of immunoaffinity purification of Wuchereria bancrofti specific antibodies from human sera; Kannan K et al.; Immunoaffinity column using Setaria digitata antigens coupled to cyanogen bromide activated Sepharose 4B beads were developed to purify antibodies from sera of filarial patients . Chaotropic (KSCN) ion elution was more efficient for purifying specific antibodies from the column in comparison to }c elution . Dot blot analysis indicated that purified antibodies showed a high degree of reactivity with cattle filarial antigen and recombinant filarial protein but not with bacterial proteins of E . coli suggesting that the antibodies are specific.

Biochim Biophys Acta, 1998 Jan 21, 1395(2), 192 - 201
Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase; Fukuoka SI et al.; Mammalian quinolinate phosphoribosyltransferase (QPRTase) (EC 2.4.2.19) is a key enzyme in catabolism of quinolinate, an intermediate in the tryptophan-nicotinamide adenine dinucleotide (NAD) pathway . Quinolinate acts as a most potent endogenous exitotoxin to neurons . Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders . As the first step to elucidate molecular basis underlying the quinolinate metabolism, the cDNA encoding human brain QPRTase was cloned and characterized . Utilizing partial amino acid sequences obtained from highly purified porcine kidney QPRTase, a human isolog was obtained from a human brain cDNA library . The cDNA encodes a open reading frame of 297 amino acids, and shares 30 to 40% identity with those of bacterial QPRTases . To confirm that the cDNA clone encodes human QPRTase, its functional expression was studied in a bacterial host . Introduction of the human cDNA into a QPRTase defective (nadC) E . coli strain brought about an abrupt increase in QPRTase activity and allowed the cells to grow in the absence of nicotinic acid . It is concluded that the cloned cDNA encodes human QPRTase which is functional beyond the phylogenic boundary.

Biochim Biophys Acta, 1998 Jan 21, 1395(2), 181 - 91
Two types of chicken 2',5'-oligoadenylate synthetase mRNA derived from alleles at a single locus; Yamamoto A et al.; We have isolated two types of chicken 2',5'-oligoadenylate synthetase cDNAs, A and B, which encode predicted proteins of 508 amino acids (58316 Da) and 476 amino acids (54336 Da), respectively . The region of A-protein comprising 33 amino acid residues from 385Ala to 417Cys is substituted by a single amino acid 385Tyr in B-protein . The homology between chicken and mammalian 2',5'-oligoadenylate synthetases is 49.5% over the amino-terminal 337 residues . Proteins expressed from A- and B-cDNAs in E . coli cells were both active in synthesizing 2',5'-oligoadenylate . However, the activity of B-protein was 10-15% of that of A-protein . Southern blotting hybridization indicated that the chicken synthetases are encoded by a single gene . RT-PCR and PCR analyses of RNA and DNA of chicken erythrocytes together with the sequence data of the PCR products showed that A- and B-mRNAs are derived from alleles at a single locus encoding chicken 2',5'-oligoadenylate synthetase, designated as OAS * A and OAS * B . Chickens carrying OAS * A/B produce two types of synthetase with molecular masses of 58 and 54 kDa, and those carrying OAS * A/A produce only a single type of 58 kDa.

Biochim Biophys Acta, 1998 Jan 21, 1395(2), 135 - 40
Characterisation of the mob locus from Rhodobacter sphaeroides required for molybdenum cofactor biosynthesis; Palmer T et al.; A clone carrying the mob locus from Rb . sphaeroides WS8 has been isolated from a cosmid library by Southern blotting with a probe covering the mob genes of Escherichia coli . The mob DNA has been subcloned and partially restores molybdoenzyme activities when transformed into E . coli mob strains . DNA sequence analysis of the subclone carrying the mob genes predicted at least 2 open reading frames . The mobA gene encodes protein FA whilst mobB encodes a nucleotide binding protein which has at least one extra domain relative to its E . coli counterpart.

Brain Res, 1998 Jan 5, 780(1), 119 - 28
In vivo gene transfer into the adult mammalian central nervous system by continuous injection of plasmid DNA-cationic liposome complex; Imaoka T et al.; Previously reported methods of liposome-mediated direct in vivo gene transfer have been inefficient, especially when performed with highly differentiated, quiescent cells of the adult mammalian central nervous system (CNS) . We have therefore improved these procedures based upon a novel concept . Following continuous injection of plasmid DNA-cationic liposome complex which contained a reporter gene encoding E . coli beta-galactosidase into the striatum of adult rats, the expression of transgene was dramatically elevated without any adverse effects . This new technique may enable a wide application of liposome-mediated gene transfer technology not only to basic analysis of gene functions in the brain but also for clinical treatment of certain CNS disorders.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 302 - 6
Cloning and correct expression in Escherichia coli of the petE and petJ genes respectively encoding plastocyanin and cytochrome c6 from the cyanobacterium Anabaena sp . PCC 7119; Molina-Heredia FP et al.; The genes coding for plastocyanin (petE) and cytochrome c6 (petJ) from Anabaena sp . PCC 7119 have been cloned and properly expressed in Escherichia coli . The recombinant proteins are identical to those purified from the cyanobacterial cells . The products of both the petE and petJ genes are correctly processed in E . coli, as deduced from their identical N-terminal amino acid sequences as compared with those of the metalloproteins isolated from the cyanobacterium . Physicochemical and functional properties of the native and recombinant protein preparations are also identical, thereby confirming that expression of petE and petJ genes in E . coli is an adequate tool to address the study of the structure/function relationships in plastocyanin and cytochrome c6 from Anabaena by site-directed mutagenesis.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 273 - 6
Characterisation of a new human and murine member of the DnaJ family of proteins; Silins G et al.; We report the characterisation of a human gene, designated MCG18 (multiple endocrine neoplasia type 1 candidate gene 18), that encodes a new member of the DnaJ family of proteins . Database searches indicate that MCG18 also has the locus name HSPF2 . MCG18 lies 250bp centromeric of the VRF/VEGFB gene on chromosome 11q13 . The MCG18 cDNA is predicted to encode a 241 amino acid product that has partial homology to Escherichia coli dnaJ in that it contains the J domain . However, MCG18 has greatest similarity to a functionally undefined protein from Caenorhabditis elegans, both of which are predicted to have a membrane-spanning region adjacent to their J domains . The cDNA encoding the murine homolog (Mcg18) was also cloned and sequenced, and the encoded protein shares 81% similarity to MCG18 . The coding region of MCG18 is interrupted by 4 introns and the mRNA is expressed as a 1.4kb message in all tissues examined, including those derived from the breast, ovary, bladder, lung and keratinocytes.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 153 - 7
Abnormal proteins enhance stress-induced cell death; Kim SJ et al.; The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides . Escherichia coli methionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability . The polypeptide chain was split by linker insertion and expressed as two fragments . Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity . The in vitro activities of these split mutants were enhanced by the presence of chaperonin, GroESL . However, cells containing these split polypeptides became sensitive to conditions that induce GroESL . The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 90 - 5
A stimulation factor for hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli; Kurokawa K et al.; Hydrolysis of ATP bound to DnaA protein by its intrinsic ATPase activity negatively controls chromosomal DNA replication in Escherichia coli . We developed a new in vitro assay system for ATP hydrolysis, which makes feasible a search for factors affecting the ATPase activity of DnaA protein . A crude cell extract enhanced the hydrolysis of ATP bound to DnaA protein, in a dose-dependent manner . Gel-filtration analyses revealed a single entity of the stimulation factor for the ATP hydrolysis and an apparent molecular mass of 170 kDa . The stimulation activity for ATP hydrolysis coeluted with the inactivation activity for DnaA protein initiating an oriC DNA replication, as determined by anion-exchange and gel-filtration column chromatographies . Activity of the stimulation factor required DNA and ATP . These observations suggested that IdaA protein, a previously described negative factor for DnaA protein, inactivated DnaA protein through stimulation of the hydrolysis of ATP bound to DnaA protein.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 6 - 12
Retroviral coexpression of thymidylate synthase and dihydrofolate reductase confers fluoropyrimidine and antifolate resistance; Fantz CR et al.; Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents . We show that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents . Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold) . Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30- to 140-fold) . A L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold) . These results show that recombinant retroviruses can be used to transfer the cDNA that encodes both TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents.

Plasmid, 1998, 39(1), 77 - 83
Unwinding of the Escherichia coli origin of replication (oriC) can occur in the absence of initiation proteins but is stabilized by DnaA and histone-like proteins IHF or HU; Polaczek P et al.; The unwinding of the origin of replication (oriC) is a critical step in initiation of DNA replication in Escherichia coli . Previous observations indicate that efficient unwinding of supercoiled plasmid templates containing oriC sequences requires the DnaA initiation protein and one or more accessory factors . The precise contribution of each protein to this process is unknown . Here, we demonstrate that unwinding can occur under physiological conditions at the same bases in oriC, in either the presence or the absence of initiation proteins, as detected by a single-strand specific nuclease, P1 . This suggests that oriC unwinding is a spontaneous event determined solely by DNA sequence . DnaA and IHF, as part of a large nucleoprotein complex, may function to stabilize the DNA strand opening prior to initiation of DNA replication.

Plasmid, 1998, 39(1), 48 - 62
Replication regulation of ColE1-like plasmids in amino acid-starved Escherichia coli; Wrobel B et al.; Differential replication of various ColE1-type plasmids in stringent (relA+) and relaxed (relA-) strains of Escherichia coli starved for particular amino acids was reported previously . A role for the plasmid-encoded Rom protein in the stringent control of ColE1 replication has also been demonstrated . Here we have studied the efficiency of replication of five ColE1-type plasmids in E . coli relA+ and relA- strains starved for five amino acids to find the differential replication of each plasmid in cells starved for each amino acid . The efficiency of replication was found to be in positive correlation with the homology between nucleotide sequences of particular loops of RNA I or RNA II and anticodon loops of tRNA molecules corresponding to the kind of the amino acid deprived . Efficient plasmid DNA replication was observed under conditions for which we predicted (on the basis of theoretical calculations) relatively strong interactions between tRNA molecules, expected to occur in high concentrations in an uncharged from, and RNA I or RNA II . When the theoretical possibility of the tRNA-RNA I or tRNA-RNA II interactions was very small, the observed plasmid DNA replication was negligible . Replication of ColE1-like plasmids during the stringent response was observed only in the absence of a functional rom gene . We observed plasmid replication in the amino acid-starved pcnB relA double mutant . We propose a model for regulation of ColE1 replication in the amino acid-starved E . coli cells based on interactions between uncharged tRNA molecules and RNA I or RNA II . During starvation for different amino acids, different kinds of uncharged tRNA molecules appear in cells (they are much more abundant, however, in relA- mutants than in relA+ hosts) leading to various efficiencies of replication initiation . The Rom protein may modulate the effect of tRNA(s) by enhancing RNAI-RNA II, but not tRNA-RNA I and tRNA-RNA II, interactions.

Plasmid, 1998, 39(1), 41 - 7
An IS4 insertion at the glnA control region of Escherichia coli creates a new promoter by providing the -35 region of its 3'-end; Camarena L et al.; An insertion element (IS)4 insertion selected as suppressor of the rpoN73::Tn5 alelle was located inside the control region of the glnA gene in Escherichia coli . In the rpoN73::Tn5 background the IS4 insertion promotes glnA transcription at a low constitutive level sufficient to sustain glutamine-independent growth . The IS4 insertion mutation in either rpoN73::Tn5 or wild-type backgrounds promotes glnA transcription from a new start site located two bases downstream of the glnAp2 start site . Analysis of sequences flanking the insertion point showed a promoter sequence whose -35 region was located inside the IS4 sequence and the -10 region was inside the glnA control region . Site-directed mutagenesis of relevant nucleotide residues of the newly created promoter impaired transcription of a reporter gene . The results support our contention that IS4 carries a -35 promoter region that is able to create functional hybrid promoters . We propose that this mechanism could be one of the molecular reasons of the suppressor activity previously reported for IS4.

Plasmid, 1998, 39(1), 21 - 34
Expression in Escherichia coli of Y5 mutant and N-terminal domain-deleted DNA gyrase B proteins affects strongly plasmid maintenance; Brino L et al.; Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA) . Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F) . Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid . This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used . Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number . By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria . Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins . In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions . Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities . Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo . This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits . This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo . Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.

Viral Immunol, 1997, 10(4), 213 - 20
A novel method to assay herpes simplex virus neutralizing antibodies using BHKICP6LacZ-5 (ELVIS) cells; Ashley RL et al.; A novel method for determining neutralizing serum antibody titers to herpes simplex virus type 2 (HSV-2) was developed based on reduction of infectivity in BHKICP6LacZ-5 (ELVIS) cells; baby hamster kidney (BHK) cells that have been genetically engineered to contain the Escherichia coli LacZ gene under the control of an inducible herpes simplex virus type 1 (HSV-1) promoter . The test has a semiautomated, colorimetric readout resulting in rapid, objective readings of infectivity reduction . Extent of neutralization is calculated against a calibration curve of virus infectivity generated in each run . HSV-2 neutralizing activity can be detected with serum dilutions in excess of 1:5120.

Eur J Pharmacol, 1997 Nov 27, 339(2-3), 215 - 21
Effect of the phosphodiesterase III inhibitor amrinone on cytokine and nitric oxide production in immunostimulated J774.1 macrophages; Nemeth ZH et al.; The level of intracellular cyclic nucleotides is a regulatory factor in a variety of immune processes . Increases in intracellular cyclic AMP (cAMP) and/or cyclic GMP (cGMP) concentration by the inhibition of phosphodiesterase have been shown to modulate the inflammatory response . Amrinone is a clinically used positive inotropic agent which elevates intracellular cAMP and cGMP levels by selective inhibition of the phosphodiesterase III isoenzyme . In the current study, we investigated the effect of various concentrations (1-300 microM) of amrinone on lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and of nitric oxide (NO) in vitro . In cultured murine J774.1 macrophages, 1 ng/ml-10 microg/ml of lipopolysaccharide from Escherichia coli O55:B5 induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-10, and nitrite (breakdown product of NO) . Pretreatment of cells with amrinone caused a dose-dependent suppression of TNF-alpha production in the concentration range of 1-100 microM . Furthermore, this drug suppressed NO production in the range of 30-300 microM . Similarly to the results in the J774.1 cells, amrinone also inhibited TNF-alpha and NO production in the range of 10-100 microM in primary rat peritoneal macrophages . At 300 microM, but not at lower concentrations, amrinone inhibited interleukin-10 production in lipopolysaccharide-treated J774.1 macrophages . Pretreatment of the macrophages with 100 and 300 microM amrinone increased the lipopolysaccharide-elicited translocation of nuclear factor-kappa B . Taken together, our results indicate that the phosphodiesterase III inhibitor amrinone modulates the activation/production of many pro- and anti-inflammatory factors in endotoxin-stimulated cells . It remains to be further investigated how such immunomodulatory effects contribute to the clinical profile of the agent.

