Escherichia coli

Methods Enzymol, 1995, 258, 278 - 303
Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E . coli ribonucleotide reductase; Bollinger JM Jr et al.; The SF-Abs, RFQ-EPR, and RFQ-Moss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary . Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent . A number of problems can be associated with the RFQ approach . For example, isopentane could conceivably interfere with or alter the chemistry to be studied . A second potential problem involves temperature-dependent equilibria among different intermediate species . This problem has been encountered by Dooley et al . with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers . This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general . Each of the three methods employed can yield time-resolved quantitation of reaction components . In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis . Obvious advantages to the RFQ-Moss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron . In our hands, quantitation by RFQ-EPR was most problematic and least reproducible . This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density . As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator . Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands . Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem . Time-resolved Moss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available . All of the iron centers can be examined simultaneously . The problems associated with Moss arise from its extreme insensitivity . It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Arch Virol, 1995, 140(4), 687 - 702
Serological recognition of feline infectious peritonitis virus spike gene regions expressed as synthetic peptides and E . coli fusion protein; Suiter BT et al.; Cats exposed to feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) cannot be differentiated by serological analysis . Three synthetic peptides and an E . coli recombinant fusion protein generated from FIPV 79-1146 spike gene sequence were produced . Coronavirus positive cat sera reacted to peptide aa 950-990 but were non-reactive to aa137-151 and aa 150-180 peptides as demonstrated by ELISA . Amino acid sequence 97-222 expressed as a galk fusion protein in E . coli was tested against coronavirus positive cat sera by western blot analysis . Only sera from cats exposed to the FIPV type-II strains DF-2 or 79-1146 that were exhibiting signs of FIP recognized the fusion protein . Sera from FECV exposed cats did not recognize the 97-222 fusion protein in western blot analysis.

Vaccine, 1995 Jan, 13(1), 17 - 21
Adjuvant activity of QS-21 for experimental E . coli 018 polysaccharide vaccines; Coughlin RT et al.; Three types of experimental vaccines containing O-side-chain polysaccharide from the enterotoxigenic strain Escherichia coli 018 were evaluated . The immunogenicity of free O-polysaccharide (PS), a polysaccharide-diphtheria toxoid conjugate (PS-conj), and detoxified lipopolysaccharide (dLPS) was tested in female ICR mice, either alone or in combination with QS-21, a purified saponin adjuvant derived from the bark of the tree Quillaja saponaria Molina . Both the number of individual mice responding and the titres of O-polysaccharide specific antibodies in pools of sera were increased by the addition of QS-21 . The immune response to both O-specific polysaccharide and carrier was primarily IgM and IgG1 . The addition of QS-21 not only increased the level of IgG1, but also had a significant adjuvant effect on antigen-specific IgG2a, IgG2b and IgG3.

Vaccine, 1995 Jan, 13(1), 11 - 6
Vaccination with a formalin-killed P-fimbriated E . coli whole-cell vaccine prevents renal scarring from pyelonephritis in the non-human primate; Roberts JA et al.; A formalin-killed P-fimbriated Escherichia coli serotype O4 vaccine was evaluated for protective efficacy in monkeys in an experimental pyelonephritis model following urethral bacterial inoculation . The vaccination did not protect against initial colonization and there were no significant differences in the time of bacteriuria after experimental infection in the two groups of animals . The whole-cell vaccine offers a limited protection against renal dysfunction and scarring (p = 0.002) and less renal involvement (p = 0.04), results that are quite similar to those given by a synthetic O-antigen-specific saccharide-protein conjugate vaccine previously tested.

In Vitro Cell Dev Biol Anim, 1995 Jan, 31(1), 71 - 6
Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by the E . coli lac repressor; Izquierdo RE et al.; Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency . In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation . In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest . We demonstrate that the Escherichia coli ldc operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture . A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter . The latter components were positioned between two synthetic lac operators . Lac repressor expression was driven by a simian cytomegalovirus promoter . In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-beta-D-thiogalactoside (IPTG) . In the absence of IPTG, hGH expression was almost fully repressed . These results show that the components of the E . coli lac operon provide a stringent regulatory system for use in myoblasts . The system might prove to be useful for the regulation of transferred genes in animals.

Bioessays, 1995 Jan, 17(1), 63 - 70
Are there DNA damage checkpoints in E . coli?
Bridges BA.
The concept of regulatory 'checkpoints' in the eukaryotic cycle has proved to be a fruitful one . Here, its applicability to the bacterial cell cycle is examined . A primitive DNA damage checkpoint operates in E . coli such that, after exposure to ultraviolet light, while excision repair occurs, chromosome replication continues very slowly with the production of discontinuous daughter strands . The slower the rate of excision of photoproducts, the greater the delay before the normal rate of DNA replication is restored, the additional time for repair ensuring that normal survival is maintained . A model is proposed in which replication rate is controlled by the ratio of RecA-coated to uncoated single stranded regions of DNA in the replication fork . There are also two cell division inhibitors SulA (= SfiA) and SfiC under the control of the SOS system and sensitive to DNA damage, but they are irrelevant to the survival of wild-type bacteria under normal conditions . In strains where SulA and SfiC do not operate, inhibition is not influenced by the rate of excision repair and so fails one of the criteria for a DNA damage checkpoint, namely the monitoring of the DNA for the level of residual damage.

Nippon Jinzo Gakkai Shi, 1995 Jan, 37(1), 74 - 80
{An adult case of hemolytic uremic syndrome (HUS) after pathogenic Escherichia coli (E . coli) infection}; Arai T et al.; In recent years, it has been revealed that verocytotoxin-producing E . coli (VTEC) infection is one of the leading causes of HUS and the molecular aspects of its pathophysiology have also been studied extensively . We report a case of a 56-year-old man who developed HUS after E . coli (O 26 strain) infection with diarrhea . The characteristic laboratory findings in this case included hypergammaglobulinemia, hypocomplementemia and a high level of immune complex in addition to the common findings of HUS . The light microscopic findings of the first renal biopsy performed before treatment revealed extensive interstitial changes with remarkable mononuclear cell infiltrations as well as mild mesangial proliferation with crescent formation . Subendothelial electron-dense deposits within the glomerular capillary walls and mesangial area were also detected by electron microscopic examination . The diagnostic possibilities of infectious endocarditis and collagen diseases, such as Sjogren syndrome, were reasonably ruled out by the appropriate examinations . After 2-month prednisolone therapy, proteinuria and deteriorated renal functions as well as the abnormal immunological parameters described above were remarkably improved . The second renal biopsy after treatments showed clearly diminished subendothelial deposits and interstitial mononuclear cell infiltrations . This case report might provide information on the unique features of renal interstitial damage and immunological abnormalities in VTEC-induced adult HUS.

Adv Biophys, 1995, 31, 133 - 47
A new type of E . coli recombinational hotspot which requires for activity both DNA replication termination events and the Chi sequence; Horiuchi T et al.; In E . coli rnh- mutants we identified chromosome-derived, specific DNA fragments termed Hot DNA . When the DNA in the ccc form is integrated into the E . coli genome by homologous recombination to form a directly repeated structure, a striking enhancement of excisional recombination between the repeats occurs . We obtained 8 groups of such Hot DNA, 7 of which were clustered in a narrow region called the replication terminus region (about 280 kb) on the circular E . coli genome . A Ter site can impede the replication fork in a polar fashion . The six Ter sites are approximately symmetrical in the terminus and surrounding region . To block the fork at the Ter site, a protein factor, Ter binding protein encoded in the tau (or tus) gene, is required . In tau- cells, Hot activity of HotA, B, and C DNAs disappears, thereby indicating that the Hot activity is fork arrest-dependent . Other Hot activities were tau-independent . In addition, for at least HotA activity, the presence of Chi, and E . coli recombinational hotspot sequence, is required; the Chi dependent HotA activity was detected in a wild type strain but to a lesser extent than that in the rnh- mutant . To explain the HotA phenomenon at the molecular level, we propose a model in which a ds-break occurs at the replication fork arrested at the Ter site . Our recent data that HOT1, a yeast recombinational hotspot, may also depend on the fork blocking event for activity, suggests that a similar ds-break occurs in both eucaryotes and procaryotes.

Life Sci, 1995, 57(13), PL147 - 52
Effects of inhibition of nitric oxide synthesis on TNF alpha serum levels in E . coli endotoxemic rats; Boczkowski J et al.; We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNF alpha serum levels in rats inoculated with E . Coli endotoxin (30 mg/kg i.v.) Pre-treatment of endotoxemic rats with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNF alpha serum levels . By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modify serum TNF alpha . These results suggest that, in E . Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNF alpha synthesis.

Mutat Res, 1995 Jan, 326(1), 17 - 27
Correlation of doxorubicin footprints with deletion endpoints in lacO of E . coli; Sedwick WD et al.; This study explored the possibility that the sequence location of doxorubicin-induced deletion endpoints might relate to DNA structural alterations caused by doxorubicin binding to DNA . The 3'-OH endpoints of doxorubicin-induced deletions terminating in the 35-bp region of lacO appear to distribute differently from spontaneous deletion endpoints . Doxorubicin-induced deletions focus in the 26-bp palindrome which is separated by a 9-bp region with no reverse complementary, whereas spontaneous deletion 3'-OH endpoints are found distributed throughout the operator region . In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA labeled at the 5' end of each of the complementary DNA strands encompassed by lacO . Doxorubicin protected the 9-bp region between the palindromic sequences from DNase I cutting and caused enhanced DNase I cleavage at symmetrical sites in the palindrome, which were inherently resistant to the nuclease in the absence of the drug . These symmetrical sites also define regions in which the occurrence of deletion endpoints is enhanced 6-fold in the presence of doxorubicin . This enhanced cutting and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sites of the drug . Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adjacent to regions where doxorubicin appeared to inhibit cutting by DNase I . These results suggest that the binding of doxorubicin in the palindrome directs both the frequency and the specificity of deletion formation in this gene region.

Cell, 1994 Dec 2, 79(5), 853 - 64
Genetic recombination in E . coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro; Shah R et al.; The E . coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair . Using recombination intermediates made by RecA protein, we have identified specific "hotspots" for RuvC resolution . Characterization of these sites reveals a common tetranucleotide sequence, with the consensus 5'-A/TTT decreases G/C-3' . The correct orientation of the resolution site is required for cleavage . These observations suggest that the strand bias of this sequence will affect the outcome of recombinational crosses by directing resolution to either "patch" or "splice" recombinant products . Mutation of the consensus site in synthetic Holliday junctions abolishes or significantly reduces the efficiency of cleavage, although binding is unaffected, demonstrating that junction recognition and incision are biochemically separable events . We propose that efficient RuvC resolution requires the translocation of Holliday junctions to specific cleavage sites, thus providing a biochemical basis for the similar genetic defects observed in ruvA, ruvB, and ruvC mutants.

EMBO J, 1994 Dec 1, 13(23), 5764 - 71
Purification and characterization of the human Rad51 protein, an analogue of E . coli RecA; Benson FE et al.; In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene . A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity . The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity . Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S . Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining . With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments . Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex . In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S . These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

Arch Biochem Biophys, 1994 Dec, 315(2), 489 - 94
Baculovirus expression of bovine cytochrome P450c17 in Sf9 cells and comparison with expression in yeast, mammalian cells, and E . coli; Barnes HJ et al.; Expression of bovine 17 alpha-hydroxylase cytochrome P450 (P450c17) in insect Sf9 cells using baculovirus is the fourth heterologous expression system developed for studying this enzyme . The enzyme in Sf9 cell membranes has all the expected activities and closely resembles that from the other systems: COS1 cells, yeast, and Escherichia coli . One curious feature of baculovirus expression of this hemoprotein is that the immunodetectable P450c17 is present in an insoluble pellet when produced in the absence of added hemin and in a 10,000 g supernatant when expressed in the presence of added hemin . When comparing the level of expression of bovine P450c17 in these different expression systems, E . coli produces the largest quantity per liter culture, while baculovirus produces the most molecules of P450c17 per cell.