Eur J Pharmacol, 1997 Nov 27, 339(2-3), 201 - 9
Influence of endotoxin on contractility in the rat gastric fundus; Lefebvre RA; The influence of in vivo treatment with E . coli lipopolysaccharide endotoxin on the contractility of the rat gastric fundus was studied . Four h after lipopolysaccharide treatment (20 mg/kg i.p.), the contractile responses to prostaglandin F2alpha in longitudinal muscle strips from the gastric fundus were not different from those in control animals, while the well-known decreased response to noradrenaline in rings of the thoracic aorta was confirmed . Incubation of the tissues with L-arginine did not depress the response to prostaglandin F2alpha in fundus strips of lipopolysaccharide-treated rats . Twelve h after lipopolysaccharide treatment (6.7 mg/kg i.p.), the prostaglandin F2alpha-induced contractions were consistently depressed . The impairment of the prostaglandin F2alpha-induced responses by lipopolysaccharide treatment was not reversed by the nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA, 10(-4) M), NG-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M), aminoguanidine (10(-4) M) and L-N6-l-iminoethyl-lysine (L-NIL, 10(-4) M) nor by the cyclooxygenase inhibitor indomethacin (10(-5) M) . The impairment was prevented by pretreating the animals with dexamethasone (5 mg/kg i.p.), which had no effect per se on the contractile response to prostaglandin F2alpha . Lipopolysaccharide treatment did not influence the contractile responses to KCl and serotonin . The nonadrenergic noncholinergic relaxant responses to transmural electrical stimulation were not influenced 4 h after lipopolysaccharide treatment but were moderately reduced after 12 h . The results illustrate that the selective impairment of prostaglandin F2alpha-induced contractions in the rat gastric fundus by lipopolysaccharide treatment is not mediated via generation of nitric oxide; downregulation of the prostaglandin F2alpha-receptor by lipopolysaccharide treatment might be involved.

J Bacteriol, 1998 Feb, 180(4), 998 - 1001
Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12; Andrianopoulos K et al.; GDP-L-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP-D-mannose . L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains . We previously sequenced the E . coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd . We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.

J Bacteriol, 1998 Feb, 180(4), 994 - 7
The StpA protein functions as a molecular adapter to mediate repression of the bgl operon by truncated H-NS in Escherichia coli; Free A et al.; The mechanism of repression of the beta-glucoside utilization (bgl) operon of Escherichia coli by a carboxy-terminally truncated derivative of the nucleoid-associated protein H-NS which is defective in DNA binding was investigated . The DNA-binding function of the H-NS-like protein StpA was found to be necessary for repression, which is consistent with a role for StpA as a DNA-binding adapter for mutant derivatives of H-NS.

J Bacteriol, 1998 Feb, 180(4), 989 - 93
Mismatch repair in Escherichia coli cells lacking single-strand exonucleases ExoI, ExoVII, and RecJ; Harris RS et al.; In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease . We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates . ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

J Bacteriol, 1998 Feb, 180(4), 985 - 8
Both acetate kinase and acetyl coenzyme A synthetase are involved in acetate-stimulated change in the direction of flagellar rotation in Escherichia coli; Barak R et al.; Escherichia coli strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins . With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response . Thus, a response was observed when one of these enzymes was missing but not when both were absent.

J Bacteriol, 1998 Feb, 180(4), 938 - 44
Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12; Aono R et al.; Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA . The TolC level was regulated positively by MarA, Rob, or SoxS . A possible mar-rob-sox box sequence was found upstream of the tolC gene . These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions . When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level . The tolerance level was restored by transformation of the transductants with a wild-type tolC gene . Therefore, it is evident that TolC is essential for E . coli to maintain organic solvent tolerance.

J Bacteriol, 1998 Feb, 180(4), 914 - 20
Chimeric chemoreceptors in Escherichia coli: signaling properties of Tar-Tap and Tap-Tar hybrids; Weerasuriya S et al.; The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of Escherichia coli together mediate chemotactic responses to dipeptides . Tap is a low-abundance receptor . It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr . Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run) . To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains . The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator . Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain . Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap . The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability . The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation . However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody . Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg . The hybrid, truncated, and extended receptors exhibit various levels of methylation . However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected . We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.

J Bacteriol, 1998 Feb, 180(4), 881 - 4
Roles of FtsA and FtsZ in activation of division sites; Begg K et al.; Increasing FtsZ induces the formation of minicells at cell poles but does not increase the frequency or timing of central divisions . A coordinate increase in both FtsZ and FtsA, however, increases the frequency of both polar and central divisions.

J Bacteriol, 1998 Feb, 180(4), 862 - 70
Identification and characterization of alcR, a gene encoding an AraC-like regulator of alcaligin siderophore biosynthesis and transport in Bordetella pertussis and Bordetella bronchiseptica; Beaumont FC et al.; A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance . The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source . In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein . A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant . Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR . Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site . The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels . In-frame alcR deletion mutants of B . pertussis and B . bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins . The cloned alcR gene provided in trans restored these siderophore system activities to the mutants . Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.

J Bacteriol, 1998 Feb, 180(4), 846 - 54
Roles of DnaK and RpoS in starvation-induced thermotolerance of Escherichia coli; Rockabrand D et al.; DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli . Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein . Because these dnaK mutant phenotypes closely resemble those of rpoS (sigma38) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30 degrees C in strains with genetically altered DnaK content . A starvation-specific effect of DnaK on RpoS abundance was observed . During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold . Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal . RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS . Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS . RpoH (sigma32) suppressors of the dnaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance . The results suggest that DnaK coordinates sigma factor levels in glucose-starved E . coli.

J Bacteriol, 1998 Feb, 180(4), 831 - 9
CpxP, a stress-combative member of the Cpx regulon; Danese PN et al.; The CpxA/R two-component signal transduction system of Escherichia coli can combat a variety of extracytoplasmic protein-mediated toxicities . The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP . However, other factors are also employed by the Cpx system for this stress-combative function . In an effort to identify these remaining factors, we screened a collection of random lacZ operon fusions for those fusions whose transcription is regulated by CpxA/R . Through this approach, we have identified a new locus, cpxP, whose transcription is stimulated by activation of the Cpx pathway . cpxP specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein . In addition, we show that cpxP transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that cpxP and cpx mutant strains display hypersensitivity to growth in alkaline conditions.

J Bacteriol, 1998 Feb, 180(4), 785 - 92
Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification; Van Dyk TK et al.; The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM) . To further understand the physiological consequences of flux alterations at this point in metabolism, Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought . Plasmid-based fusions of random E . coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM . From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified . The lux reporter genes were joined to seven uncharacterized open reading frames, f253a, f415, frvX, o513, o521, yciG, and yohF, and five known genes, inaA, IdcC, osmY, poxB, and sohA . Inactivation of the rpoS-encoded sigma factor, sigmaS, reduced basal expression levels of six of these fusions 10- to 200-fold . These six genes defined four new members of the sigmaS regulon, f253a, IdcC, yciG, and yohF, and included two known members, osmY and poxB . Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the sigmaS-controlled genes and inaA . The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the sigmaS-dependent stress response.

J Bacteriol, 1998 Feb, 180(4), 767 - 72
Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids; Roe AJ et al.; During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm . This study was undertaken to determine the impact of anion accumulation on cellular pools . At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+ and Na+ levels . At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion . However, at high osmolarity, glutamate compensated for over half of the accumulated acetate . Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.

Hum Gene Ther, 1998 Jan 20, 9(2), 235 - 48
Targeting strategy for gene delivery to carcinoembryonic antigen-producing cancer cells by retrovirus displaying a single-chain variable fragment antibody; Konishi H et al.; Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy . Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach . Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies . Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase . The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEA-producing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells . These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy.

J Gen Virol, 1998 Feb, 79 ( Pt 2), 347 - 52
Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells; Drexler I et al.; Although desirable for safety reasons, the host range restrictions of modified vaccinia virus Ankara (MVA) make it less applicable for general use . Propagation in primary chicken embryo fibroblasts (CEF) requires particular cell culture experience and has no pre-established record of tissue culture reproducibility . We investigated a variety of established cell lines for productive virus growth and recombinant gene expression . Baby hamster kidney cells (BHK), a well-characterized, easily maintained cell line, supported MVA growth and as proficient expression of the E . coli lacZ reporter gene as the highly efficient CEF, whereas other cell lines were non-permissive or allowed only very limited MVA replication . Importantly, no virus production occurred in patient-derived infected primary human cells . These results emphasize the safety and now improved accessibility of MVA for the development of expression vectors and live recombinant vaccines.

Patol Fiziol Eksp Ter, 1997 Oct-Dec, (4), 11 - 3
{Components of the kinin system in the lymph and blood, flowing from the liver and intestines in dogs in endotoxic shock}; Zamechnik TV et al.; The role of the kinin system (KS) in pathogenesis of endotoxin shock was investigated . In the lymph and blood flowing out of the small intestine and in the liver the activity of KS was measured 30, 60, 90 and 120 min after injection of the sublethel dose of endotoxin E coli 0 111 B4 . The results suggest that in the lymph and blood flowing from the intestine KS activity occurs during the first minutes of endotoxin shock . In the liver KS activity was observed during the second hour of shock development followed by enhancing synthesis of KS components in the presence of KS higher activity and kinin developing function exhaustion to the end of the third hour of shock duration.

J Biotechnol, 1997 Dec 3, 58(3), 157 - 66
Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite; Eis C et al.; Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E . coli, was substantially improved by a novel approach using ceramic hydroxyapatite . Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries . At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes . The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix . The interaction of E . coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity . Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

Biochemistry, 1998 Jan 13, 37(2), 734 - 40
Trimeric inorganic pyrophosphatase of Escherichia coli obtained by directed mutagenesis; Velichko IS et al.; Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits . Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis . The results of such measurements, together with the results of activity measurements as a function of {Mg2+} and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface . The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate . The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer . Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation . The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.

Biochemistry, 1998 Jan 13, 37(2), 608 - 14
Mg2+ coordination in catalytic sites of F1-ATPase; Weber J et al.; Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated . From the X-ray structure of the mitochondrial enzyme {Abrahams, J . P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628}, it may be inferred that the hydroxyl of betaThr-156 is a direct ligand of Mg2+, whereas the carboxyls of betaGlu-181, betaGlu-185, and betaAsp-242 might contribute via intervening water molecules . Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that betaThr-156, betaGlu-185, and betaAsp-242 are all involved in Mg2+ coordination, whereas betaGlu-181 is not . A derived structural model for the octahedral coordination around the Mg2+ ion is presented . The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity . Correct Mg2+ coordination is required for catalytic activity at physiological rates . Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.

Biochemistry, 1998 Jan 13, 37(2), 596 - 607
Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone; Russell R et al.; DnaK, the prototype Hsp70 protein of Escherichia coli, functions as a molecular chaperone in protein folding and protein disassembly reactions through cycles of polypeptide binding and release that are coupled to its intrinsic ATPase activity . To further our understanding of these processes, we sought to obtain a quantitative description of the basic ATPase cycle of DnaK . To this end, we have performed steady-state and pre-steady-state kinetics experiments and have determined rate constants corresponding to individual steps in the DnaK ATPase cycle at 25 degrees C . Hydrolysis of ATP proceeds very slowly with a rate constant (khyd approximately 0.02 min-1) at least 10-fold smaller than the rate constant for any other first-order step in the forward reaction pathway . The ATP hydrolysis step has an activation energy of 26.2 +/- 0.4 kcal/mol and is rate limiting in the steady-state under typical in vitro conditions . ATP binds with unusual strength to DnaK, with a measured KD approximately 1 nM . ADP binds considerably less tightly than ATP and dissociates from DnaK with a koff of approximately 0.4 min-1 (compared with a koff of approximately 0.008 min-1 for ATP) . However, in the presence of physiologically relevant concentrations of inorganic phosphate (Pi), the release of ADP from DnaK is greatly slowed, approximately to the rate of ATP hydrolysis . Under these conditions, the ADP-bound form of DnaK, the form that binds substrate polypeptides most tightly, was found to represent a significant fraction of the DnaK population . The slowing of ADP release by exogenous Pi is due to thermodynamic coupling of the binding of the two ligands, which produces a coupling energy of approximately 1.6 kcal/mol . This result implies that product release is not strictly ordered . In the absence of exogenous inorganic phosphate, Pi product, by virtue of its higher koff, is released prior to ADP . However, at physiological concentrations of inorganic phosphate, the alternate product release pathway, whereby ADP dissociates from a ternary DnaK.ADP.Pi complex, becomes more prominent.

Biochemistry, 1998 Jan 13, 37(2), 571 - 9
Rate of incision of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts by UvrABC nuclease is adduct- and sequence-specific: comparison of the rates of UvrABC nuclease incision and protein-DNA complex formation; Mekhovich O et al.; The UvrABC nuclease, the nucleotide excision repair complex from Escherichia coli, is able to incise a variety of types of DNA damage and the repair efficiency of this enzyme complex appears to be influenced by the structure of the damage and the sequence context within which the damage is positioned . In order to better establish these relationships, we have constructed two DNA sequences each containing a site-specifically positioned N-2-aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF) adduct and have determined both the kinetics of UvrABC nuclease incision and the kinetics of UvrABC nuclease-substrate complex formation . It is well established that these two adducts induce very different structures in the DNA and that these structures also depend on the sequence context . We have found that the rate of incision of both AAF- and AF-DNA adducts is significantly faster when they are positioned in the mutation hotspot NarI sequence (5-GGCG*CC-3') than when located in a normal or non-NarI sequence (5'-GATG*ATA-3') and that the rate of incision for AAF-DNA adducts is faster that for AF adducts in both sequences . Most siginificantly, we find that the rate of UvrB and UvrBC-substrate complex formation correlates with the rate of UvrABC nuclease incision.

Biochemistry, 1998 Jan 13, 37(2), 558 - 63
Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals; Reddy SG et al.; We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation . Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*) . The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation . Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734 . These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical . The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein . Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium . A mechanism that accounts for the formation of the radical intermediates is proposed.

Biochemistry, 1998 Jan 13, 37(2), 486 - 95
Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands; Cetin R et al.; Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis . To analyze the function of domains 2 {middle (M) domain} and 3 {C-terminal (C) domain}, EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified . Circular dichroism and thermostability showed that both constructs have conserved organized structures . Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities . Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP . EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu . Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule . The interaction with aa-tRNA of both mutants was >90% reduced . As a major result, their GDP-bound form could efficiently respond to EF-Ts . All four EF-Tu-specific antibiotics {kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa} retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM) . GTP . Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts . Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.

Biochemistry, 1998 Jan 13, 37(2), 476 - 85
Specificity in protein-protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase-Im9 complex; Wallis R et al.; The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein . The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude . DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins . Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase . In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9 . Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix . We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding . In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues . This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction . The generality of such a dual recognition mechanism in other systems is also discussed.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 376 - 81
A transcription activation system for regulated gene expression in transgenic plants; Moore I et al.; A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines . The promoter, pOp, consists of lac operators cloned upstream of a minimal promoter . No expression was detected from this promoter when placed upstream of a beta-glucuronidase (GUS) reporter gene in transgenic plants . Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4 . This factor comprised transcription-activation domain-II from Gal4 of Saccharomyces cerevisiae fused to a mutant lac-repressor that binds its operator with increased affinity . When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter . The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed . The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention . It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 346 - 51
Gsalpha-selective G protein antagonists; Hohenegger M et al.; Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the Galpha subunit, i.e., the exchange of GDP for GTP . Here, we have searched for analogues that are selective for Gsalpha . Two compounds have been identified: NF449 (4,4',4",4'"-{carbonyl-bis{imino-5,1,3-benzenetriyl bis-(carbonylimino)}}tetrakis-(benzene-1,3-disulfonate) and NF503 (4, 4'-{carbonylbis{imino-3,1-phenylene-(2, 5-benzimidazolylene)carbonylimino}}bis-benzenesulfonate) . These compounds (i) suppress the association rate of guanosine 5'-{gamma-thio}triphosphate ({35S}GTP{gammaS}) binding to Gsalpha-s but not to Gialpha-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc- membranes (deficient in endogenous Gsalpha) by exogenously added Gsalpha-s, and (iii) block the coupling of beta-adrenergic receptors to Gs with half-maximum effects in the low micromolar range . In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A1-adenosine receptor with its cognate G proteins (Gi/Go) at concentrations that are >30-fold higher than those required for uncoupling of beta-adrenergic receptor/Gs tandems; similarly, the angiotensin II type-1 receptor (a prototypical Gq-coupled receptor) is barely affected by the compounds . Thus, NF503 and NF449 fulfill essential criteria for Gsalpha-selective antagonists . The observations demonstrate the feasibility of subtype-selective G protein inhibition.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 102 - 7
Supercoiling affects the accessibility of glutathione to DNA-bound molecules: positive supercoiling inhibits calicheamicin-induced DNA damage; LaMarr WA et al.; DNA superhelical tension, an important feature of genomic organization, is known to affect the interactions of intercalating molecules with DNA . However, the effect of torsional tension on nonintercalative DNA-binding chemicals has received less attention . We demonstrate here that the enediyne calicheamicin gamma1I, a strand-breaking agent specific to the minor groove, causes approximately 50% more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity . Furthermore, we show that the decrease in damage in positively supercoiled DNA is controlled at the level of thiol activation of the drug . Our results suggest that supercoiling may affect both the activity of nonintercalating genotoxins in vivo and the accessibility of glutathione and other small physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.