Mo Med, 1994 Dec, 91(12), 730 - 3
E . coli O157:H7 is an emerging pathogen in Missouri; Skala MF; Dramatic media reports of recent outbreaks of illness caused by Escherichia coli O157:H7 have drawn national attention to this emerging pathogen . In 1993, a widely publicized outbreak of over 500 culture-confirmed cases was traced to fast-food hamburgers in Washington, Idaho, Nevada and California . Closer to home, the first (and still the largest) reported waterborne outbreak of this disease occurred in Cabool, Missouri in 1989 . That outbreak affected 243 people, of whom 32 were hospitalized, 2 had hemolytic uremic syndrome, and 4 died . E . coli O157:H7 was added to the list of reportable diseases in Missouri in mid-1992, but its importance is still not widely recognized . This article reviews the results of state and national surveillance for the disease, and suggests methods for improving diagnosis and reporting.

Biochem Mol Biol Int, 1994 Dec, 34(6), 1245 - 51
Protease activity of outer membrane protein OmpT in clinical E.coli isolates--studies using translation initiation factor IF2 as target protein; Steffensen S et al.; During purification of the translation initiation factor IF2 from ompT+ strains of Escherichia coli the IF2 is partially degraded in the presence of membrane debris during the first steps of purification . This is a result of proteolytic cleavage by outer membrane protease OmpT {1} . Here we have investigated the activity of OmpT in 51 clinical E . coli isolates of human origin, by a time dependent OmpT activity assay using IF2 as target protein . The activity of OmpT in the outer cell membrane is highly variable among wild type E.coli strains, ranging from no detectable activity in 65% of the strains to a very high activity in 5 strains (10%) . The OmpT activity is closely related to the assay temperature and to the growth temperature of the cells, and can be reduced or even eliminated by lowering the temperature of growth . The results open the possibility of using non-denaturing gel electrophoresis of crude cell lysates as a screening method in population genetic studies of initiation factor IF2 and other cytoplasmic proteins which are cleaved by OmpT.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4943 - 6
Expression of the E . coli fpg gene in mammalian cells reduces the mutagenicity of gamma-rays; Laval F; The E . coli fpg gene encodes the formamido-pyrimidine-DNA-glycosylase (FPG protein) which specifically removes the formamido-pyrimidine and C8-oxoGuanine residues from gamma-irradiated DNA . The fpg gene was ligated in the psV2 vector and transfected into the Chinese hamster CHO and V-79 cells . The transfected cells expressed a formamido-pyrimidine-DNA-glycosylase activity 30 to 40-fold over the constitutive level . The resistance of CHO and V-79 cells to the lethal effect of gamma-rays was similar in control and transfected cells . Furthermore CHO cells expressing the fpg gene had the same resistance to the lethal effect of hydrogen peroxide as control cells . However, the sensitivity to the mutagenic effect of gamma-rays, measured as 6-thioguanine resistance, decreased both in CHO and V-79 transfected cells . Since the lethal effect of gamma-rays was not modified in cells overproducing the FPG protein, the results suggest that this protein protects the cells against the mutagenic lesions formed by ionizing radiations, and among them C8-oxoguanine.

FEBS Lett, 1994 Nov 14, 354(3), 248 - 50
Effect of single-point mutations Phe41-->His and Phe143-->Glu on folding and catalytic properties of recombinant horseradish peroxidase expressed in E . coli; Gazaryan IG et al.; Wild-type recombinant and Phe41-->His and Phe143-->Glu mutant forms of horseradish peroxidase have been expressed in E . coli and reactivated from inclusion bodies with a yield of about 25% . The purified homogeneous preparations have been studied in the reaction of ABTS oxidation . The effect of mutations on heme entrapment and kinetics of ABTS oxidation demonstrates the essential role of the replaced residues in providing the hydrophobic crevice for the non-covalent heme binding.

Nucleic Acids Res, 1994 Nov 11, 22(22), 4768 - 78
A hidden Markov model that finds genes in E . coli DNA; Krogh A et al.; A hidden Markov model (HMM) has been developed to find protein coding genes in E . coli DNA using E . coli genome DNA sequence from the EcoSeq6 database maintained by Kenn Rudd . This HMM includes states that model the codons and their frequencies in E . coli genes, as well as the patterns found in the intergenic region, including repetitive extragenic palindromic sequences and the Shine-Delgarno motif . To account for potential sequencing errors and or frameshifts in raw genomic DNA sequence, it allows for the (very unlikely) possibility of insertions and deletions of individual nucleotides within a codon . The parameters of the HMM are estimated using approximately one million nucleotides of annotated DNA in EcoSeq6 and the model tested on a disjoint set of contigs containing about 325,000 nucleotides . The HMM finds the exact locations of about 80% of the known E . coli genes, and approximate locations for about 10% . It also finds several potentially new genes, and locates several places were insertion or deletion errors/and or frameshifts may be present in the contigs.

Biophys Chem, 1994 Nov, 52(3), 227 - 49
A model for the binding of E . coli single-strand binding protein to supercoiled DNA; Clendenning JB et al.; A model is proposed for the binding of E . coli single strand binding protein (SSB) to supercoiled DNA . The basic tetrameric binding units of SSB are assumed to bind in pairs to the complementary single strands of a locally melted region . The cooperativity of the binding includes contributions from both protein-protein and base-pair stacking interactions . Each bound SSB tetramer is assumed to unwind l = 34 bp, which implies an unwinding angle of 3.27 turns . The resulting loss of superhelical strain is the essential driving force for binding SSB to supercoiled DNAs . All molecular parameters entering into the theory are estimated from available data, except for the composite binding constant (Ka), which is adjusted to best-fit the theory to the fluorescence quenching (FQ) and diffusion coefficient (D0) data of Langowski et al . Very good fits are obtained with optimum values of Ka that are consistent with estimates from other data . This binding model predicts several noteworthy features . (1) SSB binds essentially always in a single contiguous stack on a supercoiled plasmid, and relative fluctuations in stack length are quite small, in agreement with results of electron microscopy studies . (2) The progressive loss of superhelical strain with increasing bound ligand decreases the affinity of the DNA for SSB . This anti-cooperativity offsets the cooperativity of the binding and causes apparent saturation of the binding at rather low binding ratios . Consequently, over the limited span of the measurements, the FQ data can also be satisfactorily fitted by a non-cooperative model comprising a small number of independent sites . (3) When SSB binds to a population of different topoisomers, the distribution of linking differences of the resulting complexes is extremely narrow . Thus, SSB acts to level any differences in superhelical strain in a population of topoisomers . Finally, the effects of restricting binding to a region comprising only part of the plasmid are assessed.

Scand J Immunol, 1994 Nov, 40(5), 564 - 72
Identification of the site of uptake of the E . coli heat-labile enterotoxin, LTB; Lindner J et al.; The results of this study demonstrate that the B subunit of the E . coli heat labile toxin (LTB) binds to the brush border of intestinal epithelial cells in a highly specific, lectin-like manner . Uptake of LTB and transcytosis to the basolateral side of the enterocytes can be observed within 1 h after feeding, and occurs through both the villous epithelial cells and the epithelial cells overlying lymphoid follicles and Peyer's patches . Binding and uptake most probably occur via receptor-mediated endocytosis, with GM1 ganglioside and galactoproteins on the enterocyte cell surface acting as specific ligands to which the LTB binds . Cell ELISA data, together with the observed distribution of immunocompetent cells and the localization of LTB binding, suggest that LTB which is taken up by the villous enterocytes enters the circulation and subsequently generates an IgG immune response in the spleen . At the same time, LTB which is taken up via the patch associated epithelium generates a local IgG and IgA immune response within the Peyer's patches and intestinal lymphoid follicles.

Bioorg Khim, 1994 Nov, 20(11), 1218 - 25
{Interaction of RNAase H from E . coli with modified hybrid duplexes . I . Duplexes modified at the carbohydrate residue}; Krynetskaia NF et al.; The influence of oligodeoxyribonucleotide probes containing 1-(D-beta-2'-deoxythreo-pentofuranosyl)thymine or 1-(D-beta-2'-deoxy-2'-fluoro-pentofuranosyl)uracil on the ability of the hybrid duplexes to interact with RNase H from E . coli was studied . A kinetic approach was used to measure of the modification effect . The hybrid duplex, prA18/d(TTflU)6TT, was shown not to interact with RNase H, whereas prA18/d(xTTT)6 inhibited the RNase H activity (Ki = 0.67 mkM) . The thermostability of the modified duplexes was estimated . The present technique may lead to the use of some modified oligonucleotides as antisences.

Vaccine, 1994 Nov, 12(14), 1270 - 4
Enteral immunization and challenge of volunteers given enterotoxigenic E . coli CFA/II encapsulated in biodegradable microspheres; Tacket CO et al.; The development of a safe and effective vaccine against enterotoxigenic Escherichia coli (ETEC) would be useful for travellers and for young children in endemic areas . A feasibility study of an enteral ETEC vaccine prototype consisting of colonization factor antigen II (CFA/II), containing two component antigens CS1 and CS3, encapsulated in biodegradable polymer microspheres (BPM) was conducted in healthy volunteers . Ten adult volunteers swallowed intestinal tubes on days 0, 7, 14 and 28; after collection of jejunal fluid samples, 1 mg of CFA/II in BPM was administered via the tube . Volunteers kept a diary of symptoms after each dose . Secretory IgA in jejunal fluids, serum responses and circulating antibody-secreting cells (ASC) were measured before and after vaccination . The vaccine was well tolerated . Five of ten volunteers developed IgA anti-CFA/II ASC by 7 days after the last dose of vaccine; these same five vaccinees had IgA anti-CS3 ASC, and three of these five vaccinees had IgA anti-CS1 ASC . Five of ten vaccinees developed rises in jejunal fluid sIgA anti-CFA/II with peak GMT of 1:42 . About 8 weeks after the first dose of vaccine, ten vaccinees and ten unvaccinated control volunteers underwent challenge with 10(9) c.f.u . ETEC E24377A (O139:H28 LT+ST+CS1+CS3+) . Ten of ten controls and seven of ten vaccinees developed diarrhoea (p = 0.11, 30% vaccine efficacy) . Two of the three protected vaccinees had the highest numbers of ASC and highest sIgA titres during the course of immunization, suggesting that these responses were protective and that this vaccine development strategy has merit . Future studies with higher dosages and a different dosing schedule are planned.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 945 - 56
Characterization of a hemA/hemE mutant of E . coli and regulation of hemE; Pido S et al.; Uroporphyrinogen III is the committed intermediate common to heme and siroheme biosynthesis in E . coli . Uroporphyrinogen III decarboxylase is the first enzyme at the branch point which commits to heme synthesis . A hemin-permeable hemA mutant which could grow on 5-aminolevulinic acid (ALA) or hemin, was mutagenized to give a double mutant, 10L2-1 . The second mutation which was identified as hemE because it was mapped to 90.1 min . by F' and Hfr mapping and P1 transduction, accumulated uroporphyrin and had no uroporphyrinogen decarboxylase activity . This mutation could be complemented with a plasmid harboring the hemE gene of Synechococcus . The complemented strain could grow on ALA and accumulated coproporphyrin and protoporphyrin but not uroporphyrin . The E . coli hemE gene was cloned by transducing 10L2-1 with an E . coli genomic library in lambda gt11 . hemE with upstream regions of various sizes was cloned in front of a promoterless CAT gene . Good growth on chloramphenicol (25-75 micrograms/ml) depended on a promoter within 152 bp upstream of the hemE structural gene start of translation site . In addition, this construct could complement the hemE requirement of 10L2-1 as well as allow it to grow on chloramphenicol . Addition of hemin did not inhibit this growth and therefore it appears that it does not affect the hemE promoter . The hemE structural gene alone allowed good growth on 10 micrograms/ml but poor growth on 25 micrograms/ml chloramphenicol, suggesting that there is a weak promoter within hemE for a downstream ORF . Quantitation of CAT protein in these strains showed a weak promoter within hemE, a promoter 152 bp upstream of hemE and another promoter within 1.3 kb upstream of hemE . The 1.3 kb region contains an ORF 40 bp upstream of hemE, thus suggesting that hemE is part of an operon.