EMBO J, 1997 Dec 15, 16(24), 7361 - 71
The novel SAR-binding domain of scaffold attachment factor A (SAF-A) is a target in apoptotic nuclear breakdown; Gohring F et al.; The scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes . Evidence from previous experiments had suggested that SAF-A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism . We now show that the protein has a novel scaffold-associated region (SAR)-specific bipartite DNA-binding domain which is independent from the previously identified RNA-binding domain, the RGG box . During apoptosis, but not during necrosis, SAF-A is cleaved in a caspase-dependent way . Cleavage occurs within the bipartite DNA-binding domain, resulting in a loss of DNA-binding activity and a concomitant detachment of SAF-A from nuclear structural sites . On the other hand, cleavage does not compromise the association of SAF-A with hnRNP complexes, indicating that the function of SAF-A in RNA metabolism is not affected in apoptosis . Our results suggest that detachment of SAF-A from SARs, caused by apoptotic proteolysis of its DNA-binding domain, is linked to the formation of oligonucleosomal-sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells.

EMBO J, 1997 Dec 15, 16(24), 7351 - 60
Reconstitution of a chloroplast protein import channel; Hinnah SC et al.; The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane . Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Lambda = 145pS . Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient . Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening . In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75 . The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (dporeapproximately equal to 8-9 A) . Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.

EMBO J, 1997 Dec 15, 16(24), 7297 - 304
The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events; van der Wolk JP et al.; SecA is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade, and is bound at the membrane surface to the integral membrane domain of the preprotein translocase . Preproteins are thought to be translocated in a stepwise manner by nucleotide-dependent cycles of SecA membrane insertion and de-insertion, or as large polypeptide segments by the protonmotive force (Deltap) in the absence of SecA . To determine the step size of a complete ATP- and SecA-dependent catalytic cycle, translocation intermediates of the preprotein proOmpA were generated at limiting SecA translocation ATPase activity . Distinct intermediates were formed, spaced by intervals of approximately 5 kDa . Inhibition of the SecA ATPase by azide trapped SecA in a membrane-inserted state and shifted the step size to 2-2.5 kDa . The latter corresponds to the translocation elicited by binding of non-hydrolysable ATP analogues to SecA, or by the re-binding of partially translocated polypeptide chains by SecA . Therefore, a complete catalytic cycle of the preprotein translocase permits the stepwise translocation of 5 kDa polypeptide segments by two consecutive events, i.e . approximately 2.5 kDa upon binding of the polypeptide by SecA, and another 2.5 kDa upon binding of ATP to SecA.

EMBO J, 1997 Dec 15, 16(24), 7241 - 9
Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2; Medici R et al.; Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction . We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes . To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed . This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented . This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition . Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently . It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal . To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

EMBO J, 1997 Dec 15, 16(24), 7219 - 30
The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters; Auerbach G et al.; Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases . We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP . The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices . Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied . Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N . The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization . Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible . The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

Nucleic Acids Res, 1998 Jan 1, 26(1), 332 - 3
Current status of the SWISS-2DPAGE database; Hoogland C et al.; The SWISS-2DPAGE database consists of two-dimensional polyacrylamide gel electrophoresis images, as well as textual descriptions of the proteins that have been identified on them . The current release contains 15 reference maps from human biological samples, as well as from Saccharomyces cerevisiae , Escherichia coli and Dictyostelium discoideum origin . These reference maps have 2088 identified spots, corresponding to 410 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence.

Nucleic Acids Res, 1998 Jan 1, 26(1), 280 - 4
Expanded versions of the 16S and 23S ribosomal RNA mutation databases (16SMDBexp and 23SMDBexp)
Triman KL, Peister A, Goel RA.
Expanded versions of the Ribosomal RNA Mutation Databases provide lists of mutated positions in 16S and 16S-like ribosomal RNA (16SMDBexp) and 23S and 23S-like ribosomal RNA (23SMDBexp) and the identity of each alteration . Alterations from organisms other than Escherichia coli are reported at positions according to the E.coli numbering system . Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation, (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (iii) relevant literature citations . The databases are available via ftp and on the World Wide Web at the following URL: //tml

Nucleic Acids Res, 1998 Jan 1, 26(1), 198 - 9
Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents; Cariello NF et al.; We have created databases and software applications for the analysis of DNA mutations at the human p53 gene, the human hprt gene and both the rodent transgenic lacI and lacZ loci . The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers with Microsoft Windows . Each database has a separate software analysis program . The software created for these databases permit the filtering, ordering, report generation and display of information in the database . In addition, a significant number of routines have been developed for the analysis of single base substitutions . One method of obtaining the databases and software is via the World Wide Web . Open the following home page with a Web Browser: html . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu . There is no password required to enter the system . The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations . Two other programs are available at the site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Nucleic Acids Res, 1998 Jan 1, 26(1), 187 - 9
The Database of Ribosomal Cross links (DRC); Baranov PV et al.; The Database of Ribosomal Cross-links (DRC) provides a complete collection of all the published data produced by cross-linking studies on the Escherichia coli ribosome, as well as on its components and functional ligands . The DRC currently includes data on 986 cross-links from >100 research papers, yielded by >40 different reagents . For each cross-link, information is given concerning its location in the ribosome, the chemical or photochemical reagent applied, a brief description of the method(s) used to locate the cross-link, and the literature reference . The DRC is freely available via the World Wide Web at: or at see text}baranov/DRC/

Nucleic Acids Res, 1998 Jan 1, 26(1), 166 - 7
The tmRNA database (tmRDB); Zwieb C et al.; This first release of the tmRNA database (tmRDB) contains 19 tmRNA sequences, a tmRNA sequence alignment with emphasis of base pairs that are supported by comparative sequence analysis, and a tabulation of tmRNA-encoded tag peptides . The tmRNADB also offers an RNA secondary structure diagram of the Escherichia coli tmRNA, as well as PDB-formatted coordinates for three-dimensional modeling . The data are available on the World Wide Web at edu/tmRDB/tmRDB.html

Nucleic Acids Res, 1998 Jan 1, 26(1), 55 - 9
RegulonDB: a database on transcriptional regulation in Escherichia coli; Huerta AM et al.; RegulonDB is a DataBase that integrates biological knowledge of the mechanisms that regulate the transcription initiation in Escherichia coli , as well as knowledge on the organization of the genes and regulatory signals into operons in the chromosome . The operon is the basic structure used in RegulonDB to describe the elements and properties of transcriptional regulation . The current version contains information around some 500 regulation mechanisms, essentially for sigma 70 promoters.

Nucleic Acids Res . 1998 Jan 1;26(1):54.
Genes and proteins of Escherichia coli K-12; Riley M; GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each . Also present are data on sequence similarities among E.coli proteins, representing groups of paralogous genes, with PAM values, percent identity of amino acids, length of alignment and percent aligned . GenProtEC can be accessed at the URL http://www.mbl.edu/html/ecoli.html

Nucleic Acids Res, 1998 Jan 1, 26(1), 50 - 3
EcoCyc: Encyclopedia of Escherichia coli genes and metabolism; Karp PD et al.; The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli . The database describes 3030 genes of E.coli , 695 enzymes encoded by a subset of these genes, 595 metabolic reactions that occur in E.coli, and the organization of these reactions into 123 metabolic pathways . The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways . EcoCyc can be thought of as an electronic review article because of its copious references to the primary literature, and as a (qualitative) computational model of E.coli metabolism . EcoCyc is available at URL http://ecocyc.PangeaSystems.com/ecocyc/

Nucleic Acids Res, 1998 Jan 1, 26(1), 46 - 9
Compilation of DNA sequences of Escherichia coli K12: description of the interactive databases ECD and ECDC; Kroger M et al.; We have compiled the DNA sequence data for Escherichia coli K12 available from the GenBank and EMBL data libraries and independently from the literature . We provide the most definitive version of the ECD Escherichia coli database now exclusively via the World Wide Web System . Our database encloses the completed genome sequence recently published by two competing groups and an assembled set of all elder sequences . The organisation of the database allows precise physical location of each individual gene or regulatory region, even taking into consideration discrepancies in nomenclature . The WWW program allows to the user to branch into the original EMBL and SWISS-PROT datafiles . A number of links to other WWW servers dealing with E . coli is provided . A FASTA and BLAST search may be performed online . Besides the WWW format a flat file version may be obtained via ftp . A number of discrepancies between the two systematic sequence determinations and/or the literature have not yet been resolved . However, our database may serve as a reference source for resolution and/or the assignment of strain difference.

Nucleic Acids Res, 1998 Jan 1, 26(1), 16 - 20
DNA Data Bank of Japan at work on genome sequence data; Tateno Y et al.; We at the DNA Data Bank of Japan (DDBJ) have recently begun receiving, processing and releasing EST and genome sequence data submitted by various Japanese genome projects . The data include those for human, Arabidopsis thaliana, rice, nematode, Synechocystis sp . and Escherichia coli . Since the quantity of data is very large, we organized teams to conduct preliminary discussions with project teams about data submission and handling for release to the public . We also developed a mass submission tool to cope with a large quantity of data . In addition, to provide genome data on WWW, we developed a genome information system using Java . This system can in theory be used for any genome sequence data . These activities will facilitate processing of large quantities of EST and genome data.

Curr Biol, 1997 Dec 1, 7(12), R745 - 9
Recombination initiation: easy as A, B, C, D.. . chi?
Eggleston AK, West SC.
The octameric Chi (chi) sequence is a recombination hotspot in Escherichia coli . Recent studies suggest a singular mechanism by which chi regulates not only the nuclease activity of RecBCD enzyme, but also the ability of RecBCD to promote loading of the strand exchange protein, RecA, onto chi-containing DNA.

Magn Reson Med, 1998 Feb, 39(2), 190 - 7
Effects of endotoxin lung injury on NMR T2 relaxation; Cutillo AG et al.; The effects of endotoxin injury on lung NMR relaxation times (T1, CPMG T2, and Hahn decay constant (Hahn T2)) were studied in excised unperfused rat lungs . Blinded histologic examination showed no clear-cut separation between endotoxin and control lungs . Morphometric lung tissue volume density and gravimetric lung water content did not differ significantly between the two groups . In contrast, the values of the fast, intermediate, and slow T2 components, obtained by multiexponential analysis of the CPMG decay curve, increased markedly after endotoxin administration, with minimal overlap between endotoxin and control values . The response of Hahn T2 was, in general, in the same direction as that of CPMG T2; however, Hahn T2 may be more affected by measurement errors and may be less sensitive to the presence of lung injury . T1 showed minimal changes after injury . The present data suggest that CPMG T2 measurements can consistently detect the presence of lung injury even when conventional histologic, morphometric, and gravimetric studies provide negative or equivocal results, and that the CMPG T2 method is superior, in this respect, to the Hahn decay method . T1 does not appear to be sensitive to lung injury in the absence of significant lung water accumulation.

Pharmacotherapy, 1998 Jan-Feb, 18(1), 170 - 4
Effect of sustained endotoxemia on alpha1-adrenergic responsiveness in parenterally fed rats; Dickerson RN et al.; We investigated the effect of endotoxemia on alpha1-adrenergic receptor-mediated smooth muscle contraction as measured by mean arterial pressure (MAP) in response to incremental doses of a vasopressor . Twelve male Sprague-Dawley rats were randomized to receive parenteral nutrition alone (PN) or in combination with a continuous infusion of endotoxin (PN-LPS) for 48 hours . Incremental doses of phenylephrine were given and peak MAP response was recorded . The endotoxin group had a decreased rise in MAP with the same dose of phenylephrine compared with the control group (59 +/- 14 and 99 +/- 12 mm Hg, respectively, p<0.001) . However, the baseline MAP was higher in the endotoxin group (102 +/- 18 and 71 +/- 7 mm Hg, respectively, p<0.002) . The overall maximum effect was the same for both groups (161 +/- 16 and 170 +/- 8 mm Hg, respectively, p=NS) . These data indicate that sustained endotoxemia does not result in desensitization of alpha1-adrenergic responsiveness . Other mechanisms are responsible for the ineffectiveness of vasopressors during advanced sepsis.

J Immunol, 1998 Feb 15, 160(4), 1598 - 605
Creating CTL targets with epitope-linked beta 2-microglobulin constructs; Uger RA et al.; Eliciting a strong CTL response is dependent upon displaying suitably high levels of specific class I MHC/peptide complexes at the cell surface . In an effort to enhance the presentation of defined CTL target structures, two unique peptide-linked beta 2-microglobulin (beta 2m) molecules were constructed . The first, designated NP(366-374)-L8-h beta 2m, links the carboxyl terminus of the H-2Db-restricted influenza nucleoprotein (NP) epitope NP(366-374) to the amino terminus of h beta 2m through an eight-amino acid glycine/serine linker . The second molecule, designated NP(147-155)-L12-h beta 2m, similarly couples the H-2Kd-restricted influenza NP epitope NP(147-155) to h beta 2m via a 12-residue polypeptide linker . Transfection of the NP(366-374)-L8-h beta 2m vector into H-2b-expressing cell lines sensitized these cells for lysis by NP(366-374)-specific CTLs . Free NP peptide could not be detected when class I bound peptides were acid-extracted from the surface of NP(366-374)-L8-h beta2m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to h beta 2m and not by a degradation by-product . CTL target structure formation was also achieved by an exogenous presentation pathway . H-2d-expressing target cells were sensitized for lysis when pulsed with NP(147-155)-L12-h beta 2m protein derived from an Escherichia coli cell lysate . The effect of recombinant NP(147-155)-L12-h beta 2m was inhibited by competitor wild-type h beta 2m, indicating that the active peptide-h beta 2m fusion protein remained intact . The observation that beta 2m with covalently attached peptide can effectively create CTL target structures in vitro offers new possibilities for the in vivo induction of epitope-specific CTL responses by either DNA immunization or injection of the purified epitope-linked beta 2m.

Biophys Chem, 1997 Oct, 68(1-3), 147 - 59
Biophysical investigations on the myb-DNA system; Hosur RV et al.; The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation . It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions . The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats . We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences . The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E . coli BL21 (DE3) . Circular dichroism of the protein has revealed that the domain is largely helical in nature . Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior . The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases . Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different . Most of the conserved tryptophans are associated with the specific DNA binding site . Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein . Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent . Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein . In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization . The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

Digestion, 1998, 59(1), 33 - 9
Helicobacter pylori stimulates DNA synthesis in a small intestinal cell line in vitro; Brannstrom J et al.; BACKGROUND: Helicobacter pylori, which causes gastritis and peptic ulcer, seems to be an important factor in the pathogenesis of gastric cancer and MALT lymphoma . Thus our aim was to examine whether H . pylori influences DNA synthesis in epithelial cells in vitro . METHODS: Sonicated and water extracts of H . pylori (cytotoxic strains NCTC 11637, 88-23 and A5, and a noncytotoxic isogenic mutant of A5, A5 vac A) were diluted to a final concentration of 1/1,000, 1/100, 1/50 and 1/10 . Water extracts of Escherichia coli were used as reference . IEC-6 cells were incubated during 24 h with fragments of H . pylori or extracts of the concentrations described above . The cells were labeled with 3H-methylthymidine for 4 h and processed for autoradiography . DNA synthesis was evaluated by the labeling index (LI) . RESULTS: The LI% of controls was 15.6 +/- 5.1% . All the water extracts and sonicated strains of H . pylori increased the LI% in a dose-dependent manner (p < 0.001) . The highest concentrations of the sonicated strains tended to reduce the LI%, although these values were still higher than those of the control group . The water extracts of E . coli increased the LI% in a dose-dependent manner (p < 0.0001) . CONCLUSION: H . pylori stimulates DNA synthesis in epithelial cells in vitro, but no association was found with the presence of cytotoxin production . Our results suggest that hitherto unknown components of H . pylori may contribute to the increase in cell proliferation observed in gastritis and to the development of MALT lymphoma and gastric cancer.

FEBS Lett, 1998 Jan 16, 421(3), 237 - 42
Growth phase dependent stop codon readthrough and shift of translation reading frame in Escherichia coli; Wenthzel AM et al.; Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E . coli . The strains used carry plasmid constructs with a translation assay reporter gene . This reporter gene contains an internal stop codon or a run of U-residues . Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products . Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase . In early exponential phase, about 7% of -1 frameshift at a U9 slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase . A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease . Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.

Crit Care Med, 1998 Feb, 26(2), 338 - 43
Effects of growth hormone and insulin-like growth factor I on opsonin receptor expression on local and systemic phagocytes in a lethal peritonitis model; Inoue T et al.; OBJECTIVE: To investigate the effects of pretreatment with growth hormone (GH) and insulin-like growth factor I (IGF-I) on phagocyte exudation and bacterial clearance, focusing on CD11b and CD32/CD16 expression on local and systemic phagocytes, in a lethal peritonitis model . DESIGN: Prospective randomized experimental study . SETTING: Research laboratory in a university hospital . SUBJECTS: Balb/c mice (n = 21) . INTERVENTIONS: Mice were challenged intraperitoneally with 1 x 10(8) Escherichia coli, after 6 days of pretreatment with saline (control), GH (4.8 mg/kg/day), or IGF-I (24 mg/kg/day) . Samples were harvested at 4 hrs after the challenge . MEASUREMENTS AND MAIN RESULTS: Viable bacterial counts in peritoneal lavage fluid (PLF) and blood were determined . Peritoneal exudative cells and peripheral blood leukocytes were counted and analyzed for receptor expressions by flow cytometry . GH reduced viable bacterial counts in PLF, as compared with the saline control . GH (three-fold) and IGF-I (two-fold) increased the number of peritoneal exudative neutrophils (PENs), as compared with the saline control . The number of PENs showed an inverse correlation with PLF viable bacterial counts . By contrast, there were no differences in peripheral blood neutrophil (PN) counts among the three groups, nor was there a correlation between PN and PEN counts . CD11b expression was greater on PENs than on PNs in all three groups . CD11b expression on PNs did not differ among the three groups . However, GH increased CD11b expression on PENs, as compared with saline and IGF-I, and this expression showed a positive correlation with PEN numbers and an inverse correlation with PLF viable bacterial counts . CD11b expression on peritoneal macrophages and peripheral blood monocytes did not differ among the three groups . There were no differences in phagocyte CD32/CD16 expression among the three groups . CONCLUSIONS: GH pretreatment enhanced CD11b expression on PENs, but not PNs, possibly in association with enhanced neutrophil recruitment, phagocytosis, and bacterial elimination by PENs, without activation of PNs . GH prophylaxis may be useful for reducing the frequency rate and severity of septic complications, via modulation of CD11b expression on local and systemic neutrophils.

Microbiology, 1998 Jan, 144 ( Pt 1), 177 - 82
An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter; Davagnino J et al.; RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate . The beta, beta', alpha and omega subunits were approximately the same size as those of Escherichia coli, and the identity of the omega subunit was confirmed by N-terminal sequence analysis . N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be sigma factors, a 40 kDa polypeptide that shared identity with sigma 32 homologues, and a 97 kDa polypeptide that shared identity with sigma 70 homologues in other bacteria . The 97 kDa polypeptide did not cross-react with antibody to E . coli sigma 70 . The same complement of putative sigma factors was found in RNAP purified from M . extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible sigma factor is present in this strain . Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1997 Nov, 118(3), 301 - 4
Effect of dietary ascorbic acid on the hepatic microsomal mixed function oxidase system in liver of chicks treated with Escherichia coli lipopolysaccharide; Takahashi K et al.; We determined whether dietary ascorbic acid (0.3 or 3 g/kg diet) modulates hepatic microsomal mixed function oxidase (MFO) system and plasma alpha 1 acid glycoprotein (AGP) concentration in chicks treated with Escherichia coli lipopolysaccharide (LPS) . Injection of LPS (250 micrograms/kg body weight every other day) intraperitoneally for 14 days decreased cytochromes P450 and b, content and NADPH-cytochrome c reductase activity in hepatic microsomes in male broilers . Content of cytochromes P450 and b5 was negatively correlated with plasma AGP concentration . Feeding ascorbic acid partly alleviated the reduction of cytochromes P450 and b5 in males . Plasma AGP concentration also increased with the LPS injection and was partly lowered by feeding ascorbic acid . The results indicate that dietary ascorbic acid modulates the responses of the microsomal MFO system and of plasma AGP concentration against repeated injection of LPS in male broiler chicks.

Vet Hum Toxicol, 1998 Feb, 40(1), 14 - 8
Effects of indomethacin on lipopolysaccharide-induced plasma PGE2 concentrations and clinical pathological disorders in experimental endotoxemia; Andonova M et al.; The effects of indomethacin, a cyclooxygenase inhibitor, upon plasma concentrations of prostaglandin E2 (PGE2), the febrile response, and metabolic and hematological alterations induced by lipopolysaccharide (LPS) were studied . Experimental endotoxemia was provoked via i.p . injection of 1.0 mg E coli LPS/kg in rats (group A) . Indomethacin was introduced/os (2.5 mg/kg) 30 min prior to LPS challenge (group B) . Pretreatment with this medication completely inhibited the hyperthermic response to LPS and eliminated the LPS-induced non-specific symptoms of anorexia, adipsia, reduced locomotory activity and gastrointestinal troubles . Plasma PGE2 concentrations increased as early as the 2nd h after the LPS challenge but were blocked when endotoxin application was preceded by indomethacin treatment . Indomethacin did not significantly influence hematological parameters . The dynamics of hematocrit and erythrocyte counts were similar in both groups with a decrease up to the 2nd h followed by an increase to maximum at post-treatment day 3 . Pretreatment with indomethacin did not influence the endotoxin-induced leukopenia observed at the 2nd h or the accompanying neutropenia and left shift . Cyclooxygenase inhibition affected total protein concentrations; they were decreased in the early hours of the study (hours 4-6) in both groups . The later tendency towards increase in total protein concentrations was more expressed in animals from group B . Changes in blood glucose were characterized by a permanent tendency towards decrease after hour 2 of LPS challenge up to day 6 (group A) . In group B, a similar tendency was observed, but glucose concentrations decreased between hours 2-6 and then returned to initial values.

J Mol Biol, 1998 Jan 30, 275(4), 695 - 714
Escherichia coli cytidine deaminase provides a molecular model for ApoB RNA editing and a mechanism for RNA substrate recognition; Navaratnam N et al.; ApoB RNA-editing enzyme (APOBEC-1) is a cytidine deaminase . Molecular modeling and mutagenesis show that APOBEC-1 is related in quaternary and tertiary structure to Escherichia coli cytidine deaminase (ECCDA) . Both enzymes form a homodimer with composite active sites constructed with contributions from each monomer . Significant gaps are present in the APOBEC-1 sequence, compared to ECCDA . The combined mass of the gaps (10 kDa) matches that for the minimal RNA substrate . Their location in ECCDA suggests how APOBEC-1 can be reshaped to accommodate an RNA substrate . In this model, the asymmetrical binding to one active site of a downstream U (equivalent to the deamination product) helps target the other active site for deamination of the upstream C substrate.

J Mol Biol, 1998 Jan 23, 275(3), 503 - 13
Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine beta-trypsin; Krokoszynska I et al.; Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5-Cys55, Cys14-Cys38 and Cys30-Cys51 . Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI . Oxidative refolding starts with the formation of single disulfide intermediates . All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli . In each variant the remaining four cysteine residues were replaced by alanine . Four of these variants are shown here to inhibit bovine beta-trypsin: three of them contain native and one non-native (Cys5-Cys51) disulfide . All but one (Cys5-Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low . Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI . Remarkably, in some of the variants the binding constants were found to be higher for the reduced rather than for the oxidized form of the variant . Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value . Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific . In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin . In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra . In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein . This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein . This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster . On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.

J Mol Biol, 1998 Jan 16, 275(2), 245 - 53
Antibody scFv fragments without disulfide bonds made by molecular evolution; Proba K et al.; We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL . This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display . Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation . Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm . We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.

J Mol Biol, 1998 Jan 16, 275(2), 171 - 6
Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3; Bainbridge G et al.; Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution . Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore . These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement . We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography . These channels have altered single-channel conductances but unimpaired voltage-gating . The data show that L3 movement is not required for voltage-gating.

J Mol Biol, 1998 Jan 16, 275(2), 165 - 70
Genomic position analyses and the transcription machinery; Perez-Rueda E et al.; Position analyses have been devised to extract additional transcriptional information from rapidly expanding genomic data bases . The locations of promoter regulatory sites and also the locations of transcription factor DNA-binding domains are analyzed . Strongly preferred positions of activator binding sites occur in both Escherichia coli and eukaryotes, suggesting specific common features of transcription in the two systems . In both systems, regulatory proteins are found to have their DNA-binding domains near termini and the data suggest an evolutionary analysis that complements a phylogenetic analysis based on sequence alignments . The results indicate that positional information can be an important adjunct to sequence comparisons in analyzing genomic information.

Biochimie, 1997 Oct, 79(9-10), 599 - 606
Overexpression of kin17 protein forms intranuclear foci in mammalian cells; Kannouche P et al.; We used antibodies against E coli RecA protein to identify in mouse cells a 45-kDa DNA-binding protein called kin17, which has an active zinc finger and a nuclear localisation signal . Kin17 protein produced in E coli binds preferentially to the curved DNA of a bacterial promoter in vivo and in vitro, suggesting a transcriptional regulation activity . The fact that in rodent cells kin17 protein levels increase after gamma-irradiation suggests its participation in a cellular response to ionising radiation . We raised polyclonal antibodies against the whole kin17 protein and against its derived synthetic peptides . We report the detection of kin17 protein and of truncated forms of the protein by Western blot or by immunocytochemistry after transient overexpression in cultured human cells . Our results indicate that the cross-reactivity with the anti-RecA antibodies is due to an antigenic determinant located in the core of kin17 protein, between residues 129 and 228 . The kin17 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm . The RecA homologous region seems to play an essential role in the localisation of kin17 protein since the deletion of this particular region dramatically changes the form and the distribution of the intranuclear foci . We hypothesise that these dot-like structures reflect nuclear metabolism compartmentalization.

Biochimie, 1997 Oct, 79(9-10), 587 - 92
Mammalian Rad51 protein: a RecA homologue with pleiotropic functions; Vispe S et al.; During the last years, homologues of E coli RecA have been cloned in numerous species including man . These Rad51 proteins share sequence as well as functional homologies with the bacterial protein . Human Rad51 (HsRad51) is able to catalyze strand exchange in vitro between homologous DNAs, but with a lower efficiency compared to that of RecA . This suggests the requirement of additional factors . A very interesting feature of Rad51 is its essential role in mouse which could mean that it has gained an essential function in cell growth . The interaction of HsRad51 with several tumor suppressor genes namely p53, BRCA1 and BRCA2 implies possible role(s) of this protein in tumorigenesis . Thus, the continued study of Rad51 should bring important insights not only into homologous recombination mechanisms but also into cell proliferation regulation.

J Infect Dis, 1998 Feb, 177(2), 388 - 94
Limulus antilipopolysaccharide factor prevents mortality late in the course of endotoxemia; Roth RI et al.; Limulus antilipopolysaccharide factor (LALF) can neutralize bacterial endotoxin, but its ability to prevent mortality following prolonged endotoxemia is unknown . Mice were challenged with an LD50 dose of intraperitoneal E . coli lipopolysaccharide (LPS) and then received LALF at various times after administration of LPS . Survival at 72 h was significantly improved by the administration of LALF at 4, 10, and even 24 h after LPS (73%, 78%, and 65% survival, respectively, vs . 15% survival in controls) . Following intravenous administration of LALF at either 10 or 24 h after LPS, plasma levels of biologically active LPS abruptly fell (> 1000-fold lower than pre-LALF levels) . Plasma LALF concentrations fell much more gradually in LPS-treated mice (t1/2 = 120 min) than in control mice (t1/2 = 2.5 min) . In conclusion, LALF markedly decreased plasma concentrations of biologically active LPS and protected mice from lethality even when LALF was not administered until long after the onset of continuous endotoxemia.

J Infect Dis, 1998 Feb, 177(2), 293 - 300
Chronic uveitis in guinea pigs infected with varicella-zoster virus expressing Escherichia coli beta-galactosidase; Cohen JI et al.; There is no small animal model that replicates chickenpox and herpes zoster, which are caused by varicella-zoster virus (VZV) . Therefore, to detect VZV in tissues of infected animals, the Escherichia coli beta-galactosidase gene was inserted into the viral genome . Intravitreal inoculation of guinea pigs with virus-infected cells resulted in a chronic uveitis, with mononuclear cells in the vitreous cavity of the eye of nearly all animals . Staining with X-gal demonstrated the presence of VZV in the ciliary body or iris of approximately 40% of the animals and in retinal pigmented epithelial cells in 4 animals . X-gal staining showed VZV in the eye of 1 animal 140 days after inoculation . These experiments indicate that VZV expressing beta-galactosidase is useful for detecting virus in tissues and that VZV can cause a chronic uveitis in which virus can be detected in some animals for up to 4 months.