Prep Biochem, 1994 Nov, 24(3-4), 297 - 304
A rapid method for the purification of large amounts of an alpha-complementing peptide derived from beta-galactosidase (E . coli); Gallagher CN et al.; A DNA segment coding for residues 6-44 of beta-galactosidase was ligated to a vector with the glutathione-S-transferase gene which also contained a sequence coding for a thrombin recognition site . The fused protein, with an additional 9 amino acids coded for by the vector, was purified using a glutathione agarose affinity column . A peptide made up of residues 6-44 of beta-galactosidase and the 9 additional amino acids was then cleaved from the glutathione-S-transferase using thrombin and purified with a gel filtration column . The peptide was about 3-4 times as active for alpha-complementation as the alpha-peptide derived from CNBr digestion of wild type beta-galactosidase.

Protein Eng, 1994 Nov, 7(11), 1373 - 7
Circularly permuted dihydrofolate reductase of E . coli has functional activity and a destabilized tertiary structure; Protasova NYu et al.; Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed . Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends . Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield . The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble . Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure . Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.

Carbohydr Res, 1994 Oct 17, 263(2), 271 - 84
Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E . coli K5; Casu B et al.; O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E . coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities . SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions . The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters . Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues . Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions . Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions . These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Carbohydr Res, 1994 Oct 17, 263(2), 217 - 25
Structural comparison of the O6 specific polysaccharides from E . coli O6:K2:H1, E . coli O6:K13:H1, and E . coli O6:K54:H10; Jann B et al.; Two distinct forms of the O6 antigen (LPS) from E . coli were analysed using 1H and 13C NMR spectroscopy . Their structures were found to be {formula: see text} In the O6-specific polysaccharide from E . coli O6:K2 and O6:K13, X is beta-D-Glc p, as had previously been shown for the O6 polysaccharide from E . coli O6:K15; in the O6 specific polysaccharide from E . coli O6:K54, X is beta-D-Glc pNAc.

EMBO J, 1994 Oct 3, 13(19), 4653 - 61
Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E . coli inner membrane protein proW; Whitley P et al.; The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100-residue-long N-terminal tail protruding into the periplasmic space . We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW-PhoA and ProW-Lep fusion proteins, both in wild-type cells and in cells with an impaired sec machinery . Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N-terminal tail . This suggests that the sec machinery can act properly only on domains located on the C-terminal side of a translocation signal, and that the N-terminal tail is driven through the membrane by a mechanism that involves the proton motive force.

Br J Radiol, 1994 Oct, 67(802), 983 - 7
The effects of static 3.0 T and 0.5 T magnetic fields and the echo-planar imaging experiment at 0.5 T on E . coli; Mahdi A et al.; Various mutant strains of Escherichia coli have been exposed to a homogeneous static magnetic field of either 0.5 T or 3.0 T and to the time varying magnetic fields found in echo-planar imaging experiments . No evidence of increased DNA damage was detected, even with bacterial strains disabled for DNA repair.

Res Microbiol, 1994 Oct, 145(8), 621 - 32
Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E . coli strains with multiple adhesive factors; Jallat C et al.; A total of 335 Escherichia coli strains were isolated from sporadic cases of aqueous diarrhoea in patients hospitalized in Clermont-Ferrand, France, during 1991 and 1992 . Many of these strains belonged to the diffusely adhering E . coli (DAEC) group, since 51 of them (15.2%) hybridized with the daaC probe corresponding to the accessory gene of the F1845 adhesin and 13 (3.9%) with the AIDA-I (adhesin involved in diffuse adhesion-I) structural gene . The other pathogenic E . coli groups were weakly represented: 0.6% (2 strains) of enterotoxigenic E . coli (ETEC), 0.6% (2 strains) of enterohaemorrhagic E . coli (EHEC) and 3.9% (13 strains) of enteroaggregative E . coli (EAggEC) . Neither enteropathogenic E . coli (EPEC) nor enteroinvasive E . coli (EIEC) were isolated in our study period . Among the DAEC strains studied, we described two major surface proteins of 16 and 29 kDa . We showed that the 16-kDa protein (CF16K) was involved in adhesion in vitro to Caco-2 and HEp-2 cells . Pretreatment of bacteria with anti-CF16K serum or of Caco-2 cells with purified CF16K greatly decreased the adhesion of the E . coli CF1085 strain producing the CF16K protein to both cell types . The CF16K adhesive factor was found in 9.5% (33 strains) of the 335 E . coli strains studied by colony immunoblot assays with anti-CF16K serum . Twelve strains producing CF16K hybridized with the daaC probe, indicating that the CF16K is not related to the Dr family adhesins which recognized the Dr blood group antigen as receptor . The 29-kDa protein, isolated from 9 strains out of the 335 studied (5.1%), was identified as the CS31A antigen by Western blot assay using anti-CS31A serum and by hybridization experiments with a CS31A DNA probe . This antigen is routinely observed in septicaemic or enterotoxigenic bovine E . coli strains . We showed that a single diarrhoeogenic E . coli strain could harbour at least two adhesive factors, since 36% of CF16K E . coli strain producers and 68.4% of CS31A E . coli strain producers hybridized with the daaC DNA probe.

J Vet Med Sci, 1994 Oct, 56(5), 823 - 6
Expression and purification of recombinant Marek's disease virus serotype 1 specific phosphorylated protein pp38 in E . coli; Endoh D et al.; Phosphorylated protein pp38 is the only protein that is detected in the Marek's disease (MD) lymphoma caused by MD virus serotype 1 (MDV-1) and lymphoblastoid cell lines . In this study, a recombinant protein coded for by the almost entire open reading frame of the MDV-1 pp38 cDNA was produced in E . coli and purified by affinity chromatography . Prior to the expression and purification of the protein, cDNA containing the entire coding region for pp38 was cloned and its nucleotide sequence was determined . Immunoblot analysis indicated that the expressed recombinant protein electrophoresed close to that of the pp38 in infected cells . The difference in mobility of the purified recombinant and the pp38 in infected cells corresponded to a fusion peptide . The recombinant pp38 may be of interest for function analyses and the diagnotic use of pp38.

Protein Eng, 1994 Oct, 7(10), 1239 - 47
Glycine 85 of the trp-repressor of E . coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches; Komeiji Y et al.; Experimental and computational analyses were performed on the corepressor (L-tryptophan) binding site of the trp-repressor of Escherichia coli to investigate the ligand-protein interactions . Gly85, one of the residues forming the hydrophobic pocket of the binding site, was systematically replaced with Ala, Val, Leu and Trp by cassette mutagenesis . Biochemical characterization showed that all these mutations caused significant decreases in tryptophan binding activity . Free energy perturbation calculations were performed for the mutants and were consistent with the experimental results . The lack of a side chain at position 85 was concluded to be essential for binding the corepressor; the structure of the binding pocket was suggested to be tight in the vicinity of Gly85.

Nihon Kyobu Shikkan Gakkai Zasshi, 1994 Oct, 32(10), 956 - 62
{Effect of E . coli endotoxin and D-galactosamine on pathophysiology in rat lungs}; Aritomi T et al.; The effects of repeated intravenous injection of E . coli endotoxin (ETX) and intraperitoneal injection of D-galactosamine (GAL), which decreases the circulating level of alpha 1-antitrypsin, on the pathophysiology of chronic lung injury was studied in rats . Four groups were prepared as follows for 8 weeks . Group 1 (control): Intravenous injection of saline . Group 2: Intravenous injection of ETX (2 mg/kg) once a week . Group 3: Intraperitoneal injection of GAL (200 mg/kg), 2 times daily on 3 consecutive days each week . Group 4: Injection of both ETX and GAL, at the same dosages as used in groups 2 and 3 . Total lung capacity and static lung compliance divided by weight were high in the ETX group and the ETX + GAL group, comparative when compared with those in the control and GAL groups, even though weight gain rates in the ETX + GAL group was less than in other groups . Mean linear intercept of rats in the ETX + GAL group was significantly greater than in other groups . These results suggest that ETX + GAL-treated rats have more emphysematous changes in pulmonary function and structure.

Berl Munch Tierarztl Wochenschr, 1994 Oct, 107(10), 331 - 4
{Detection of verotoxin-producing E . coli in field isolates from domestic and agricultural animals in Sachsen-Anhalt}; Gallien P et al.; A report is given on the detection of verotoxin-producing E . coli (VTEC) strains from field isolates of healthy or ill cattle (n = 141), pigs (n = 306), sheep (n = 15), cats (n = 29) and dogs (n = 25) in the region of the new federal land Sachsen-Anhalt . 5% of the strains isolated from cattle, 32% from pigs, 20% from sheep, 4% from dogs and 0% from cats have shown VTEC . The E . coli-strains were checked for the presence of other factors of virulence, too . A good correlation (82%) was found between the colonization factor F107 and SLT 2/2v-containing strains from pigs in the region of Sachsen-Anhalt, too . Enterohemolysin was not found in SLT 2/2v-positive strains . 91% of the VTEC, isolated from pigs, produced alpha-Hemolysin . The correlation of SLT-containing strains and the production of enterohemolysin was confirmed for ruminants, only . Plasmidprofilings of VTEC from pigs showed mainly a 60 MDa or a 68 MDa plasmid or both, too . The occurrence of heat labile (LT) and in some cases of heat stable (ST) toxin was also checked, to differentiate the VTEC-strains from the enterotoxigenic E . coli strains (ETEC) . These investigations showed, that VTEC produce SLT almost without exception . Correlations and conclusions on the pathogenicity for humans are discussed.

Zentralbl Veterinarmed A, 1994 Oct, 41(8), 640 - 4
Influence of the oestrous cycle on experimental intrauterine E . coli infection in the sow; de Winter PJ et al.; The influence of the oestrous cycle on the onset of endometritis in the sow was studied . Ten pubertal, unmated gilts of the Belgian Negative Landrace were used . Nine gilts were inoculated into the uterus by laparotomy with a suspension of an E . coli strain isolated from the uterus of a discharging sow from a herd having many problems with vaginal discharge and a lowered fertility . One gilt was as a control inoculated with 2 ml of a PBS-solution . All sows inoculated during dioestrus developed clinical symptoms, but only 1 of the 5 gilts inoculated at standing oestrus developed a vaginal discharge . These data confirm the hypothesis that the stage of the oestrous cycle has an important influence on the onset of endometritis . The resistance to E . coli infections was higher when the gilts were inoculated during oestrus.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3846 - 53
Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E . coli and in vitro; Pletsa V et al.; Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors . After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT . Taken together, these observations suggest reduced AGT repair at 12G2 . Kinetic analysis of in vitro DNA replication in the same sequences using E . coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis . By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.

Cell, 1994 Sep 23, 78(6), 1063 - 72
Atomic structure of the RuvC resolvase: a holliday junction-specific endonuclease from E . coli; Ariyoshi M et al.; The crystal structure of the RuvC protein, a Holliday junction resolvase from E . coli, has been determined at 2.5 A resolution . The enzyme forms a dimer of 19 kDa subunits related by a dyad axis . Together with results from extensive mutational analyses, the refined structure reveals that the catalytic center, comprising four acidic residues, lies at the bottom of a cleft that nicely fits a DNA duplex . The structural features of the dimer, with a 30 A spacing between the two catalytic centers, provide a substantially defined image of the Holliday junction architecture . The folding topology in the vicinity of the catalytic site exhibits a striking similarity to that of RNAase H1 from E . coli.

Cell, 1994 Sep 23, 78(6), 1051 - 61
DNA replication triggered by double-stranded breaks in E . coli: dependence on homologous recombination functions; Asai T et al.; Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E . coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site . RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi . Chi is dispensable when cells are induced for the SOS response or contain a recD mutation . recBC sbcA mutant cells are also capable of RDR induction . A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells . RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively . Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1217 - 23
High level expression in E . coli and purification of yeast transcription factor IIIA; Ottonello S et al.; Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form . High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E . coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species . Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.

FEBS Lett, 1994 Sep 12, 351(3), 401 - 4
Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E . coli; Chen B et al.; We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin-labelled probe synthesized with the polymerase chain reaction . The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M(r) = 6,276) . The ferredoxin gene was expressed in aerobically grown E . coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein . EPR analysis of recombinant ferredoxin revealed the presence of a {3Fe-4S} cluster which is characteristic of native D . gigas ferredoxin II.

Nucleic Acids Res, 1994 Sep 11, 22(18), 3737 - 41
Inefficient excision of uracil from loop regions of DNA oligomers by E . coli uracil DNA glycosylase; Kumar NV et al.; Kinetic parameters for uracil DNA glycosylase (E . coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined . Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides . This is mainly because of the favourable Vmax value of the enzyme for single-stranded substrates . More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient . The poor efficiency with which uracil is excised from loop regions is a result of both increased Km and lowered Vmax values . This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins . In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.

Nucleic Acids Res, 1994 Sep 11, 22(18), 3708 - 14
Role of conserved nucleotides in building the 16S rRNA binding site of E . coli ribosomal protein S8; Allmang C et al.; Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained . Despite its restricted size, the precise conformation of this region remains a question of debate . Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region . These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site . The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove . In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone . Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization . The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove . The third adenine (A595) is not a direct recognition site but must adopt a bulged position . The U598-U641 pair should not be directly in contact with the protein.

Cell, 1994 Sep 9, 78(5), 845 - 53
SecA protein is exposed to the periplasmic surface of the E . coli inner membrane in its active state; Kim YJ et al.; E . coli cells harboring pCG169 containing the secD secF locus possessed SecA protein almost entirely in an integral membrane form in which it displayed normal protein translocation activity . These results imply that integral membrane SecA is the catalytically active form of this enzyme and that products of the secD secF locus regulate SecA association with the inner membrane . Protease and biotinylation accessibility studies of right side-out and inside-out membrane vesicles derived from this strain revealed that SecA was exposed to the periplasmic surface of the inner membrane . These studies suggest a model of bacterial protein secretion, whereby insertion of SecA into the inner membrane and its association with SecY/E/G promotes assembly of active protein-conducting channels comprised in part of integral membrane SecA protein, and products of the secD secF locus regulate the channel assembly-disassembly reaction by modulating the SecA insertion-deinsertion step.

Zentralbl Veterinarmed A, 1994 Sep, 41(7), 530 - 47
Metabolic, endocrine and haematological responses to intravenous E . coli endotoxin administration in 1-week-old calves; Kinsbergen M et al.; Responses to i.v . injected E . coli endotoxin (E), followed by saline infusion, as compared with saline infusion alone, were studied for 24 h in 1-week-old calves . After administration of E, respiratory rate (RR), heart rate (HR), rectal temperature (RT), serum iron, insulin, (I), cortisol and tumor necrosis factor-alpha, transiently, and urea, continuously, increased . Isoleucine and leucine became elevated at 24 h, whereas white-blood-cell number, free fatty acids (FFA) and triglycerides (TG) increased after an initial fall . After administration of E, packed-cell volume, erythrocyte number, haemoglobin, glucose (G), cholesterol, phospholipids (PL), lysine, arginine, proline, citrulline, calcium (Ca), inorganic phosphorus, insulin-like growth factor I (IGF-I) and 3,5,3'-triiodothyronine (T3) concentrations and alkaline phosphatase (AP) and gamma-glutamyl transferase (gamma GT) activities increased significantly while growth hormone decreased non-significantly . When saline was infused alone, G, TG, PL, Ca, AP, gamma GT, I, IGF-I and T3 decreased, while FFA, urea and sodium increased, but, changes of G, urea, AP, IGF-I and T3 were less marked than after injection of E . Potassium, total protein and albumin concentrations, and glutamyl dehydrogenase and glutamate oxalacetate transaminase activities were not significantly affected by either treatment . In conclusion, metabolic and endocrine changes during saline infusion alone were typical for food withdrawal . Changes of variables after administration of E were transient, biphasic or sustained, thus expressing complex interactions between metabolic parameters, endocrine factors and cytokines.

J Bacteriol, 1994 Sep, 176(17), 5494 - 504
Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E . coli and transposase suppressor mutations; Weinreich MD et al.; Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli . This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp . Instead, it was strictly correlated with the presence of a wild-type N terminus . Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect . This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation . Four E . coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci . These suppressor mutations map near essential genes involved in cell division and DNA segregation . One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min . A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E . coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell . Furthermore, the N terminus of Tnp is necessary for this interaction . The possible significance of this phenomenon for the transposition process is discussed.

J Mol Recognit, 1994 Sep, 7(3), 207 - 9
Identification and analysis of a template-primer (ds-DNA) binding cleft in E . coli DNA polymerase I: an electrostatic potential contour pattern of the modeled structure; Yadav PN et al.; In the modeled structure of the Klenow fragment of E . coli DNA polymerase I, we have identified a distinct region that exhibits a strong electropositive potential contour . The examination of the distribution of the electropositive and negative potential across the two-dimensional slices of the modeled structure revealed that the positive potential was concentrated around the cleft . The approximate size and shape of the region appears well suited to accommodate eight base pairs of duplex DNA and is consistent with the position of the dsDNA binding cleft reported in the crystal structure {Beese et al., Science (1993) 260, 352-355}.

Curr Microbiol, 1994 Sep, 29(3), 177 - 84
Identification and characterization of Mycobacterium paratuberculosis recombinant proteins expressed in E . coli; el-Zaatari FA et al.; Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants, and it has also been isolated and identified from patients with Crohn's disease, an inflammatory bowel disease . The control of Johne's disease has been hampered by the lack of a reliable diagnostic test because of the large degree of antigenic cross-reactivity between mycobacterial and non-mycobacterial species . To help identify specific antigen(s) or epitope(s), an M . paratuberculosis expression library was screened with antibodies and DNA probes . In total, 54 clones were randomly picked, purified, and characterized by DNA probes and monoclonal antibodies with known specificity to individual mycobacterial antigens . Four clones carrying the heat shock protein 65K-, two representing the secreted protein 32K-, three representing the 21K-, and 20 clones representing the specific insertion element of M . paratuberculosis (IS900)-encoding genes and their gene products were identified and characterized . Well-defined recombinant antigens and/or epitopes representing M . paratuberculosis may facilitate the development of specific diagnostic tests and the investigation of their role in these chronic diseases.

J Physiol, 1994 Aug 15, 479 ( Pt 1), 159 - 69
Neurally maintained hypersecretion in undernourished rat intestine activated by E . coli STa enterotoxin and cyclic nucleotides in vitro; Nzegwu HC et al.; 1 . The electrogenic secretory responses of stripped jejuna and ilea from chronically undernourished rats (50% control diet for 21 days) to the bacterial enterotoxin Escherichia coli STa, measured as the short-circuit current in vitro, show an enhanced maximum secretion (ISC, max) with a prolonged duration compared with fed intestine . 2 . The ISC, max is unaffected by pretreatment of the intestine in vitro with hexamethonium, atropine, procaine or indomethacin, or by desensitization to 5-hydroxytryptamine (5-HT), while the prolonged duration is unaffected by atropine, indomethacin or 5-HT desensitization but is reduced by hexamethonium and procaine . 3 . Both 8-bromo-cyclic GMP and dibutyryl cyclic AMP added serosally activate the enhanced ISC, max and its maintenance . Pretreatment with tetrodotoxin had no effect on the initial ISC, max but prevented its maintenance . 4 . Bethanechol, dimethyl phenyl piperazinium, vasoactive intestinal polypeptide, 5-HT and luminal propionate all induced the characteristic hypersecretory activity in the undernourished intestine compared with the fed state, but none could activate the maintenance circuit to prolong their transient responses . 5 . Maintenance of the induced hypersecretory activity is the first example of induction of the neural control of intestinal secretion by the dietary intake level and illustrates the plasticity of the enteric nervous system.

FEBS Lett, 1994 Aug 8, 349(3), 354 - 8
Artificial antifreeze proteins can improve NaCl tolerance when expressed in E . coli; Holmberg N et al.; A chemically synthesized DNA fragment encoding an artificial antifreeze protein was expressed in E . coli as a translational fusion with a truncated protein A . Two constructions were made, with two and four antifreeze domains, respectively . The fusion proteins stimulated the growth of their bacterial host cells at inhibitory NaCl concentrations . The fusion protein carrying four antifreeze domains also conferred improved tolerance towards freezing.

J Vet Med Sci, 1994 Aug, 56(4), 799 - 801
Construction of recombinant infectious laryngotracheitis virus expressing the LacZ gene of E . coli with thymidine kinase gene; Okamura H et al.; We constructed the recombinant infectious laryngotracheitis virus (ILTV), CE strain, containing the LacZ gene of E . coli in the thymidine kinase gene . The growth property of the recombinant virus was almost the same as parental CE strain in chicken embryo fibroblasts . The recombinant CE strain of ILTV could be used as a live vaccine vector.

Protein Expr Purif, 1994 Aug, 5(4), 337 - 45
Expression of active human GRO beta and GRO gamma neutrophil chemotactic proteins in E . coli; Zagorski J et al.; Human GRO alpha, GRO beta, and GRO gamma are neutrophil chemoattractants structurally related to IL-8 and compete with IL-8 for binding to IL-8 receptors on neutrophils . These proteins are part of a large superfamily of chemotactic cytokines, the "chemokines," members of which share striking structural similarities . We have expressed GRO cDNA's in Escherichia coli as fusions to the MalE gene product, maltose-binding protein (MBP), in a way that allows separation of GRO and MBP moieties by factor Xa cleavage . GRO beta and GRO gamma expressed in bacteria were active in in vitro chemotaxis assays and were as effective as IL-8 in inducing chemotactic migration of neutrophils . Recombinant GRO beta was chemotactic rather than chemokinetic when tested by checkerboard analysis while GRO gamma showed evidence of chemokinetic as well as chemotactic activity . The activities of GRO beta and GRO gamma were not species-specific as both proteins were active on rat as well as human neutrophils and were inhibitable by antibodies raised against CINC, the rat GRO homolog . These data indicate that the MBP fusion protein expression system provides a rapid and simple method for obtaining large quantities of members of the chemokine protein family for biological uses.

Protein Eng, 1994 Aug, 7(8), 945 - 51
Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E . coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM; Kishan R et al.; The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution . The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E . coli TIM with the equivalent unit of chicken TIM . This replacement involves 10 sequence changes . One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine . The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7) . No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities . The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8) . From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM.

Wei Sheng Wu Xue Bao, 1994 Aug, 34(4), 266 - 70
{The cloning of threonine operon in vivo and mutagenesis in vitro in E . coli}; Wang A et al.; Report here was the selection of the E . coli strain producing 1.2% threonine . The wild type threonine operon was cloned in vivo and subcloned in vitro . The subclone was mutagenizied in vitro and a clone pTHR12-9-1 producing threonine was obtained . Also the effect of pTHR12-9-1 on threonine yield of hosts producing different amount of threonine was studied . We found that pTHR12-9-1 could promote the production of threonine in lower yield hosts, but it reduced the production of threonine in higher yield hosts . The phenomenon was discussed.