Shock, 1998 Jan, 9(1), 33 - 9
Comparison of hypertonic with isotonic saline hydroxyethyl starch solution on oxygen extraction capabilities during endotoxic shock; Maciel F et al.; Hypertonic colloid solutions reportedly exert protective effects on the microcirculation . The present study investigated the effects of a hypertonic saline hydroxyethyl starch (HES) solution on the oxygen extraction capabilities in an endotoxic shock model in the dog . Fourteen anesthetized and mechanically ventilated dogs received 2 mg/kg of Escherichia coli endotoxin before being randomly divided into two groups to receive a 4 mLkg infusion in 10 min of either hypertonic (7.5%, n = 7), or isotonic (.9%, n = 7) HES solution . Thereafter, each animal received isotonic HES titrated to restore cardiac index to baseline levels, followed by a constant infusion of normal saline at 20 mLkg throughout the study . The amount of fluid required to restore cardiac index to baseline levels was approximately one-half in the hypertonic saline HES group as compared with the isotonic group (123+/-12 vs . 291+/-62 mLkg, p < .05) . The two groups of dogs had similar mean arterial pressure and cardiac index values . Hypertonic saline HES-treated animals had a higher sodium concentration than the control group (144.4+/-4.0 vs . 138.7+/-3.1 mM/L, p < .05) . There were no significant differences in blood gases or lactate concentrations between the groups . When cardiac tamponade was induced to study the tissue oxygen extraction capabilities, hypertonic saline HES-treated dogs had a slightly lower critical oxygen delivery (11.1+/-1.6 vs . 14.2+/-3.3 mL/kg x min, p = NS), and a significantly higher critical oxygen extraction ratio (61.9+/-17.1 vs . 44.0+/-11.5%, p < .05) than the isotonic group . We conclude that during endotoxic shock in dogs, hypertonic saline HES solution can increase whole body oxygen extraction capabilities, probably by an improvement in microvascular perfusion in septic conditions.

J Pharm Pharmacol, 1997 Dec, 49(12), 1239 - 41
Effects of endotoxin on neurally-mediated gastric acid secretion in the rat; Ramirez C et al.; The effects of a peripheral administration of E . coli endotoxin on neurally-mediated gastric acid secretion and the role of endogenous opioids or PAF receptors in endotoxin effects have been evaluated in the continuously perfused stomach of the anaesthetized rat . Gastric acid secretion stimulated by distension (20 cm H2O) was reduced dose-dependently by single intravenous bolus injection of endotoxin (0.1-10 microg kg(-1)) . Doses of 5 microg kg(-1) induced a peak reduction of distension-stimulated acid output and significantly reduced the secretory response induced by an intravenous bolus of 2-deoxy-D-glucose (150 mg kg(-1)) . This dose of endotoxin did not significantly modify mean systemic arterial blood pressure throughout the experimental period . Pretreatment with the opioid receptor antagonist naloxone (1 mg kg(-1) , i.v.) or the platelet-activating factor (PAF) receptor antagonist WEB 2086 (2 mg kg(-1), i.v.) did not reverse the inhibitory effects of endotoxin (5 microg kg(-1) , i.v.) on acid secretion stimulated by both distension and 2-deoxy-D-glucose . These findings suggest that endotoxin-induced acute inhibition of neurally-mediated acid responses, stimulated by gastric distension or administration of 2-deoxy-D-glucose, do not involve the activation of endogenous opioids or PAF receptors.

Endocr J, 1997 Oct, 44(5), 739 - 44
Effect of renal denervation on phosphate excretion in endotoxemic rats; Hiki N et al.; Our previous studies showed that endotoxin (Et) administration causes hypophosphaturia in the presence of PTH . In this study, we tested the hypothesis that enhanced renal nerve activity during endotoxemia is responsible for hypophosphaturia . Two weeks after bilateral renal denervation, phosphate excretion was examined in endotoxemic Wistar rats (300 g body weight) . Renal clearance studies were performed before and after 4 mg/kg body weight Escherichia coli Et administration . Et administration resulted in a marked fall in glomerular filtration rates of innervated rats (n=12, from 2.09 +/- 0.11 ml/min to 0.89 +/- 0.15 ml/min, P<0.005) compared to saline-treated innervated rats (n=7, from 1.98 +/- 0.19 ml/min to 1.76 +/- 0.16 ml/min) . The glomerular filtration rate of renal denervated rats was the same for saline-treated rats (n=9, from 2.67 +/- 0.92 ml/min to 1.69 +/- 0.12 ml/min) and Et-treated rats (n=10, from 2.37 +/- 0.19 ml/min to 1.52 +/- 0.07 ml/min) . Fractional phosphate excretion was significantly reduced after Et challenge in innervated rats (from 24.0 +/- 3.3% to 11.8 +/- 2.2%, P<0.0001) compared to saline injection in innervated rats (from 26.9 +/- 3.9% to 33.0 +/- 1.6%) . Although renal denervation improved the hypophosphaturia in comparison to the innervated rats, fractional phosphate excretion was still lower in Et-treated rats (from 28.8 +/- 5.0% to 18.0 +/- 4.7%, P<0.005) than in saline-treated rats (from 30.2 +/- 6.1% to 38.7 +/- 4.2%) . In conclusion, our data did not support the hypothesis that renal nerves have an important role in reducing renal phosphate excretion during acute endotoxemia.

Mol Microbiol, 1998 Jan, 27(1), 197 - 208
Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE; McNicholas PM et al.; Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate . These changes are mediated by the transcription factors Fnr and NarL respectively . Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon . To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed . The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE . In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E . coli ModE operator sites . Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation . In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site . We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity . Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo . This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

Mol Microbiol, 1998 Jan, 27(1), 187 - 95
Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor Müller; Kim EJ et al.; A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp . The gene for NiSOD (sodN) was cloned from S . coelicolor Muller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme . It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins . The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing . The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa) . The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis . Ni2+ regulates the synthesis of NiSOD polypeptide . S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription . Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S . lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed . Expression of the full-length sodN gene in E . coli produced NiSOD polypeptide without any SOD activity even in the presence of Ni2+ . However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E . coli, indicating that N-terminal processing is required to produce active NiSOD . These results reveal the unique role of nickel as a multifaceted regulator in S . coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.

Mol Microbiol, 1998 Jan, 27(1), 143 - 57
Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli; Bouveret E et al.; Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target . The N-terminal domain of colicins is involved in the import process . The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB . We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the 'TolA box', was important for colicin A import but was not involved in the colicin A-TolA interaction . It was, however, involved in the colicin A-TolB interaction . The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated . Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm . This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells . Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB . The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB.

Mol Microbiol, 1998 Jan, 27(1), 41 - 50
DNA-binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker; Jorgensen CI et al.; The Escherichia coli CytR regulator belongs to the LacI family of sequence-specific DNA-binding proteins and prevents CRP-mediated transcription in the CytR regulon . Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP-CRP complex . Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family . In this report we examined the effect of changing the spacing between CytR half-operators on CytR regulation in vivo and on CytR binding in vitro . Maximum repression was seen with the short spacing variants: repression peaks when the half-operators lie on the same face of the DNA helix . Repression was retained for most spacing variants with centre separations of half-operators < or = 3 helical turns . Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co-operative binding with CRP . Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding . We conclude that CytR does not use hinge alpha-helices for minor groove recognition . Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.

Mol Microbiol, 1998 Jan, 27(1), 1 - 8
The fats of Escherichia coli during infancy and old age: regulation by global regulators, alarmones and lipid intermediates; DiRusso CC et al.; The fluidity and phase state of bacterial lipid bilayers commonly change in response to ambient environmental conditions to maintain the critical functions of the envelope as a semipermeable and selective boundary . A special, and intricate, set of alterations in membrane lipid metabolism is elicited by conditions causing growth arrest . Under such conditions, specific alterations in the membrane lipid-fatty acid composition are required for survival of the cell and, concurrently, the membrane lipids are suggested to serve as endogenous reserves providing carbon/energy for maintenance requirements . It appears that the global regulator FadR is required for both of these activities to be performed properly and that the FadR regulon is interconnected to the universal stress response of Escherichia coli . FadR, in conjunction with long-chain fatty acyl-CoA, long-chain acyl-ACP, ppGpp and cAMP, are key players in regulating the activities of enzymes and expression of genes involved in fatty acid and phospholipid metabolism in dividing and ageing E . coli cells.

Mutat Res, 1997 Dec 12, 395(2-3), 93 - 9
Effects of gender and species on spectra of mutation induced by 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine in the lacI transgene; Okonogi H et al.; Feeding of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) to F344 rats induces colon tumors specifically in male rats . Mutant frequencies and mutational spectra of the lacI transgene were studied in male and female Big Blue transgenic rats after feeding 400 ppm of PhIP in the diet for 60 days . Mutant frequencies in the colon mucosa were increased 20-25 times compared with those of the control rats, being 661.4 +/- 33.3 x 10(-6) and 718.2 +/- 16.9 x 10(-6) in males and females, respectively . No significant differences in types and distribution of the mutations were detected between males and females . One-base deletions were the most frequent mutation, including the characteristic guanine deletion at 5'-GGGA-3' which is also seen in the Apc gene of rat colon cancers induced by PhIP . Comparison of the lacI mutations in the rat colon with those previously identified in the mouse colon showed that the rate of G to T transversions was significantly higher in the mouse . This is the first report stating that there exist differences in the mutation specificity on the same gene, among mammalian species . However, the characteristic guanine deletion was recovered in both the mouse and the rat . These findings do not offer a mechanistic explanation of the gender specificity of PhIP-induced colon cancer in rats, though the universality of the guanine deletion suggests that this alteration may prove a useful indicator of human exposure.

World J Surg, 1998 Jan, 22(1), 6 - 11
Influence of enteral nutrition-induced splanchnic hyperemia on the septic origin of splanchnic ischemia; Kazamias P et al.; The purpose of this experimental study was to investigate whether enteral nutrition-induced postprandial intestinal hyperemia has a beneficial effect on the splanchnic ischemia due to sepsis . Fourteen dogs, after exposure to Escherichia coli endotoxin via portal vein administration were grouped according to whether they were fed enterally via a jejunostomy or given a placebo . Systemic hemodynamics; portal vein, hepatic, and superior mesenteric artery blood flow; hepatic and intestinal microcirculation; hepatic tissue PO2; intestinal pHi; and hepatic energy charge were assessed before, during, and after endotoxin infusion as well as during and after enteral or placebo feeding . All splanchnic hemodynamic parameters revealed a statistically significant decline (p = 0.001) during the endotoxin shock period relative to the baseline . After enteral feeding all parameters exhibited a statistically significant increase (p = 0.001) relative to the placebo group . The results of this study led us to suggest that enteral nutrition reverses the lipopolysaccharide infusion-induced splanchnic ischemia.

Genomics, 1998 Jan 1, 47(1), 7 - 11
Enrichment for loci identical-by-descent between pairs of mouse or human genomes by genomic mismatch scanning; McAllister L et al.; Mapping genes that underlie complex genetic traits, including genes that determine susceptibility to common diseases, requires an efficient method for high-resolution genotyping . Single-nucleotide differences between pairs of allelic sequences from unrelated individuals occur approximately once in every kilobase . Genomic mismatch scanning (GMS), by analyzing numerous single-nucleotide polymorphisms in a single genome-wide step, offers a potentially powerful and efficient approach to linkage analysis . GMS, originally developed in a yeast system, is shown here to be applicable to the more complex mouse and human genomes.

Genomics, 1998 Jan 1, 47(1), 1 - 6
Genomic mismatch scanning identifies human genomic DNA shared identical by descent; Cheung VG et al.; Genomic mismatch scanning (GMS) is a high-throughput, high-resolution identity by descent mapping technique that enriches for genomic DNA fragments that are shared between related individuals . In GMS, DNA heteroduplexes are formed from restriction-digested genomic DNA fragments from two relatives . Mismatch-free DNA heteroduplexes, likely representing DNA shared identical by descent between the two individuals, are relatively purified by depleting the mismatch-containing heteroduplexes using the Escherichia coli mismatch repair proteins and exonuclease . Here, we demonstrate using quantitative microsatellite genotyping that, despite the complexity of the human genome, GMS can enrich the majority of restriction fragments that are identical by descent between two related humans . As the entire genome is selected in GMS, an extraordinarily dense set of markers (up to 200,000 markers) may be screened in parallel . The demonstration of the molecular enrichment of identical DNA fragments in the context of the whole human genome establishes conditions for the application of GMS to human genetics . This forms a frame-work for the further development of GMS as a hybridization-based mapping technique that utilizes DNA microarray technology to map the selected identical by descent DNA fragments.

Protein Eng, 1997 Sep, 10(9), 1085 - 97
Production of human normal adult and fetal hemoglobins in Escherichia coli; Shen TJ et al.; A hemoglobin expression system in Escherichia coli is described . In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter . We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields . The globin genes can be derived from either synthetic genes or human globin cDNAs . The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase . The heme is inserted correctly into the expressed alpha-globin from our expression plasmids . A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin . However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule . We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra . Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E . coli and in high yields . Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Environ Mol Mutagen, 1998, 31(1), 82 - 91
Influence of DNA repair by (A)BC excinuclease and Ogt alkyltransferase on the distribution of mutations induced by n-propyl-N-nitrosourea in Escherichia coli; Ferrezuelo F et al.; In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increment in mutation induction by N-propyl-N-nitrosourea (PNU) . Irrespective of the presence or the absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM PNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system . Some increased susceptibility to mutagenesis by PNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity . These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease . Forward mutations induced by 6 mM PNU within the initial part of the lacl gene were recovered from Uvr+ Ogt-, Uvr- Ogt+, and Uvr- Ogt- bacteria . A total of 439 independent mutations were characterized by DNA sequence analysis . The PNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents . Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)) . We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base and DNA-strand associated with the PNU-induced G:C-->A:T transitions . Preferences different from those previously reported for the ethylating (ENU) and methylating (MNU) analogs were detected . We indicate that these differences might be caused by the PNU possibility of giving iso-propyl adducts, in addition to the expected n-propyl adducts, and by possible preferences in the initial distribution of these lesions as well as in their repair by the (A)BC excinuclease and the Ogt ATase of E . coli.

Environ Mol Mutagen, 1998, 31(1), 32 - 40
The butadiene metabolite, 1,2:3,4-diepoxybutane, induces micronuclei but is only weakly mutagenic at lacI in the Big Blue Rat2 lacI transgenic cell line; Saranko CJ et al.; 1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites, 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB) . We investigated the mutagenicity and induction of micronuclei by DEB in vitro in Rat2 lambda/lacI transgenic fibroblasts (Big Blue Rat2 cells, Stratagene, LaJolla, CA) . Assays for mutagenicity and micronuclei induction were carried out at concentrations of 0, 2, 5, or 10 microM DEB for 24 hours . Exposure of cells to these concentrations of DEB resulted in approximately 100, 50, and 10% survival, respectively, compared with media controls . In independent replicate experiments, no statistically significant increase in lacI mutant frequency was observed in Rat2 cells at any of the DEB exposure concentrations when compared to media or solvent controls . However, regression analyses indicated a trend toward increasing mutant frequency with increasing DEB exposure concentration . Experiments to examine the induction of micronuclei by DEB revealed a concentration-dependent increase in micronuclei in Rat2 cells following exposure to DEB . These results indicate that DEB induces micronuclei in the absence of detectable gene mutation at lacI in Big Blue Rat2 cells . The induction of micronuclei but only weak mutagenicity at the lacI transgene is likely due to the poor recovery of deletions using this lambda shuttle vector system, demonstrating the need to investigate multiple endpoints of genotoxicity when considering the mutational activity of a compound.

Eur J Immunol, 1997 Dec, 27(12), 3447 - 55
A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice; Lamarre A et al.; The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration . After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies . The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag . Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level) . To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose . A small delay in mortality was observed for the scFv-treated animals . Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min . Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.

Protein Eng, 1997 Sep, 10(9), 1099 - 100
A three-step PCR protocol for construction of chimeric proteins; Grandori R et al.; A general method is described for creating chimeric proteins by transposition of subdomain-sized gene fragments . The method uses three sequential PCR steps to circumvent several limitations inherent in transposition of small insertions and has been optimized to avoid purification of intermediate products . The sequence-independence of the method permits flexibility in the choice of host molecule and insertion site.