Ann N Y Acad Sci, 1994 Jul 29, 726, 223 - 34; discussion 234-5
Structure and function of the DNA repair enzyme exonuclease III from E . coli; Kuo CF et al.; The three-dimensional structure of exonuclease III, the major AP DNA repair endonuclease of Escherichia coli, has been determined using x-ray crystallographic methods at 2.7 A resolution . The atomic model was fit to an electron density map calculated with phases obtained from three isomorphous heavy atom derivatives . The overall chain fold of exonuclease III is that of a compact alpha,beta-protein of dimensions 55 by 50 by 45 A . The pair of extended beta-pleated sheets pack against each other in an approximately parallel fashion to form the hydrophobic core of a four-layered sandwich structure . These beta sheets are flanked by four alpha-helices that form the outer two layers of the fold . The individual strands of the beta-sheets are in a mostly antiparallel configuration and are linked by extensive loop regions that connect adjoining strands . The structure contains internal symmetry with the two extended beta-sheets and four alpha-helices related by a pseudo-twofold axis running approximately down the center of the two sheets . This internal symmetry is not mirrored in the structure of the loop regions, nor is it detectable within the amino acid sequence . There is a "groove" between the beta-sheets at one end of the molecule that is bordered by several of the exposed loop regions and may be significant for DNA binding.

Biochim Biophys Acta, 1994 Jul 29, 1186(3), 243 - 6
Complementation of Escherichia coli uncD mutant strains by a chimeric F1-beta subunit constructed from E . coli and spinach chloroplast F1-beta; Burkovski A et al.; ATP-synthesizing F0F1-ATPases are complex enzymes consisting of at least eight different subunits . These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues . It was surprising to find that some of the less well conserved subunits like delta and epsilon could replace their E . coli counterparts, whereas the highly conserved beta subunit, which carries the active site, in the E . coli enzyme could not be substituted by spinach chloroplast beta (Lill et al . (1993) Biochim . Biophys . Acta 1144, 278-284) . We constructed a chimeric F1-beta subunit consisting of spinach beta in which the 96 N-terminal amino acids were replaced by the respective residue sequence from E . coli beta . Whereas spinach beta did not complement E . coli uncD mutant strains, the chimeric beta subunit restored growth under conditions of oxidative phosphorylation.

FEBS Lett, 1994 Jul 18, 348(3), 233 - 8
Formation of SRP-like particle induces a conformational change in E . coli 4.5S RNA; Lentzen G et al.; E . coli P48 protein is homologous to the SRP54 component of the eukaryotic signal recognition particle . In vivo, P48 is associated with 4.5S RNA which shares a homology with eukaryotic SRP RNA . To study the interaction between P48 and 4.5S RNA in vitro, we used 4.5S RNA with fluorescein coupled to the 3'-terminal ribose . Upon binding of P48, the fluorescent 4.5S RNA shows a substantial decrease in fluorescence . Fluorescence quenching as well as anisotropy measurements reveal that the effect is not due to a direct interaction of P48 with the dye . This suggests that the binding of P48 induces a conformational change in 4.5S RNA which affects the structure at the 3' end of the RNA . From equilibrium titrations with fluorescent 4.5S RNA, a dissociation constant of 0.15 microns is obtained for the RNA.protein complex . The formation of the complex is not affected by GTP binding to or hydrolysis by P48.

EMBO J, 1994 Jul 15, 13(14), 3348 - 55
Quality and position of the three lac operators of E . coli define efficiency of repression; Oehler S et al.; Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation . We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome . We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators . A short distance of 92 bp allows effective repression by two very weak operators (O3, O3) . The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration . This increase in concentration depends on flexible DNA which allows loop formation.

Nucleic Acids Res, 1994 Jul 11, 22(13), 2538 - 46
Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E . coli ribosomal protein S15; Philippe C et al.; Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site . This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state . In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex . The results were compared to in vivo expression and repression rates . The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.

J Biol Chem, 1994 Jul 8, 269(27), 17863 - 71
Expression, purification, and kinetic characterization of the mannitol transport domain of the phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli . Kinetic evidence that the E . coli mannitol transport protein is a functional dimer; Boer H et al.; The overexpression of the membrane-bound C domain of the mannitol transport protein EIIMtl of Escherichia coli has been achieved . This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity . Gel filtration experiments showed that purified IICMtl was a dimer, confirming that the interaction within the EIIMtl dimer occurs between the membrane-bound portions of the protein . IICMtl in combination with a chimeric protein consisting of the membrane-bound EIIGlc C domain and the cytoplasmic EIIMtl BA domain could restore both phosphoenolpyruvate-dependent phosphorylation and mannitol/mannitol-P exchange activity . The interaction in this complex was comparable to that of IICMtl with soluble IIBAMtl in as much as there appeared to be no specific interaction between IICMtl and the membrane-bound EIIGlc C domain; the Km of IICMtl for the chimer was so low that saturation could not be achieved . In contrast, a very high affinity with a Km of 2 nM was measured between purified IICMtl and purified EIIMtl . This interaction was manifested in a IICMtl-dependent stimulation of the EIIMtl catalyzed phosphoenolpyruvate-dependent mannitol phosphorylation reaction and the mannitol/mannitol-P exchange reaction . The high affinity of IICMtl for the wild type enzyme can be explained by the formation of heterodimers consisting of a IICMtl monomer and an EIIMtl monomer which interact at the level of the membrane-bound domains . The 2-fold increase in mannitol phosphorylation activity of the hetero- versus homodimer is an indication that the individual subunits in the homodimer are functionally coupled and work at only half their maximum rate . It is known that the EIIMtl dimer, but not the monomer, catalyzes the mannitol/mannitol-P exchange reaction . Since the heterodimer also catalyzes this reaction, it appears that only one functional B domain is required per dimer.

FEBS Lett, 1994 Jul 4, 348(1), 41 - 5
Calorimetric studies of the energetics of protein-DNA interactions in the E . coli methionine repressor (MetJ) system; Cooper A et al.; Calorimetric measurements of binding of a specific DNA fragment and S-adenosyl methionine (SAM) co-repressor molecules to the E . coli methionine repressor (MetJ) show significant differences in the energetics of binary and ternary protein-DNA complexes . Formation of the MetJ:SAM:DNA ternary complex is significantly more exothermic (delta H congruent to -99 kJ.mol-1) than either MetJ:DNA or MetJ:SAM binary complexes alone (delta H congruent to -10 kJ.mol-1 each) . The protein is also significantly more stable to unfolding (delta Tm congruent to 5.4 degrees C) when bound to DNA . These observations suggest that binding of SAM to the protein-DNA complex leads to a significant reduction in dynamic flexibility of the ternary complex, with considerable entropy-enthalpy compensation, not necessarily involving any overall conformational change.

Protein Eng, 1994 Jul, 7(7), 823 - 30
The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution; Kankare J et al.; The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9% . The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini . Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology . The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues . Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases . Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions . The others, more spreadout and more positively charged, presumably bind substrate . Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently . Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.

Brain Res Mol Brain Res, 1994 Jul, 24(1-4), 145 - 52
A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E . coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice; Huh SO et al.; Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells . Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders . To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E . coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice . The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice . A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus . In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice . These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.

Proteins, 1994 Jul, 19(3), 183 - 98
The closed conformation of a highly flexible protein: the structure of E . coli adenylate kinase with bound AMP and AMPPNP; Berry MB et al.; The structure of E . coli adenylate kinase with bound AMP and AMPPNP at 2.0 A resolution is presented . The protein crystallizes in space group C2 with two molecules in the asymmetric unit, and has been refined to an R factor of 20.1% and an Rfree of 31.6% . In the present structure, the protein is in the closed (globular) form with the large flexible lid domain covering the AMPPNP molecule . Within the protein, AMP and AMPPNP, and ATP analog, occupy the AMP and ATP sites respectively, which had been suggested by the most recent crystal structure of E . coli adenylate kinase with Ap5A bound (Muller and Schulz, 1992, ref . 1) and prior fluorescence studies (Liang et al., 1991, ref . 2) . The binding of substrates and the positions of the active site residues are compared between the present structure and the E . coli adenylate kinase/Ap5A structure . We failed to detect a peak in the density map corresponding to the Mg2+ ion which is required for catalysis, and its absence has been attributed to the use of ammonium sulfate in the crystallization solution . Finally, a comparison is made between the present structure and the structure of the heavy chain of muscle myosin.

Nature, 1994 Jun 30, 369(6483), 761 - 6
Three-dimensional structure of beta-galactosidase from E . coli; Jacobson RH et al.; The beta-galactosidase from Escherichia coli was instrumental in the development of the operon model, and today is one of the most commonly used enzymes in molecular biology . Here we report the structure of this protein and show that it is a tetramer with 222-point symmetry . The 1,023-amino-acid polypeptide chain folds into five sequential domains, with an extended segment at the amino terminus . The participation of this amino-terminal segment in a subunit interface, coupled with the observation that each active site is made up of elements from two different subunits, provides a structural rationale for the phenomenon of alpha-complementation . The structure represents the longest polypeptide chain for which an atomic structure has been determined . Our results show that it is possible successfully to study non-viral protein crystals with unit cell dimensions in excess of 500 A and with relative molecular masses in the region of 2,000K per asymmetric unit . Non-crystallographic symmetry averaging proved to be a very powerful tool in the structure determination, as has been shown in other contexts.

FEBS Lett, 1994 Jun 27, 347(2-3), 169 - 72
Positively charged residues influence the degree of SecA dependence in protein translocation across the E . coli inner membrane; Andersson H et al.; The sec machinery catalyzes the translocation of nascent polypeptide chains across the inner membrane of E . coli, yet some inner membrane proteins depend only weakly or not at all on an intact sec function for membrane insertion even though they have stretches of chain protruding into the periplasmic space . Earlier work has demonstrated that the length of a periplasmic loop correlates with its degree of sec-dependence . We now show that the content of positively charged residues in a translocated loop also correlates with the degree of dependence on SecA function, suggesting that arginines and lysines may be inherently difficult to move across the membrane during sec-dependent translocation.

Nucleic Acids Res, 1994 Jun 25, 22(12), 2399 - 403
The 'endo-blue method' for direct cloning of restriction endonuclease genes in E . coli; Fomenkov A et al.; A new E . coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methylation-dependent restriction systems (McrA-, McrBC-, Mrr-) . This strain has been used to clone restriction endonuclease genes directly into E . coli . When E . coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response . The SOS-induced cells form blue colonies on indicator plates containing X-gal . Using this method the genes coding for the thermostable restriction enzymes Taql (5'TCGA3') and Tth111l (5'GACNNNGTC3') have been successfully cloned in E . coli . The new strain will be useful to clone other genes involved in DNA metabolism.

Nucleic Acids Res, 1994 Jun 25, 22(12), 2344 - 50
Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter; Gaal T et al.; DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro . This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold . A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription . However, the precise position of the curvature has not been determined . We address here whether this bend is in fact related to activation of rRNA transcription . Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site . Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e . downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription . Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 866 - 70
Site directed substitutions suggest that His-418 of beta-galactosidase (E . coli) is a ligand to Mg2+; Roth NJ et al.; Site directed mutagenesis was used to replace His-418 of beta-galactosidase with Phe (H418F) or Glu (H418E) . Kinetic analysis revealed that H418F beta-galactosidase was not significantly affected by the presence of Mg2+ whereas H418E beta-galactosidase retained its sensitivity to Mg2+ . H418F had a kcat similar to that of Mg(2+)-free wild type beta-galactosidase . Its pH profile was shifted 1.0 pH unit lower on the alkaline side as compared to wild type beta-galactosidase (with Mg2+) . This was similar to the shifting of the wild type beta-galactosidase pH profile when Mg2+ was absent . H418E beta-galactosidase was inactivated (rather than activated) by Mg2+ binding . Equilibrium dialysis studies indicated that H418E and wild type beta-galactosidase bind Mg2+ tightly whereas H418F does not . The results indicate that His-418 is probably a ligand to Mg2+.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 820 - 8
Intrinsic fluorescence of E . coli RNA polymerase as a probe for its conformational changes during transcription initiation; Sen R et al.; A simple fluorimetric assay based on internal fluorescence of tryptophan residues of E . Coli RNA polymerase has been developed to ascertain the number of steps during conversion of closed complex of the polymerase-promoter (trp promoter cloned in plasmid pDR720) to open complex . Our results from measurement on relative ratio of fluorescence at 340 nm (lambda ex = 295 nm) for free and promoter-bound RNA polymerase as a function of temperature, within the range 4 degrees C to 37 degrees C, indicate following equilibria for the above conversion: R+P<-->RPc<-->RPi1<-->RPi2<-->RPo . Apart from detection of one more intermediate in terms of conformational states of the bound RNA polymerase, second feature of our studies is the examination of conformational state of the polymerase using accessibility of fluorophor, tryptophan residues, to a neutral quencher, acrylamide, as the probe . We observe that in terms of accessibility of tryptophan residues in protein, intermediate complex, RPi2, is conformationally most perturbed in comparison to free polymerase . Implications of these results are discussed and compared with the available reports from footprinting and gel retardation assays of RNA polymerase-promoter interactions.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 546 - 51
Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E . coli and the thermodynamic analysis of its antigen-binding properties; Tsumoto K et al.; In order to physically stabilize the Fv fragment of anti-lysozyme monoclonal antibody, HyHEL10, the variable domains were linked covalently with a flexible linker . A marked difference in the level of expression in E . coli was observed between VH-linker-VL (scFvHL) and VL-linker-VH (scFvLH) . The highly expressed scFvLH was purified by a single step of affinity chromatography from the culture supernatant with a typical yield of 3-5 mg per liter of culture . This HyHEL10 scFvLH showed reduced binding activity toward its antigen, HEL, in comparison with Fv . Thermodynamic study showed that this reduced activity was due to entropic loss upon binding to its antigen, although this interaction between scFvLH and its antigen was enthalpically favorable.

Nucleic Acids Res, 1994 Jun 11, 22(11), 2158 - 65
Analysis of E.coli promoter structures using neural networks; Mahadevan I et al.; Backpropagation neural network is trained to identify E.coli promoters of all spacing classes (15 to 21) . A three module approach is employed wherein the first neural net module predicts the consensus boxes, the second module aligns the promoters to a length of 65 bases and the third neural net module predicts the entire sequence of 65 bases taking care of the possible interdependencies between the bases in the promoters . The networks were trained with 106 promoters and random sequences which were 60% AT rich and tested on 126 promoters (Bacterial, Mutant and Phage promoters) . The network was 98% successful in promoter recognition and 90.2% successful in non-promoter recognition when tested on 5000 randomly generated sequences . The network was further trained with 11 mutated non-promoters and 8 mutated promoters of the p22ant promoter . The testing set with 7 mutated promoters and 13 mutated non-promoters of p22ant were identified . The network was upgraded using total 1665 data of promoters and non-promoters to identify any promoter sequences in the gene sequences . The network identified the locations of P1, P2 and P3 promoters in the pBR322 plasmid . A search for the start codon, Ribosomal Binding Site and the stop codon by a string search procedure has also been added to find the possible promoters that can yield protein products . The network was also successfully tested on a synthetic plasmid pWM528.

FEBS Lett, 1994 Jun 6, 346(1), 69 - 72
Sec-independent protein insertion into the inner E . coli membrane . A phenomenon in search of an explanation; von Heijne G; Translocation of proteins through the inner membrane of E . coli is normally catalyzed by the so-called sec-machinery . Yet, many integral inner membrane proteins appear not to require a fully functional sec-machinery for proper insertion, in spite of the fact that sometimes quite sizable domains have to be translocated to the periplasmic side . This review will focus on recent studies of sec-independent translocation events in an attempt to pin-point the main differences between sec-dependent and sec-independent translocation.

Protein Expr Purif, 1994 Jun, 5(3), 233 - 41
Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E . coli; Gustafson ME et al.; Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway . Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein . In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E . coli using the tac promoter expression system . High-level expression in recombinant E . coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies . Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea . Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Lymphokine Cytokine Res, 1994 Jun, 13(3), 191 - 6
Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E . coli; Zucker K et al.; We have utilized the reverse transcription polymerase chain reaction (RT-PCR) to clone the protein coding region of canine tumor necrosis factor (TNF-alpha) cDNA . The gene displays 90% sequence homology to the corresponding human TNF-alpha cDNA . The predicted initial translation product is 233 amino acids and shows 88% homology to the human counterpart, and 92% homology with the human putative mature TNF-alpha protein . The canine TNF-alpha clone was used to engineer bacteria to express large amounts of the mature form of recombinant protein . A monoclonal antibody against human TNF-alpha cross-reacted with canine rTNF-alpha using Western blot and ELISA analysis . The purified canine rTNF-alpha had a cytotoxic effect on WEHI 164 clone 13 cells as well as increasing the cell surface expression of major histocompatibility class II antigens on canine kidney cortical cell line (MDCK) in vitro . The availability of canine rTNF-alpha will allow further studies on its role in immunoregulatory mechanisms in the canine transplantation model, both by itself and in conjunction with the already available canine specific recombinant interferon-gamma.

Sci China B, 1994 Jun, 37(6), 667 - 76
Synthesis and expression of a gene from kringle-2 domain of tissue plasminogen activator in E . coli; Hua ZC et al.; The DNA fragment corresponding to the tissue plasminogen activator (tPA) sequence 174-262 (Kringle-2 domain) has been synthesized by using the solid phase phosphotriester method . The Kringle-2 domain of human tPA was expressed in Escherichia coli by secretion into the periplasmic space using the Lpp-Lac promoter and PIN-III OmpA2 signal sequence . About two thirds of the expression product was secreted into the periplasmic space, and purified with ammonium sulfate fractionation, affinity chromatography on Lysine-Sepharose, and FPLC-Mono Q exchange chromatography . The amino acid composition observed from the Kringle-2 purified from E . coli is identical with that expected for the 174-262 fragment of human tPA . Radio binding assay shows that the recombinant Kringle-2 domain possesses the activity of fibrin binding.

Hybridoma, 1994 Jun, 13(3), 191 - 7
Purification of E . coli-synthesized Pan proteins and development of a Pan-specific monoclonal antibody; Vierra CA et al.; The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan-2 (E12), are produced by the mechanism of alternative transcript splicing . Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification scheme was developed . Purified Pan-2 was used to immunize Smith-Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2 . Deletion mapping and sequence transfer studies have localized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2 . This region is conserved in Pan-1 and Pan-2 . The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta . By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.

J Biotechnol, 1994 May 15, 34(2), 149 - 55
Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E . coli and its use in ELISA; Sohn MJ et al.; To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter . The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis . The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a . 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body . Recombinant gag was purified by a simple single step purification procedure . After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography . The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA) . These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Nucleic Acids Res, 1994 May 11, 22(9), 1613 - 9
Specificities of human, rat and E . coli O6-methylguanine-DNA methyltransferases towards the repair of O6-methyl and O6-ethylguanine in DNA; Liem LK et al.; The behaviour of highly purified bacterial expressed rat O6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (km6G/ke6G = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O6-methyl and O6-ethylguanine) is similar to that of E . coli 39kD Ada protein (km6G/ke6G = 1.6) . However, the human MGMT is very different (km6G/ke6G = 163) . The preferential repair of O6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine . The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed . In addition the preferential repair of O6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O6-ethylguanine . This is in contrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT.

J Theor Biol, 1994 May 7, 168(1), 1 - 12
Free DNA concentration in E . coli estimated by an analysis of competition for DNA binding proteins; Stickle DF et al.; Transcription in E . coli is often controlled by the binding of specific gene-regulatory proteins . Binding of these proteins to their specific DNA binding sites occurs in the presence of a large excess of "nonspecific" genomic DNA . Binding to a specific DNA site thus depends on the concentration of regulatory protein, on its affinities for specific and competing nonspecific binding sites, and on the free concentrations of those sites . Although it is probable that genomic DNA is largely occluded by protein binding or by condensation in vivo, the actual extent to which the DNA is available to act as a competitor for specific binding (i.e . the effective concentration of nonspecific DNA) is not known . Because many regulatory interactions occur simultaneously in a cell, it is reasonable to expect that they will have evolved to function at equilibrium with a shared concentration of competing nonspecific DNA . This premise was the basis for this study . In vitro binding data were compiled for six regulatory proteins that function in E . coli, and used to calculate theoretical equilibrium binding distributions . The calculated distributions were used to evaluate the regulatory states of promoters according to models based on the equilibrium occupancies of regulatory sites . For four proteins whose DNA-binding affinities are modulated by ligand binding (CAP, lac repressor, trp repressor and araC), regulation was assessed as the extent to which the presence of the modulator could affect the occupancy by protein of the specific sites (e.g . the difference in equilibrium occupancy by CAP of CAP binding sites between conditions of high and low concentrations of CAP's affinity modulator, cAMP) . For two proteins whose site affinities are not modulated by ligand binding (lambda repressor and lambda-cro), regulation was assessed by specific site occupancy at equilibrium . These regulation profiles were compared to determine whether a single concentration of nonspecific competing DNA is compatible with effective regulation as defined for all of the systems . For five of the six modeled systems (CAP, trp repressor, araC, lambda repressor and lambda-cro), a free nonspecific DNA concentration on the order of 10(-4) M base pairs is compatible with regulation based on equilibria of the protein-DNA interactions . The lac repressor-operator system is an exception to these results: as has been shown previously, the regulation of operator binding by low molecular weight inducers increases with increasing concentrations of nonspecific DNA (von Hippel et al., 1974 Proc . natn . Acad . Sci . U.S.A . 71, 4808-4812).(ABSTRACT TRUNCATED AT 400 WORDS)

Cell, 1994 May 6, 77(3), 401 - 12
Topological "frustration" in multispanning E . coli inner membrane proteins; Gafvelin G et al.; The topology of E . coli inner membrane proteins depends primarily on the distribution of positively charged residues in the molecule . We have constructed model proteins with four potential transmembrane stretches and have systematically explored the topological effects of lysines placed in the loops connecting the transmembrane spans . Our results indicate that membrane insertion is locally determined, with individual "helical hairpins" inserting independently of each other . Topologically "frustrated" molecules, where the charge distribution is such that different parts of the molecule would prefer to insert with incompatible orientations, adopt "leave-one-out" topologies in which only 3 of the 4 potential transmembrane stretches span the membrane . These results are relevant for our general understanding of both membrane protein biogenesis and the evolution of multi-spanning membrane proteins.

Cell, 1994 May 6, 77(3), 413 - 26
SeqA: a negative modulator of replication initiation in E . coli; Lu M et al.; In E . coli, replication initiates at a genetically unique origin, oriC . Rapidly growing cells contain multiple oriC copies . Initiation occurs synchronously, once and only once per cell cycle at all origins present . Secondary initiations are prevented by a sequestration process that acts uniquely on newly replicated origins, which are marked because they are hemimethylated at GATC sites . We report the identification of a gene required for sequestration and demonstrate that this gene, seqA, also serves as a negative modulator of the primary initiation process . All previously identified in vivo initiation factors play positive roles . Thus, precise control of replication initiation may involve a balance between positive and negative elements . We suggest that SeqA might be a cooperativity factor, acting to make the replication initiation process dependent upon cooperative interactions among components.