Protein Eng, 1997 Sep, 10(9), 1077 - 83
Genetic construction, properties and application of a green fluorescent protein-tagged ciliary neurotrophic factor; Negro A et al.; The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance . CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha) . Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha . This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF . The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli . Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography . Under non-reducing conditions GFP-CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species . Despite these discrepancies, the molecular mass of GFP-CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536 . Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain . Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield . Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other . GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons . GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF . GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat . This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner . GFP-CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.

Protein Eng, 1997 Sep, 10(9), 1057 - 60
Catalytic efficiency of signal peptidase I of Escherichia coli is comparable to that of members of the serine protease family; Suciu D et al.; A method for estimating the activity of bacterial signal peptidase I (SPase I) was used to determine its activation energy (E{act}) . Pro-OmpA-nuclease A, a hybrid secretory precursor, was purified to homogeneity under denaturing conditions and used as a substrate . This substrate was used to determine the activity of SPase I at different temperatures . The results show that the conformation of the mature domain of the substrate pro-OmpA-nuclease A has no discernible effect on the activity of SPase I . The activity data at a range of temperatures were then used to determine the activation energy using the Arrhenius equation . We have estimated E(act) to be 10.4 +/- 0.6 kcal/mol . This work indicates that SPase I is as catalytically efficient as the His-Ser-Asp family of proteases.

Protein Eng, 1997 Sep, 10(9), 991 - 7
Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin; Williams MG et al.; Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively . The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B . Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3 . Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.

Mol Immunol, 1997 Aug-Sep, 34(12-13), 891 - 906
Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies; Kim SH et al.; The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available . In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters . A group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters . In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria . The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters . Several different expression systems were examined for their ability to produce catalytically active antibodies . When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low . The secreted scFvs had higher specific activity . On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis . Levels of expression were dependent on the sequence of VL used . Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated . Both original VH and VL regions are required for hapten binding, although the VH is more crucial for catalysis . By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis . This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies.

Cancer Lett, 1998 Jan 9, 122(1-2), 107 - 13
Suicidal inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil derived from the antiviral, sorivudine; Ogura K et al.; An enzymatic study was performed to clarify the mechanism of 18 acute deaths in patients who had received the new oral antiviral drug, sorivudine (SRV), during anticancer chemotherapy with 5-fluorouracil (5-FU) prodrugs . Human dihydropyrimidine dehydrogenase (hDPD), playing a key role in the liver as the rate-limiting enzyme in catabolism of 5-FU, was expressed in E . coli, purified and incubated in the presence of NADPH with SRV or (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of SRV produced by human gut flora . hDPD was rapidly and irreversibly inactivated by BVU, but not by SRV . Radioactivity of {14C}BVU was incorporated into hDPD in the presence of NADPH in a manner reciprocal to the enzyme inactivation . In the absence of NADPH, hDPD was not inactivated by BVU, nor radiolabeled with {14C}BVU . Thus, as we demonstrated previously with studies using the rat, the acute deaths were strongly suggested to be attributable to markedly elevated tissue 5-FU levels which were responsible for irreversible inhibition of hDPD by covalent binding of a reduced form of BVU as a suicide inactivator.

Appl Environ Microbiol, 1998 Feb, 64(2), 526 - 9
High-level production of recombinant human parathyroid hormone 1-34; Suzuki Y et al.; Expression of the synthetic human parathyroid hormone 1-34 {hPTH(1-34)} gene by a gene fusion strategy was demonstrated . hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli beta-galactosidase by linker peptides containing oligohistidine of different lengths . The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease . Optimal expression and enzymatic processing were achieved in the fusion protein beta G-117S4HPT, constructed from amino acids 1 to 117 of beta-galactosidase and the linker of HHHHPGGSVKKR . The fusion protein accumulated more than 20% of the E . coli total protein . The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture . The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.

Biochem Biophys Res Commun, 1998 Jan 26, 242(3), 640 - 7
Characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the acidothermophilic archaeon, Sulfolobus sp . strain 7; Yoshida T et al.; The chaperonin from the acidothermophilic archaeon, Sulfolobus sp . Strain 7, is composed of two kinds of subunits designated as Scp alpha and Scp beta . In this study, we characterized the recombinant Scp alpha and Scp beta, which were separately expressed in Escherichia coli . Both of them were able to assemble to homo-oligomeric double-ring complexes, similar to subunits of group II chaperonins from Thermoplasma acidophilum and Thermococcus strain KS-1 . Both complexes have no or at most trace ATPase activities . However, they could arrest spontaneous refolding of chemically denatured enzyme in the same way as the purified Sulfolobus chaperonin . We found that they dissociated in the presence of 15% ethanol to monomers, which spontaneously assembled to oligomers when concentrated in the absence of ethanol . Both the reconstituted homo-oligomers were unstable, and easily dissociated to monomers . Further structural and functional characterization is necessary to elucidate if these homo-oligomers exist and if so, their function in vivo.

Z Naturforsch {C}, 1997 Nov-Dec, 52(11-12), 807 - 11
Differential processing of homologues of the small subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues; Luo C et al.; ADP-glucose pyrophosphorylase (AGPase), a two-gene-encoded enzyme, is the key component of starch synthesis in all plants . In the present study, we have used an E . coli expression system for the (over)production of proteins derived from both full length and specifically truncated cDNAs encoding small subunits of AGPase from seed endosperm (AGPase-B1) and leaves (AGPase-B2) of barley (Hordeum vulgare) . Based on immunoblot analyses, the molecular mass of the expressed AGPase-B1 (52 kD) was similar to that from endosperm extracts, whereas the expressed AGPase-B2 (56 kD) was larger than that in barley leaves (51 kD) . Expression of truncated cDNAs for both the seed and leaf proteins has allowed for a direct verification of molecular masses that were earlier proposed for mature AGPases in barley tissues . The data suggest that seed AGPase-B1 does not undergo any post-translational proteolytic processing in barley, whereas the leaf homologue is processed to a smaller protein . Possible implications of these findings are discussed.

Biochim Biophys Acta, 1998 Jan 15, 1389(2), 91 - 100
The C-terminus of phosphatidylinositol transfer protein modulates membrane interactions and transfer activity but not phospholipid binding; Tremblay JM et al.; Rat phosphatidylinositol transfer protein (PITP) is a 32 kDa protein containing 271 amino acids . It is involved in a number of cell functions including secretion and cell signaling . To further characterize structure/activity relationships of PITP, two C-terminal truncated derivatives, PITP(1-259) and PITP(1-253), were produced in Escherichia coli and purified to homogeneity . PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholine (PC) or phosphatidylinositol (PI) when transfer was measured using 95/5 mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions . Thus, amino acids 254-258 are critical for transfer activity . The transfer activity of PITP(1-259) was strongly dependent on the composition of the donor and acceptor vesicles . With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to that of PITP . The presence of 2 mol% phosphatidic acid (PA) in either donor or acceptor vesicles reduced transfer activity to between 10 and 20% that of full-length PITP under the same conditions . If both donor and acceptor contained 2% PA, PITP(1-259) was essentially inactive, though the activity of PITP was not affected significantly under these conditions . PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electrostatic interactions . Thus, the C-terminal residues modulate the affinity of PITP for vesicles and the efficiency of phospholipid transfer.

Nature, 1998 Jan 29, 391(6666), 510 - 3
Energy transduction in ATP synthase; Elston T et al.; Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase . This enzyme consists of two principal domains . The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions . The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit . This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites . The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor . It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft . Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production . Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model.

Nature, 1998 Jan 29, 391(6666), 502 - 6
Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor; Li H et al.; Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs) . These are composed of alpha and beta, or gamma and delta, polypeptide chains with variable (V) and constant (C) regions . In contrast to alphabeta TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), gammadelta TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide . Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certain gammadelta T cells . These studies indicate that antigen recognition by gammadelta TCRs may be fundamentally different from that by alphabeta TCRs . The three-dimensional structures of several alphabeta TCRs and TCR fragments, and their complexes with peptide-MHC or superantigens, have been determined . Here we report the crystal structure of the Vdelta domain of a human gammadelta TCR at 1.9 A resolution . A comparison with antibody and alphabeta TCR V domains reveals that the framework structure of Vdelta more closely resembles that of VH than of Valpha, Vbeta or VL (where H and L refer to heavy and light chains), whereas the relative positions and conformations of its complementarity-determining regions (CDRs) share features of both Valpha and VH . These results provide the first direct evidence that gammadelta TCRs are structurally distinct from alphabeta TCRs and, together with the observation that the CDR3 length distribution of TCR delta chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by gammadelta TCRs may resemble antigen recognition by antibodies.

Curr Microbiol, 1997 Nov, 35(5), 267 - 9
Cysteine 195 has a critical functional role in catalysis by isocitrate lyase from Escherichia coli; Rehman A et al.; Cysteine 195 in isocitrate lyase from Escherichia coli has been replaced by directed mutagenesis . Substitution by Ser yields enzyme with a k(cat) that is 0.03% that of wild type, and substitution by Ala, Gly, Thr, or Val yields completely inactive enzyme . The present results are consistent with a functional role of Cys 195.

Gut, 1997 Dec, 41(6), 748 - 52
Studies on the gastric mucosal microcirculation . 2 . Helicobacter pylori water soluble extracts induce platelet aggregation in the gastric mucosal microcirculation in vivo; Kalia N et al.; BACKGROUND: The exact mechanisms by which Helicobacter pylori infection results in gastric mucosal injury are unclear . AIMS: To assess in vivo whether H pylori extracts could initiate an inflammatory response in the rat gastric mucosal microcirculation . METHODS: Extracts of H pylori, Escherichia coli, or distilled water were administered topically to the gastric mucosa of anaesthetised animals . Fluorescence in vivo microscopy assessed macromolecular leakage of labelled albumin from mucosal vessels, leucocyte adherence/rolling, and platelet activity for 90 minutes . RESULTS: H pylori induced increases (p < 0.001) in adherent platelet thrombi and circulating platelet emboli after five and 15 minutes respectively . Adherent platelet thrombi (mean of four per field of view) remained significantly increased throughout the experiment, but circulating emboli (maximum of five at 30 minutes) decreased with time . Leucocyte adherence did not occur although early transient rolling was observed . An 11% increase (p < 0.02) in albumin leakage occurred after five minutes only . The induction of platelet aggregation was only observed following H pylori administration . CONCLUSION: This in vivo study demonstrated the ability of H pylori extracts to promote platelet aggregation within gastric mucosal microvessels . Recruitment of leucocytes was not observed . The results suggest that the early events associated with H pylori infection are platelet aggregation with perhaps subsequent leucocyte recruitment by activated platelets.

Phytochemistry, 1998 Feb, 47(4), 513 - 9
Barley glutamyl tRNAGlu reductase: mutations affecting haem inhibition and enzyme activity; Vothknecht UC et al.; Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem . The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes . It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem . This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell . Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme . These amino acids are thus important for the catalytic reaction or tRNA binding.

Biochemistry (Mosc), 1997 Oct, 62(10), 1128 - 34
Dimerization of recombinant horseradish peroxidase in a reversed micellar system; Klyachko NL et al.; Recombinant horseradish peroxidase reactivated from E . coli inclusion bodies was studied in a reversed micellar system of AOT in octane . The ability of the recombinant enzyme, in contrast to native horseradish peroxidase, to form a dimeric structure was found . The existence of the dimer was proved by results of sedimentation analysis . Dimer/monomer ratio in the enzyme-containing micelles and dimer catalytic activity were found to depend on the substrate used (pyrogallol, guaiacol, o-dianisidine, o-phenylenediamine) . Computer modelling was used to describe possible structures of the dimeric recombinant horseradish peroxidase.

Biochemistry (Mosc), 1997 Oct, 62(10), 1124 - 7
Peculiarities of gene expression of the EcoRII modification-restriction system; Matvienko NN et al.; The restriction-modification genes of the EcoRII system have been cloned into plasmids under control of phage-specific promoters T7 and SP6 . The transcription was induced by cell infection with the recombinant M13 phages with the corresponding genes of phage RNA-polymerases under control of the Plac-promoter in the presence of IPTG . The induction yields significant amounts of EcoRII DNA-methylase for both phage-specific promoters . In both cases no increase in EcoRII endonuclease expression could be achieved . We hypothesize that the expression of the endonuclease gene is regulated on the translational level.

Gene, 1998 Jan 5, 206(1), 127 - 35
Characterisation of two highly conserved but non-allelic cellular disintegrins from rabbit testis; Hardy CM et al.; Two novel cellular disintegrin genes (termed ADAM 6d and ADAM 6e: Gen Bank accession numbers U82750 and U82751) were isolated and characterised from a rabbit testicular cDNA library . The cDNAs have open reading frames encoding for proteins of 731 and 730 amino acids, respectively . They share an amino-acid homology of greater than 89% and a nucleotide base matching of 94% in both the coding and non-coding regions . PCR of DNA extracted from both the parents and progeny of wild rabbits was used to demonstrate that the genes are non-allelic . Recombinant ADAM 6e fused to maltose binding protein was prepared and polyclonal antibodies produced in mice . These polyclonal antibodies recognised two bands with molecular masses of 42 kDa and 46 kDa on Western blots of rabbit sperm extracts run on SDS PAGE reducing gels . The implications of the presence of these two highly conserved proteins in rabbit testis and on sperm are discussed.

Biol Chem, 1997 Dec, 378(12), 1421 - 31
An antisense RNA in IS30 regulates the translational expression of the transposase; Arini A et al.; Two functional promoters had previously been identified in the mobile genetic element IS30 of Escherichia coli . One, P30A, controls the transcription of ORF-A, whose product is the transposase; the other, located within the ORF-A sequence but on the opposite strand, is called P30C, but the nature and function of its product had remained unknown . We identified this product as an RNA about 150 nucleotides long (called RNA-C) that functions as an untranslated antisense transcript . Indeed, biochemical evidence indicates that ORF-C, which is completely contained on RNA-C, is not translated at detectable levels . Mutational analysis of P30C revealed that overproduction of RNA-C resulted in a decrease of IS30 transposition, while a reduction in the promoter strength resulted in an increase of transposition, as measured by the rate of cointegrate formation . We showed that the translation of ORF-A, but not transcription, is negatively affected by the presence of antisense RNA-C . In contrast to other antisense RNAs acting inhibitorily on translation, RNA-C does not seem to affect translation initiation . Most likely its hybridization to the transposase mRNA in the complementary region located in the central part of ORF-A inhibits the ribosomes in their progression, thus reducing the number of completely translated transposase molecules.

Eur J Biochem, 1997 Dec 15, 250(3), 800 - 7
RNA-unwinding and RNA-folding activities of RNA helicase II/Gu--two activities in separate domains of the same protein; Valdez BC et al.; The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family . It is a unique enzyme, which possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular secondary structure in single-stranded RNA . This report shows that these two enzymatic activities are distinct . ATP{S}, GTP and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity . High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity . Mg2+ is required for the helicase activity but not for the RNA-folding reaction . Affinity-purified anti-(RH-II/Gu) polyclonal Ig inhibit the RNA-unwinding activity but not the folding activity . Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA-folding activity of RH-II/Gu but completely inhibited the RNA-helicase activity . A mutant that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single-stranded RNA . The two enzymatic activities of RH-II/Gu reside in distinct domains . Amino acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity . Amino acids 646-801 fold single-stranded RNA but lack helicase activity . This report shows distinct RNA-unwinding and RNA-folding activities residing in separate domains within the same protein.