FEBS Lett, 1994 May 2, 343(3), 242 - 6
Influenza virus M2 protein modifies membrane permeability in E . coli cells; Guinea R et al.; The M2 protein of influenza virus is an integral membrane protein with ion channel activity . This protein has been expressed in E . coli cells in an inducible manner . Expression of the M2 protein causes rapid lysis of BL21(DE3) pLysS E . coli cells upon induction with IPTB . M2 protein increases membrane permeability to a number of hydrophylic molecules, such as ONPG, uridine or impermeant translation inhibitors . The behaviour of M2 in bacteria resembles that of other viral proteins, such as poliovirus 3A and Semliki Forest virus 6K.

Ann N Y Acad Sci, 1994 May 2, 721, 73 - 81
Horseradish peroxidase isozyme C . A comparative study of native and recombinant enzyme produced by E . coli transformants; Egorov AM et al.; The purification and refolding of the recombinant horseradish peroxidase produce by E . coli transformants are described . The recombinant enzyme is of 34 kDa and has an isozyme spectrum similar to Sigma type VI horseradish peroxidase . The specific activity of the refolded peroxidase is of about 2000 U/mg with ABTS as a substrate . The recombinant and native enzyme are similar with respect to their catalytic properties in the reaction of enhanced chemiluminescence . Operational and thermal stability of the refolded peroxidase is two to three times lower than for the native one.

Biol Chem Hoppe Seyler, 1994 May, 375(5), 353 - 6
A soluble immunoglobulin variable domain without a disulfide bridge: construction, accumulation in the cytoplasm of E . coli, purification and physicochemical characterization; Frisch C et al.; Two amino acid exchanges (Y32H and C23V) were introduced sequentially into the immunoglobulin REIV, a human kappa variable domain . The first exchange stabilizes the folded state of the domain by 4.6 kJ/mol (1.1 kcal/mol), the second abolishes the central disulfide bridge and destabilizes the folded domain by 17.5 kJ/mol (4.2 kcal/mol) . Introduction of the stabilizing exchange first is a necessary pre-requisite to the removal of the central disulfide bridge without collapse of the fold . The double mutant REIV-C23V/Y32H can be accumulated in the cytoplasmatic compartment of the E . coli cell, a finding that opens new possibilities in antibody engineering.

Protein Eng, 1994 May, 7(5), 605 - 12
Three-dimensional structure of a mutant E . coli aspartate aminotransferase with increased enzymic activity; Jager J et al.; The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites . Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster . In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared . It showed improved Kcat/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values . For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively . The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate . Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 A resolution respectively . They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate . The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate . It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Kms.

Cytokine, 1994 May, 6(3), 255 - 64
Production and structural characterization of amino terminally histidine tagged human oncostatin M in E . coli; Sporeno E et al.; Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression . In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells . The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells . After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector . Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column . We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay . Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry . Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.

Free Radic Biol Med, 1994 May, 16(5), 571 - 80
Prevention and induction of oxidative damage in E . coli cells by cationized proteins; Kohen R et al.; The successful prevention of oxidative damage in E . coli B cells by cationized catalase (cCAT), and the induction of oxidative stress by cationized glucose oxidase (cGO) and cationized superoxide dismutase (cSOD) is presented in this study . Exposure of E . coli cells to hydrogen peroxide and hydroxyl radical resulted in a rapid killing of the cells . Measurements of biochemical markers: cellular potassium levels and uptake and accumulation of leucin indicated membrane damage in some of the oxidants employed . Following incubation with native CAT or SOD, the cells were washed and exposed to oxidative stress . The results of this procedure did not protect the cells against the oxidative damage . In contrast, incubation of the cells with pretreated CAT with poly-L-histidine, followed by washing of the cells and the subsequent introduction of oxidative stress inducers, resulted in a pronounced protection of the cells against the oxidative stress . Employment of pretreated SOD, and exposure, after washing the cells, to oxidative stress, resulted in an enhancement of the oxidative damage in some cases . Exposure of the cells to cGO resulted in a marked killing of the cells as compared to the untreated enzyme . The use of E . coli cells as a model system for studying the effect of cationized enzymes on cell surfaces is discussed.

Zhonghua Wai Ke Za Zhi, 1994 May, 32(5), 314 - 7
{BMP1 fusion protein expressed in E . coli}; Ma QJ et al.; The expression plasmid pMS32C/BMP1 was constructed successfully by using recombinant DNA techniques . The validity of the reconstructed plasmid was confirmed by restriction map of PMS32C/BMP1 plasmid . Then the 66KD BMP1 fusion protein was highly expressed in E . coli, which accounting for 20% of total bacterial proteins . The expressed protein was purified by means of SDS-PAGE . The antiserum of BMP1 fusion protein was prepared by immunization of mice . The antisera immunoprecipitated with fusion protein and specifically bound to BMPs purified from human bone with high titer in immuno-dot assay . It is indicated that the BMP1 fusion protein contains human BMP moiety.

Mutat Res, 1994 May 1, 307(1), 193 - 200
An E . coli ada transgenic clone of Nicotiana tabacum var . Xanthi has increased sensitivity to the mutagenic action of alkylating agents, maleic hydrazide and gamma-rays; Veleminsky J et al.; Two transgenic clones X3 and X15 of Nicotiana tabacum var . Xanthi, heterozygous in two genes (a1 and a2) for chloroplast differentiation and transformed with the E . coli DNA repair gene ada cloned downstream from the 1' direction of the dual mas promoter, differed in the expression of the ada gene, in the number of copies of integrated T-DNA and in the response to the mutagenic action of alkylating and non-alkylating agents . The X3 genome contained four copies and the X15 genome one copy of T-DNA, nevertheless the expression of the ada gene, measured by the activity of O6-alkylguanine DNA alkyltransferase (ATase), was about six times higher in X15 than in X3 . ATase activity in both clones was highest in extracts from callus whereas very low (X15) or no (X3) activity was detected in leaf extracts . This may explain the lack of difference between X15 and non-transformed tobacco (NTX) in the frequency of N-methyl-N-nitrosourea (MNU)-induced somatic mutations in leaves . In contrast, the frequency of somatic mutations in X3 was about 2-5 times higher than in NTX and X15 after the same doses of MNU, methyl methanesulfonate, maleic hydrazide and gamma-rays . Alteration of plant gene(s) essential in mutation pathway(s) by insertion of T-DNA or by somaclonal variation may explain the higher sensitivity of the X3 clone.

Mutat Res, 1994 May 1, 307(1), 141 - 7
Structure-activity studies in E . coli strains on ochratoxin A (OTA) and its analogues implicate a genotoxic free radical and a cytotoxic thiol derivative as reactive metabolites; Malaveille C et al.; Ochratoxin A (OTA), its major metabolite in rodents, ochratoxin alpha, and seven structurally related substances were assayed for SOS DNA repair inducing activity in Escherichia coli strain PQ37 . At concentrations of 0.1-4 mM, OTA, chloroxine, 5-chloro-8-quinolinol, 4-chloro-meta-cresol and chloroxylenol induced SOS DNA repair in the absence of an exogenous metabolic activation system . Ochratoxin B, ochratoxin alpha, 5-chlorosalicylic acid and citrinin were inactive, but all except ochratoxin alpha were cytotoxic . Thus, the presence of chlorine at C-5 appears to be one determinant of genotoxicity in these substances . Amino oxyacetic acid, an inhibitor of the cysteine conjugate beta-lyase, decreased the cytotoxicity of OTA but did not alter its genotoxic activity, suggesting the formation of a cytotoxic thiol-containing derivative . The mechanisms by which OTA and some of its active analogues induce SOS DNA repair activity was further investigated in E . coli PQ37 and in three derived strains (PQ300, OG100 and OG400), containing deletions within the oxy R regulon . The response in strain PQ37 was measured in the absence and presence of Trolox C, a water-soluble form of vitamin E . Trolox C completely quenched the genotoxicity of OTA, and the effect was similar in the mutant and wild-type strains . These results implicate an OTA-derived free radical rather than reduced oxygen species as genotoxic intermediate(s) in bacteria.

J Biotechnol, 1994 Apr 30, 34(1), 61 - 9
Efficient expression of E . coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA; Tohda H et al.; Dihydrofolate reductase (DHFR) of Escherichia coli (E . coli) was synthesized in a cell-free translation system of E . coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of SP diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) . The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA . Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA . It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system . Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity . This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al . (1988) . Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.

Biochim Biophys Acta, 1994 Apr 13, 1205(2), 223 - 9
Role of the TIGN sequence in E . coli tryptophanyl-tRNA synthetase; Chan KW et al.; Tryptophanyl-tRNA synthetase in E . coli does not have the HIGH sequence that is normally characteristic of class I aminoacyl-tRNA synthetases (EC 6.1.1.2), but instead contains a TIGN sequence at residues 17-20, which has been suggested to be equivalent to the HIGH sequence (Jones, M.D . et al . (1986) Biochemistry 25, 1887-1891) . We have overexpressed E . coli Trp-tRNA synthetase and have used site-directed mutagenesis to mutate Thr-17 in the TIGN sequence to alanine . The mutant enzyme has the same Km values as the wild-type for tryptophan or tRNA(Trp), and a slightly increased Km for ATP, from 0.37 to 0.64 mM . On the other hand, the kcat for either the first step or the overall reaction is decreased by a factor of 30 . In comparing the Thr-17 and Ala-17 enzymes, the delta delta G for the conversion of substrate to transition state is +9.6 kJ/mol (2.3 kcal/mol) . Thr-17 is therefore important in binding the substrate in the transition state, thus supporting the suggestion that TIGN may fulfill the role of a HIGH sequence.

Nucleic Acids Res, 1994 Apr 11, 22(7), 1287 - 95
Quantitative analysis of ribosome binding sites in E.coli; Barrick D et al.; 185 clones with randomized ribosome binding sites, from position -11 to 0 preceding the coding region of beta-galactosidase, were selected and sequenced . The translational yield of each clone was determined; they varied by more than 3000-fold . Multiple linear regression analysis was used to determine the contribution to translation initiation activity of each base at each position . Features known to be important for translation initiation, such as the initiation codon, the Shine/Dalgarno sequence, the identity of the base at position -3 and the occurrence of alternative ATGs, are all found to be important quantitatively for activity . No other features are found to be of general significance, although the effects of secondary structure can be seen as outliers . A comparison to a large number of natural E.coli translation initiation sites shows the information profile to be qualitatively similar although differing quantitatively . This is probably due to the selection for good translation initiation sites in the natural set compared to the low average activity of the randomized set.

FEMS Microbiol Lett, 1994 Apr 1, 117(2), 189 - 96
The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E . coli alpha-hemolysin family; Schmidt H et al.; Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype . In this study the hemolytic activity of E . coli O157:H7 strain EDL933 was investigated . Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity . By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E . coli K-12 strains C600 and DH5 alpha became positive for hemolysin production . By transformation of recombinant plasmids carrying a 11.9 kb BamHI fragment and a 5.3 kb SalI fragment of pSK3 hemolytic activity is revealed when transformed in E . coli C600 or DH5 alpha DNA-hybridization of pO157 and subclones with the alpha-hemolysin specific DNA probe was only found under conditions of low stringency . No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes . Our results indicate that a hitherto not described hemolysin belonging to the alpha-hemolysin family is encoded by the 90 kb plasmid of E . coli O157 strains.