Eur J Biochem, 1997 Dec 15, 250(3), 794 - 9
A DNA-topoisomerase-II-binding protein with eight repeating regions similar to DNA-repair enzymes and to a cell-cycle regulator; Yamane K et al.; A two-hybrid system was used to isolate factors that interact with the C-terminal region of DNA topoisomerase IIbeta . A positive clone isolated from a HeLa cDNA library encoded 1522 amino acid residues (molecular mass 170670) . The protein, designated topoisomerase-IIbeta-binding protein 1 (TopBP1), interacted with the C-terminal region of topoisomerase IIbeta synthesized in vitro . A database search indicated that TopBP1 possessed eight regions similar to regions of Rad4, Cut5, Ect2, Rev1 and X-ray repair cross-complementing 1 (XRCC1) proteins and a region similar to auto-modification sites of poly(ADP-ribose) polymerase, suggesting that TopBP1 supported catalytic reactions of topoisomerase II through transient breakages of DNA strands.

Eur J Biochem, 1997 Dec 15, 250(3), 751 - 7
Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases; d'Ortho MP et al.; Soluble proenzyme forms of the catalytic domains of membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP and MT2-MMP) and a form of MT1-MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins . Purified, activated forms of the MT-MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan . Only MT2-MMP showed activity against laminin . MT1-MMP retaining the hemopexin domain was able to specifically cleave native type-I and type-III collagens into the 3/4-1/4 fragments typical of the specific collagenases . The catalytic domain alone did not retain this activity . The MT-MMP did not degrade interleukin-1beta, but, similarly to many other MMP, could process a pro {tumor necrosis factor (TNF) alpha} fusion protein to release mature TNF . However, the latter was subsequently degraded into smaller fragments . These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase-3/proMMP13, MT-MMP degrade a number of extracellular matrix macromolecules . Their location at the surface of cells implies that they could play a significant role in the modulation of cell-matrix interactions.

Eur J Biochem, 1997 Dec 15, 250(3), 712 - 26
Three-dimensional solution structure of human angiogenin determined by 1H,15N-NMR spectroscopy--characterization of histidine protonation states and pKa values; Lequin O et al.; Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation . Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy . 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds . The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure . The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state . There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment . The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues . The side-chain of His114 adopts two main conformations in solution . In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pKa values of 6.65 and 6.05 respectively . The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.

Eur J Biochem, 1997 Dec 15, 250(3), 646 - 52
Variability in Arabidopsis thaliana chromosomal high-mobility-group-1-like proteins; Stemmer C et al.; The vertebrate high-mobility-group (HMG) protein HMG1 is an abundant non-histone protein which is considered as an architectural element in chromatin . In the monocotyledonous plant maize, four different HMG1-like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type . We have examined here the HMG1-like proteins of the dicotyledonous plant Arabidopsis thaliana . The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1-like proteins (now termed HMG alpha, HMG beta1/2, HMG gamma, HMG delta) from Arabidopsis indicates that the expression of multiple HMG1-like proteins is a general feature of (higher) plants . The Arabidopsis HMG1-like proteins contain an HMG domain as a common feature, but outside this conserved DNA-binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes . The five HMG1-like proteins were expressed in Escherichia coli and purified . They bind with somewhat different affinity to linear double-stranded DNA . The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA . Reverse-transcribed PCR suggested that the five HMG1-like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.

J Gen Virol, 1998 Jan, 79 ( Pt 1), 117 - 24
Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis; Preston CM et al.; Herpes simplex virus type 1 (HSV-1) transcription can be arrested at the immediate early (IE) stage by continuous treatment of cells with inhibitors of protein synthesis, usually cycloheximide, from the time of infection . We have analysed the effect of cycloheximide on IE gene expression with HSV-1 mutants deficient in the production of functional levels of the three major transactivators, the virion protein (VP16) and two IE proteins (ICP0 and ICP4) . Expression from the HSV-1 IE promoters that control synthesis of ICP0 and ICP27 was, unexpectedly, stimulated by inhibition of protein synthesis . The effect was observed for the ICP0 promoter in its normal genome location and also when cloned upstream of the Escherichia coli lacZ coding sequences and inserted into the viral thymidine kinase locus . Expression from the human cytomegalovirus major IE promoter, when cloned into the genome of HSV-1 mutants, was also increased by inhibition of protein synthesis . Cycloheximide did not affect the intracellular stability of lacZ-specific RNA, suggesting that the response represented an increase in mRNA production . Activation of the ICP0 promoter was observed when protein synthesis was blocked by alternative agents . Since inhibitors of protein synthesis are known to activate cellular signal transduction pathways, our findings demonstrate new mechanisms for the regulation of HSV-1 IE gene expression which may be important during latency and reactivation . The results also highlight previously unrecognized difficulties in analysing the intrinsic activities of promoters when cloned into the HSV-1 genome.

J Gen Virol, 1998 Jan, 79 ( Pt 1), 47 - 50
An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein; Said T et al.; The gene encoding the nucleoprotein (N) and PCR-derived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in Escherichia coli BL21(DE3) transformed by recombinant expression vector pET-14b containing N and PCR-generated sub-fragment cDNAs under the control of the T7 RNA polymerase promoter . Following induction with IPTG, recombinant His-tagged proteins were expressed, purified by affinity metal chelation chromatography under denaturing conditions and renatured . Protein blots were hybridized with various radiolabelled nucleic acid probes . Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain . To confirm this observation, two more accurate approaches were undertaken: (i) a gel retardation assay of RNA and purified protein complexes was done; and (ii) RNA-protein complexes were cross-linked by UV light and analysed on a denaturing polyacrylamide gel . All these experiments led us to conclude that the middle part of N is the domain which interacts with RNA, despite the absence of homology with known consensus amino acid sequences of other RNA-binding proteins.

Eksp Klin Farmakol, 1997 Nov-Dec, 60(6), 34 - 6
{Effect of rifampicin, endotoxin, and their combination on the phagocyte activity, microsomal oxidation, and free radical processes in the liver}; Sibiriak SV et al.; Experiments were conducted on mices to study the effect of strain MRe-600 Escherichia coli endotoxin, rifampicin, and their combination at the level of cytochrome p-450, b5, aminopyrine N-demethylase and aniline-r-hydroxylase activity in the liver, absorptive activity and oxygen dependent metabolism of macrophages, and free-radical processes in the liver . It was found that rifampicin removes the endotoxin-induced depression of microsomal oxidation in the liver, but potentiates the stimulating effect of the endotoxin on macrophageal absorptive activity and the "respiratory outburst" in these cells . The oxidative equilibrium in the liver in this case does not change.

J Surg Res, 1997 Nov, 73(1), 47 - 53
Origin of endotoxemia influences the metabolic response to endotoxin in dogs; Moeniralam HS et al.; Different routes of endotoxin administration have been used to mimic inflammatory and metabolic responses observed during sepsis . Because the origin of endotoxemia may affect the reactions to endotoxin, we compared the induction of tumor necrosis factor (TNF), interleukin-6 (IL-6), hormones, and glucose production after endotoxin (1.0 microg/kg Escherichia coli 0111:B4) administration into a peripheral (n = 8) versus the portal (n = 8) vein in anesthetized dogs . Prior to endotoxin, a laparotomy was performed for cannulation of hepatic vessels . To evaluate the effects of surgery and anesthesia, we also studied the effects of peripheral endotoxin administration in six awake dogs . The rate of appearance of glucose was measured by primed continuous infusion of {6,6-2H2}glucose . In anesthetized dogs, arterial concentrations of TNF and IL-6 increased after endotoxin administration (P < 0.01 vs basal; NS between groups) . Net hepatic TNF production was increased after endotoxin administration (peripheral vs portal endotoxin administration: 533 +/- 177 vs 2135 +/- 1127 ng/min, both P < 0.05 vs basal; NS between groups) . Net hepatic IL-6 production was stimulated only after portal endotoxin delivery (from 86 +/- 129 to 4740 +/- 1899 ng/min, P < 0.05; NS between groups) . Although there were no differences in neuroendocrine activation, portal endotoxin administration resulted in decreased glucose production compared with peripheral administration (13.6 +/- 0.9 vs 16.8 +/- 1.2 micromol/kg.min, P < 0 . 05) . In contrast to anesthetized dogs, endotoxin increased glucose production considerably in awake dogs from 13.8 +/- 1.2 to 24.2 +/- 3.2 micromol/kg.min (P < 0.05; P < 0.05 vs anesthetized dogs) . The contribution of anesthesia and surgery increased the endotoxin-induced IL-6 response by approximately 350% compared with the effect of endotoxin in awake dogs (P < 0.01) . In conclusion, there are no major differences in the responses to endotoxin between peripherally treated and portally treated dogs, except for differences in glucose production . Portal delivery compared with systemic delivery of endotoxin alters hepatic metabolism through nonendocrine mechanisms, reflected in decreased glucose production . The inflammatory, endocrine, and metabolic effects of endotoxin are altered by the combination of surgery and anesthesia .

J Biol Chem, 1998 Jan 9, 273(2), 1257 - 67
Affinity purification of mammalian RNA polymerase I . Identification of an associated kinase; Hannan RD et al.; Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library . Analyses of the deduced amino acid sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the beta' and beta subunits of Escherichia coli RNA polymerase I . Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin . N1S1 cell lines expressing recombinant His-tagged A194 and FLAG-tagged A127 proteins were isolated . These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column . Immunoprecipitation of A194 from 32P metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein . Incubation of the FLAG affinity-purified RNA polymerase I complex with {gamma-32P}ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s) . One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.

J Biol Chem, 1998 Jan 9, 273(2), 1165 - 74
Initiation of DNA replication at palindromic telomeres is mediated by a duplex-to-hairpin transition induced by the minute virus of mice nonstructural protein NS1; Willwand K et al.; The linear single-stranded DNA genome of the minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate . Amplification of this RF is initiated by the folding-back of palindromic sequences serving as primers for strand-displacement synthesis and formation of dimeric RF DNA . Using an in vitro replication assay and a cloned MVM DNA template, we observed hairpin-primed DNA replication at both MVM DNA termini, with a bias toward right-end initiation . Initiation of DNA replication is favored by nuclear components of A9 cell extract and highly stimulated by the MVM nonstructural protein NS1 . Hairpin-primed DNA replication is also observed in the presence of NS1 and the Klenow fragment of the Escherichia coli DNA polymerase I . Addition of ATPgammaS (adenosine 5'-O-(thiotriphosphate)) blocks the initiation of DNA replication but not the extension of pre-existing hairpin primers formed in the presence of NS1 only . The NS1-mediated unwinding of the right-end palindrome may account for the recently reported capacity of NS1 for driving dimer RF synthesis in vitro.

J Biol Chem, 1998 Jan 9, 273(2), 756 - 62
Creation of a fully functional human chimeric DNA repair protein . Combining O6-methylguanine DNA methyltransferase (MGMT) and AP endonuclease (APE/redox effector factor 1 (Ref 1)) DNA repair proteins; Hansen WK et al.; A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow . To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells . Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis . The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage . A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination . Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity . These results demonstrate that the fusion of O6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins . These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical models of chemotherapy toxicity.

J Biol Chem, 1998 Jan 9, 273(2), 713 - 9
The interdomain connector loop of human PCNA is involved in a direct interaction with human polymerase delta; Zhang P et al.; Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase delta (pol delta) and polymerase epsilon . We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol delta, maps to residues 121-135, which overlap the interdomain connector loop of PCNA (residues 119-133) . We have mutagenized residues 122-133 of human PCNA . The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity . The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1 . Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol delta in several different assays . These mutations affected the interactions between PCNA and pol delta as determined by enzyme-linked immunosorbent assays . These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays . The findings show that the interdomain connector loop region is involved in binding of pol delta . This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol delta.

J Biol Chem, 1998 Jan 9, 273(2), 705 - 12
Interactive and dominant effects of residues 128 and 141 on cyclic nucleotide and DNA bindings in Escherichia coli cAMP receptor protein; Cheng X et al.; The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains . However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known . Natural occurring CRP mutants with different phenotypes were employed to address these issues . Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild-type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP . Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding . A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/G141Q mutants . These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP . The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP*, respectively, assumes a CRP* phenotype and has biochemical properties similar to those of the G141Q mutant . These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

J Appl Physiol, 1997 Dec, 83(6), 1941 - 6
Nitric oxide and endothelial permeability; Hinder F et al.; Nitric oxide synthase inhibition reverses systemic vasodilation during sepsis but may increase endothelial permeability . To assess adverse effects on the pulmonary vasculature, 12 sheep were chronically instrumented with lung lymph fistulas and hydraulic pulmonary venous occluders . Escherichia coli endotoxin (lipopolysaccharide; 10 ng . kg-1 . min-1) was continuously infused for 32 h . After 24 h, six animals received 25 mg/kg of Nomega-nitro-L-arginine methyl ester (L-NAME), and six received saline . All sheep developed a hyperdynamic circulatory response and elevated lymph flows by 24 h of lipopolysaccharide infusion . L-NAME reversed systemic vasodilation, increased pre- and postcapillary pulmonary vascular resistance index, pulmonary arterial pressure, and, transiently, effective pulmonary capillary pressure . Lung lymph flows were not different between groups at 24 h or thereafter . Calculated as changes from baseline, however, lung lymph flow was higher in the L-NAME group than in the control animals, with a trend toward lower lymph-to-plasma protein concentration ratio at 25 h . Permeability analysis at 32 h by the venous occlusion technique showed normal reflection coefficients and elevated filtration coefficients without differences between groups . Reversal by L-NAME of the systemic vasodilation during endotoxemia was associated with high pulmonary vascular resistance without evidence of impaired pulmonary endothelial barrier function.

Biochim Biophys Acta, 1998 Jan 19, 1368(2), 184 - 200
K+-dependence of electrogenic transport by the NaK-ATPase; Gropp T et al.; Charge translocation by the NaK-ATPase from shark rectal gland was measured by adsorption of proteoliposomes to a planar lipid membrane . The proteoliposomes were prepared by reconstitution of purified NaK-ATPase into liposomes consisting of E . coli lipids . The protein was activated by applying an ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP . K+ titrations were used to study the effect of K+ on the charge translocation kinetics of the protein . The time-dependent currents obtained after activation of the enzyme with caged ATP were analyzed with a simplified Albers-Post model (E1 (k1)-->E1ATP (k2)-->E2P (k3)-->E1) taking into account the capacitive coupling of the protein to the measuring system . The results of the K+ titrations show a strong dependence of the rate constant k3 on the K+ concentration at the extracellular side of the protein, indicating the K+ activated dephosphorylation reaction . In contrast, k1 and k2 remained constant . The K+ dependence of the rate k3 could be well described with a K+ binding model with two equivalent binding sites (E2P + 2K+ <==> E2P(K) + K+ <==> E2 P(2K)) followed by a rate limiting reaction (E2P(2K) --> E1(2K)) . The half saturating K+ concentration K3,0.5 and the microscopic dissociation constant K3 for the K+ dependence of k3 were 4.5mM and 1.9mM respectively . At saturating K+ concentration the rate constant k3 was approximately 100 s(-1) . The relative amount of net charge transported during the Na+ and the K+ dependent reactions could be determined from the experiments . Our results suggest electroneutral K+ translocation and do not support electrogenic K+ binding in an extracellular access channel . This is compatible with a model where 2 negative charges are cotransported with 3Na+ and 2K+ ions . Error analysis gives an upper limit of 20% charge transported during K+ translocation or during electrogenic K+ binding in a presumptive access channel compared to Na+ translocation.

Inflamm Res, 1997 Dec, 46(12), 486 - 90
The effects of betamethasone derivatives on endotoxin-induced uveitis in guinea pigs; Tsuji F et al.; OBJECTIVE AND DESIGN: We reported previously that the betamethasone derivative betamethasone dipropionate behaves as an anti-glucocorticoid in rat endotoxin-induced uveitis (EIU) . In the present study, we produced EIU in guinea pigs and investigated the effects of betamethasone dipropionate on the EIU . MATERIAL: Male Hartley guinea pigs were used . TREATMENT: Glucocorticoids were instilled into the eye . METHOD: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye . Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry . Prostaglandin E2 (PGE2) production after LPS injection into the anterior chamber was also examined . RESULTS: Intracameral injection of LPS (1 microgram/eye) induced cell infiltration into the anterior chamber and PGE2 production . Betamethasone dipropionate inhibited cell infiltration and PGE2 production more strongly than betamethasone . These results suggest that betamethasone dipropionate is a potent glucocorticoid in guinea pigs . CONCLUSIONS: Structure-activity relationships of glucocorticoids in the guinea pig EIL model may differ from those in the rat EIU model.

Am J Physiol, 1998 Jan, 274(1 Pt 2), H193 - 201
Relationship between plasma NOx and cardiac and vascular dysfunction after LPS injection in anesthetized dogs; Forfia PR et al.; The relationship between plasma nitrite, nitrate, and nitric oxide (NOx), cytokines, and cardiac and vascular dysfunction after lipopolysaccharide (LPS) was studied in chronically instrumented anesthetized dogs . LPS was administered (1 mg/kg i.v.), and hemodynamics were recorded at baseline, every 15 min for 1 h, and every hour for an additional 14 h . Dramatic reductions in mean arterial pressure (-48 +/- 6%), cardiac output (-40 +/- 8%), stroke volume (-42 +/- 9%), and first derivative of left ventricular pressure (LV dP/dt, -38 +/- 7%) were seen within 1 h after injection of endotoxin . Cardiac output was not different from control by 9 h, whereas mean arterial pressure (-19 +/- 7%), stroke volume (-32 +/- 8%), and LV dP/dt (-21 +/- 10%) remained significantly depressed from control . Total peripheral resistance was not significantly different from control . Therefore, the hypotension appears to be due to a reduction in cardiac function and not to vasodilation . Levels of plasma NOx were not different from control until 4 h after LPS reached levels 597 +/- 126% higher than control at 15 h . In vitro production of nitrite by coronary microvessels was also elevated, supporting our in vivo findings . In contrast, production of tumor necrosis factor-alpha and interleukin-6 occurred shortly after endotoxin injection, reaching peak levels at 45 and 150 min, respectively . Our data suggest that inducible nitric oxide synthase induction occurred after LPS injection . It is unlikely that nitric oxide contributed significantly to the hypotension and cardiac dysfunction early in our study, whereas cardiodepressive cytokines, particularly tumor necrosis factor-alpha, may be important . In contrast, the hemodynamic effects seen late after injection of endotoxin may be the result of an overproduction of nitric oxide, since there was a sixfold increase in plasma NOx levels at this time and a marked production of nitric oxide in isolated coronary microvessels in vitro.

Am J Physiol, 1998 Jan, 274(1 Pt 1), L26 - 31
Grain dust-induced lung inflammation is reduced by Rhodobacter sphaeroides diphosphoryl lipid A; Jagielo PJ et al.; To further determine the importance of endotoxin in grain dust-induced inflammation of the lower respiratory tract, we evaluated the efficacy of pentaacylated diphosphoryl lipid A derived from the lipopolysaccharide of Rhodobacter sphaeroides (RsDPLA) as a partial agonist of grain dust-induced airway inflammation . RsDPLA is a relatively inactive compound compared with lipid A derived from Escherichia coli (LPS) and has been demonstrated to act as a partial agonist of LPS-induced inflammation . To assess the potential stimulatory effect of RsDPLA in relation to LPS, we incubated THP-1 cells with RsDPLA (0.001-100 micrograms/ml), LPS (0.02 microgram endotoxin activity/ml), or corn dust extract (CDE; 0.02 microgram endotoxin activity/ml) . Incubation with RsDPLA revealed a tumor necrosis factor (TNF)-alpha stimulatory effect at 100 micrograms/ml . In contrast, incubation with LPS or CDE resulted in TNF-alpha release at 0.02 microgram/ml . Pretreatment of THP-1 cells with varying concentrations of RsDPLA before incubation with LPS or CDE (0.02 microgram endotoxin activity/ml) resulted in a dose-dependent reduction in the LPS- or CDE-induced release of TNF-alpha with concentrations of RsDPLA of up to 10 micrograms/ml but not at 100 micrograms/ml . To further understand the role of endotoxin in grain dust-induced airway inflammation, we utilized the unique LPS inhibitory property of RsDPLA to determine the inflammatory response to inhaled CDE in mice in the presence of RsDPLA . Ten micrograms of RsDPLA intratracheally did not cause a significant inflammatory response compared with intratracheal saline . However, pretreatment of mice with 10 micrograms of RsDPLA intratracheally before exposure to CDE (5.4 and 0.2 micrograms/m3) or LPS (7.2 and 0.28 micrograms/m3) resulted in significant reductions in the lung lavage concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline . These results confirm the LPS-inhibitory effect of RsDPLA and support the role of endotoxin as the principal agent in grain dust causing airway inflammation.

Helicobacter, 1996 Dec, 1(4), 227 - 8
Identification of a 23S rRNA gene mutation in clarithromycin-resistant Helicobacter pylori; Stone GG et al.; BACKGROUND: Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori . This study reports the finding that transversion mutations (A-C) can occur at 2143 as well . MATERIALS AND METHODS: Three clarithromycin-resistant H . pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene . RESULTS: Nucleotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143 . H . pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143 . CONCLUSION: This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H . pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H . pylori.

J Clin Microbiol, 1998 Jan, 36(1), 58 - 63
Multicomponent chimeric antigen for serodiagnosis of canine visceral leishmaniasis; Soto M et al.; In this work, we describe the assembly of a synthetic gene coding for several antigenic determinants found in different Leishmania infantum antigens . Selected epitopes were derived from the ribosomal proteins LiP2a, LiP2b, and LiP0 and from the histone H2A . The resulting gene was overexpressed in Escherichia coli either as a fusion protein (with the vector pMAL-c2) or alone (with the vector pQE) . In both cases, high-level bacterial production of the recombinant protein was achieved and the products were found to be stable . Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments confirmed that the corresponding epitopes are present in the engineered protein . Finally, a serological evaluation of this multiple-epitope protein by Falcon assay screening test-ELISA revealed a sensitivity of 79 to 93% and a specificity of 96 to 100% in diagnosis of canine visceral leishmaniasis, indicating that this protein represents a valuable tool for serodiagnosis.

Planta, 1997, 203(3), 362 - 72
Modification of thiol contents in poplars (Populus tremula x P . alba) overexpressing enzymes involved in glutathione synthesis; Arisi AC et al.; The hybrid poplar (Populus tremula x P . alba) was transformed to express the Escherichia coli gene for gamma-glutamylcysteine synthetase (EC 6.3.2.2: gamma-ECS) in the cytosol . Four transformed lines of poplar were obtained . These were phenotypically indistinguishable from untransformed poplars . Three lines, ggs28 (Noctor et al . 1996, Plant Physiol 112: 1071-1078), ggs11 and ggs5 possessed high levels of bacterial gene transcripts . Line ggs17 had lower transcript levels . Antisera were prepared against bacterial gamma-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS) . Using the antiserum prepared against the purified His-tagged E . coli gamma-ECS, lines ggs28, ggs11 and ggs5 were shown to possess abundant quantities of the bacterial protein, whereas ggs17 contained lower amounts . The antiserum prepared against the purified His-tagged E . coli GS was also effective in screening poplars transformed with the E . coli gene coding for this enzyme . Immunoblots of leaf extracts from poplars overexpressing GS using this antibody revealed two bands . The extractable foliar gamma-ECS activities of the gamma-ECS transformants were in quantitative agreement with the protein levels . Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher gamma-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold . The lines strongly overexpressing gamma-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), gamma-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold) . Foliar thiol contents in ggs17 were no different to those of untransformed plants.

J UOEH, 1997 Dec 1, 19(4), 255 - 64
Gene expression of trehalase during post-dormant development of the brine shrimp, Artemia: comparison of the two species; Nambu Z et al.; Based on a homology screening approach, two degenerate oligonucleotides were employed as primers in a polymerase chain reaction to amplify a fragment of DNA encoding trehalase with a template of cDNA derived from embryos of American Artemia . Sequence analysis revealed that the fragment was composed of 228 bp comprising 76 amino acids, and highly homologous to trehalases of Tenebrio molitor (mealworm beetle), rabbit, Caenorhabditis elegans, Bombyx mori (silkworm) and Escherichia coli treA and treF (58-38%, in order of description) . This fragment was used as a hybridization probe . A Northern blot analysis on American Artemia showed three transcripts of 5.0, 2.7 and 2.2 kb, and the two larger transcripts were also detected in Chinese Artemia . The developmental profile of the gene expression and the trehalase activity suggest that the transcripts of 5.0 and (or) 2.7 kb in both Artemia may be directly or indirectly related to translation of the trehalase . A Southern blot analysis on both Artemia suggested the existence of two highly homologous genes or one gene having an intron within the region where the probe binds in their haploid genome.

Heart Lung, 1997 Nov-Dec, 26(6), 501 - 3
Emphysematous pyelonephritis; Bonoan JT et al.; Emphysematous pyelonephritis, an uncommon severe necrotizing infection of the kidney, usually associated with diabetes mellitus, is a potentially fatal illness . We present a case of a 75-year-old woman with diabetes who was admitted with "pyelonephritis." The computed tomography scan of her abdomen revealed gas in the renal parenchyma, and emphysematous pyelonephritis was diagnosed . The patient had a total nephrectomy, and was treated with antibiotics . Blood cultures and operative cultures grew Escherichia coli.

J Biol Chem, 1998 Jan 16, 273(3), 1316 - 23
Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop; Lecointe F et al.; The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli . The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected . Also, the His6-tagged recombinant yeast Deg1p expressed in E . coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates . Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast . Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C) . Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy . Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments . The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied . The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E . coli (initially called hisT or PSU-I gene product).

Protein Eng, 1997 Aug, 10(8), 975 - 82
Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification; Voss S et al.; The Strep-tag, an artificial peptide ligand of streptavidin, has gained use as an affinity handle for the purification and detection of recombinant fusion proteins . In an attempt to achieve tighter complexation of the peptide, streptavidin was engineered and the amino acid residues 44-47 in the flexible loop from 44 to 53, which is close to the binding site, were subjected to random mutagenesis . A fusion between alkaline phosphatase and the Strep-tag II sequence, an improved version of the Strep-tag, was constructed as a molecular probe for peptide binding . By means of a filter-sandwich assay, two streptavidin mutants with significantly stronger binding activity for the Strep-tag II were thus identified from a library of Escherichia coli colonies . Both in an ELISA with the alkaline phosphatase fusion and in a fluorescence titration experiment with the synthetic Strep-tag II peptide, which carried an anthraniloyl group as chromophore, their affinities were found to be higher by more than one order of magnitude compared with wild-type streptavidin . The nature of the amino acid exchanges and an enhanced electrophoretic mobility of the streptavidin tetramers suggest an altered loop conformation to be part of the optimized binding mechanism . When one of the streptavidin mutants was immobilized on a chromatographic column it exhibited clearly improved performance in the purification of Strep-tag II fusion proteins, and desthiobiotin turned out to be a suitable reagent for mild competitive elution.

Protein Eng, 1997 Aug, 10(8), 959 - 66
Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting; Jung S et al.; The complementary determining regions (CDRs) from the fluorescein-binding antibody 4-4-20, which yields almost no soluble protein in periplasmic expression in Escherichia coli, were transplanted to the framework of the humanized antibody 4D5 . The resulting single-chain Fv fragment (scFv) 4D5Flu showed both a dramatic improvement in soluble expression, even at 37 degrees C, and an improved thermodynamic stability . Antigen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts . This demonstrates that the use of superior frameworks is a robust strategy to improve the physical properties of scFv fragments . We also report that the grafted version was selected in phage display over several competing variants of the same antibody with identical binding constant but poorer folding or stability properties . The selection required four panning rounds and a temperature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv molecules.

Protein Eng, 1997 Aug, 10(8), 927 - 34
Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region; Mansilla F et al.; We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid . The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA . The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity . Furthermore, arginine 7 seems to play a role in regulating the binding of GTP . The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2 . We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.

Protein Eng, 1997 Aug, 10(8), 915 - 25
Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli; Yonemoto W et al.; When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49) . Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme . To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp . The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi . When possible, isoforms were resolved and kinetic properties were measured . The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles . Ser338, which stabilizes a turn near the C-terminus, is important for stability . Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit) . Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe . Thr197 is in the activation loop near the cleft interface . Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site . Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites . In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli . Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.

Mol Endocrinol, 1997 Dec, 11(13), 1971 - 84
Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3 alpha/17 beta-HSD activity and cellular distribution; Lin HK et al.; In androgen target tissues, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5alpha-dihydrotestosterone (5alpha-DHT) (a potent androgen) and 3alpha-androstanediol (a weak androgen) . In this study, a 3alpha-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library . The human prostatic 3alpha-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3alpha-HSD and human type 1, type 2, and type 3 3alpha-HSDs, respectively, and is a member of the aldo-keto reductase superfamily . The close homology with human type 2 3alpha-HSD suggests that it is either identical to this enzyme or a structural allele . Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3alpha-HSD using this assay . Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3alpha-androstanediol oxidation over 5alpha-DHT reduction . Using {14C}-5alpha-DHT as substrate, TLC analysis confirmed that the reaction product was {14C}-3alpha-androstanediol . However, in the reverse reaction, {3H}-3alpha-androstanediol was oxidized first to {3H}-androsterone and then to {3H}-androstanedione, revealing that the expressed protein possessed both 3alpha- and 17beta-HSD activities . The 17beta-HSD activity accounted for the higher catalytic efficiency observed with 3alpha-androstanediol . These findings indicate that, in the prostate, type 2 3alpha-HSD does not interconvert 5alpha-DHT and 3alpha-androstanediol but inactivates 5alpha-DHT through its 3-ketosteroid reductase activity . Levels of 3alpha-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells . In addition, elevated levels of 3alpha-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues . Expression of 3alpha-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney . This study is the first complete characterization of recombinant type 2 3alpha-HSD demonstrating dual activity and cellular distribution in the human prostate.

Science, 1997 Dec 19, 278(5346), 2120 - 3
Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain; Huala E et al.; The NPH1 (nonphototropic hypocotyl 1) gene encodes an essential component acting very early in the signal-transduction chain for phototropism . Arabidopsis NPH1 contains a serine-threonine kinase domain and LOV1 and LOV2 repeats that share similarity (36 to 56 percent) with Halobacterium salinarium Bat, Azotobacter vinelandii NIFL, Neurospora crassa White Collar-1, Escherichia coli Aer, and the Eag family of potassium-channel proteins from Drosophila and mammals . Sequence similarity with a known (NIFL) and a suspected (Aer) flavoprotein suggests that NPH1 LOV1 and LOV2 may be flavin-binding domains that regulate kinase activity in response to blue light-induced redox changes.

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