Methods Find Exp Clin Pharmacol, 1994 Apr, 16(3), 191 - 201
E . coli endotoxin affinity at the nicotinic2 receptor: receptor theory revisited, ilumina nostros occulae; Tomera JF et al.; This paper examines the interaction of endotoxin at the nicotinic2 receptor (ACh-Rpost) with the adenylate cyclase signal transduction system in skeletal muscle . It reports the drug-receptor (DR) dissociation constant (K, 0.05020 +/- .0015 mg/kg) for E . coli endotoxin . It expands on a previous report (1) which presented the relevance of skeletal muscle cAMP as a metabolic indicator and second messenger influenced by endotoxicosis . The use of a murine endotoxic model allowed for the measuring of K for the antagonist . DR affinity measures which relied on the constrained-slope Schild plot measured it . This report assessed dose-response relations of the competitive antagonist dTC (AN) and their modification (AN') by advancing endotoxicosis . The presence of endotoxin at a fixed final concentration (i.e., 7.75 mg/kg) within the body at the end of a two week period caused a rightward shift in the dTC dose-response curve . Endotoxin desensitized cAMP and caused the dTC curve to be shifted rightward . This report differs from typical pharmacological applications where standard agonist curves are obtainable in the presence of an antagonist . This in vivo model did not allow for the measurement of agonist ACh concentration . Therefore, the use of a pair of compounds (i.e., antagonistic dTC and agonistic endotoxin) and their effects (i.e., suppression of active tension and cAMP desensitization, respectively) allowed us to quantitate K . In summary, this report supports the hypothesis linking the nicotinic2 receptor to the adenylate cyclase signal transduction system and illustrates that endotoxicosis perturbed both.

Acta Anaesthesiol Scand, 1994 Apr, 38(3), 201 - 5
Mild hypothermia during isoflurane anesthesia decreases resistance to E . coli dermal infection in guinea pigs; Sheffield CW et al.; Small changes in core temperature profoundly alter cutaneous blood flow, a major factor influencing resistance to wound infection . Furthermore, when measured in vitro, various immune functions are temperature dependent in the physiological range . Accordingly, we tested the hypothesis that mild hypothermia impairs and mild hyperthermia improves resistance to dermal infections . Thirty-two guinea pigs were anesthetized for 6 h using 1.5% (1.25 MAC) inspired isoflurane . Their core temperatures were maintained at either 39 degrees C (normal for guinea pigs, n = 11), 36 degrees C (n = 12), or 41 degrees C (n = 9) . One h after induction of anesthesia, 2 x 10(8) E . coli were injected intradermally with a 26-g needle at eight sites on each animal's back . Core temperatures were not controlled after recovery from anesthesia, and animals in each group were maintained in the same environment . Twenty-four h after injection, the area of induration surrounding each injection site was measured . This is a standard test of resistance to wound infection . Values were compared using one-way ANOVA and Scheffe's S tests . Results are presented as means +/- standard deviations; differences were considered significant when P < 0.05 . Areas of inflammation on the hypothermic animals were significantly larger (48 +/- 10 mm2) than those on normothermic (36 +/- 10 mm2) or hyperthermic (37 +/- 6 mm2) animals . These data suggest that mild hypothermia during anesthesia significantly impairs resistance to dermal infection . In contrast, mild hyperthermia does not appear to be protective.

Protein Expr Purif, 1994 Apr, 5(2), 125 - 32
Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E . coli; Lai TS et al.; The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains . We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector . A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E . coli . Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 or Met475 . We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains . JM105 harboring pKF13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium . The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 micrograms of FXIII A-chains per liter of culture . The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast . These results document that FXIII A-chains can be readily expressed and purified from E . coli culture and that they retained properties similar to those of purified human factor XIII A-chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Khim, 1994 Apr, 20(4), 367 - 81
{Guanylate kinase from bovine retina: isolation, primary structure, and expression in E . coli}; Gaidarov IO et al.; Guanylate kinase (EC 2.7.4.8), catalysing the reaction GMP+ATP = GDP+ADP, was purified to homogeneity from bovine retina . Primary structure of the enzyme was determined by parallel analyses of amino acid sequences of its peptides and nucleotide sequence of the corresponding cDNA . It is shown that the bovine retinal guanylate kinase like the analogous enzyme from yeast Saccharomyces cerevisiae contains a characteristic glycine-rich motif, involved in ATP binding . All of the amino acids, involved in GMP binding in the yeast enzyme, are conserved or conservatively substituted in the bovine retinal guanylate kinase . The bovine retinal enzyme was expressed in E . coli as a fusion protein . Data are presented on the purification of the fusion protein, its digestion by enteropeptidase, purification of the recombinant enzyme and its functional characteristics.

Mutat Res, 1994 Apr, 323(4), 173 - 7
Nucleotide sequence between recA and alaSp in E . coli K12 and the sequence change in four recA mutations; Zaitsev E et al.; The sequence of 366 nucleotides between the C-terminal trailer region of recA and the N-terminal leader region of alaS is presented . This sequence reveals an open reading frame of 166 codons we have named oraA . An NdeI restriction nuclease cleavage site also revealed by the sequence was used to clone, map and sequence three recA mutations: recA11, recA12 and recA52 . A mutation in recA (recA946), was discovered in strains originally reported to contain recH166 . The relation between recA946 and recH166 is unclear.

FEBS Lett, 1994 Mar 28, 342(1), 57 - 60
Alteration of the specificity of ecotin, an E . coli serine proteinase inhibitor, by site directed mutagenesis; Pal G et al.; The gene of ecotin, an E . coli proteinase inhibitor, was cloned, and by site-directed mutagenesis the active site residue of the protein, Met84, was mutated to Lys, Arg and Leu . The recombinant wild-type and mutant inhibitors were overexpressed in E . coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared . Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants . According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.

Nature, 1994 Mar 17, 368(6468), 265 - 8
A large-conductance mechanosensitive channel in E . coli encoded by mscL alone; Sukharev SI et al.; All cellular organisms respond to vibration, touch, gravity or changes in osmolarity, although the molecules on which such mechanosensations depend are unknown . Candidates include certain channels that gate in response to membrane stretch . Patch-clamp experiments with Escherichia coli envelope have revealed a mechanosensitive channel with very large conductance (MscL) and one with a smaller conductance (MscS) which may be important in osmoregulation . Here we have solubilized and fractionated the envelope, reconstituted the MscL activity in vitro, and traced it to a small protein, whose gene, mscL, we then cloned . Insertional disruption of mscL removes the channel activity, whereas re-expression of mscL borne on an expression plasmid restores it . MscL-channel activities were observed in material from a cell-free expression system with mscL as the only template . The mscL nucleotide sequence predicts a unique protein of only 136 amino acids, with a highly hydrophobic core and very different from porins or other known proteins.

Biochim Biophys Acta, 1994 Mar 16, 1205(1), 54 - 8
Selective modification of putative uridine-binding site of uridine phosphorylase from E . coli with fluorescein 5'-isothiocyanate; Komissarov AA et al.; A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E . coli was modified with fluorescein 5'-isothiocyanate (FITC) . Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1) . Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit . Addition of uridine prevents the enzyme inactivation by FITC . In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme . The modified protein retains the ability to bind phosphate but not uridine . According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate . The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.

Biochemistry, 1994 Mar 8, 33(9), 2531 - 7
Cloning and characterization of a new purine biosynthetic enzyme: a non-folate glycinamide ribonucleotide transformylase from E . coli; Marolewski A et al.; A novel GAR transformylase has been isolated and characterized from E . coli . The protein, a product of the purT gene, is a monomer of molecular weight 42 kDa and catalyzes the production of beta-formyl GAR from formate, ATP, and beta-GAR . As such it is an alternative to the formyl-folate utilizing purN GAR transformylase . No significant homology exists between the two transformylases . However, the purT protein shows significant homology to the purK protein, also involved in purine biosynthesis . Two different purT reactions have been characterized: one producing fGAR from ATP, beta-GAR, and formate and the other producing acetyl phosphate and ADP from acetate and ATP . The purT GAR transformylase is the first unknown de novo purine biosynthetic enzyme to be discovered in the last 30 years and represents another step forward in understanding cellular control of purine levels.

Carbohydr Res, 1994 Mar 4, 255, 87 - 101
Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E . coli K5 capsular polysaccharides; Lidholt K et al.; The E . coli K5 capsular polysaccharide is composed of 4)GlcpA(beta 1-4)GlcpNAc(alpha 1-disaccharide units . A partially N-deacetylated/N-sulfated heptasaccharide, derived from this polymer and having a nonreducing terminal GlcNAc unit, was used as acceptor for a mastocytoma microsomal GlcA-transferase involved in heparin biosynthesis . An octasaccharide with nonreducing-terminal GlcA similarly served as acceptor for the microsomal GlcNAc-transferase . Analysis of the labeled octa- and nona-saccharides formed by transfer of monosaccharide units from UDP-{14C}GlcA and UDP-{3H}GlcNAc, respectively, showed that both glycosyltransferases could utilize partially N-sulfated acceptors . The GlcA-transferase showed a marked preference for a terminal GlcNAc-GlcA-GlcNSO3-sequence, particularly when this sequence was followed by an additional N-sulfated disaccharide unit . Enzymes catalyzing the same GlcA and GlcNAc transfer reactions were solubilized from E . coli K5 membranes . The K5 capsular polysaccharide, like the heparin/heparan sulfate precursor polysaccharide, thus probably grows by stepwise, alternating addition of the two constituent monosaccharide units, from the corresponding UDP-sugars, to the nonreducing ends of the chains . Moreover, the bacterial glycosyltransferases utilized the same partially N-sulfated oligosaccharide substrates as the mammalian enzymes, and with similar preference for N-sulfate groups in certain positions.

Masui, 1994 Mar, 43(3), 339 - 45
{Effects of E . coli LPS on human platelet aggregation}; Baba K; The effects of E . coli lipopolysaccharide (LPS) on washed platelets of the healthy volunteers were studied by measuring ADP induced aggregation and intracellular ionized calcium concentration ({Ca2+}i) by fluorescent calcium indicator, quin 2 . Addition of LPS in platelets suspension in saline, caused an increased platelet aggregation . Adding LPS, however, in platelet suspension in Na(+)-citrated platelet poor plasma inhibited ADP aggregation . These trends were not affected by cyclooxygenase inhibitor, but partially antagonized by dibutyryl cyclic AMP, and verapamil . The intracellular calcium ion concentration ({Ca2+}i) was significantly increased (334 +/- 141 nM to 150 +/- 45 nM in control) on addition of LPS in platelet suspension containing ionized calcium . On the other hand, there was no significant difference observed with those suspended in calcium free solution (50 +/- 16 to 45 +/- 15 in control) . These results indicate that changes of platelet aggregation by LPS were mediated by cyclic AMP and Ca2+, but not by arachidate derivatives . The author concludes that LPS changed the mechanism of Ca2+ influx of platelet membrane and elevated {Ca2+}i of platelets . These findings, however, probably are not the cause of aggregation of platelets during DIC.

Biokhimiia, 1994 Mar, 59(3), 368 - 80
{Optimal structure of the multienzyme system of the tricarboxylic acid cycle of E . coli during growth on various carbon sources}; Drozdov-Tikhomirov LN et al.; The optimal structure (stoichiometry) of the tricarboxylic acid cycle multienzyme system of E . coli has been computed using experimental data for the E . coli biomass monomeric content and the results of the steady-state metabolic fluxes calculation . The calculation was performed for the cases of E . coli growth on acetate, glycolate, glycerate, glutamate, glucose, gluconate and pyruvate as carbon sources . The possible self-control mechanisms make it possible to optimize the cellular multienzyme system structure for each of the carbon sources under study . The relationship between the optimal structure and the maximal specific rate of the cell growth is discussed.

J Public Health Med, 1994 Mar, 16(1), 11 - 5
A severe outbreak of E . coli O157 in two psychogeriatric wards; Kohli HS et al.; In October 1990 there was a severe outbreak of Escherichia coli O157 in two psychogeriatric wards of a large psychiatric hospital in Lanarkshire . There were 11 cases (eight patients and three staff), of whom four died (all patients) . Two cases, one staff and one patient (the likely index case) were identified serologically after the outbreak was over . E . coli O157 was not cultured from any food, water or milk samples, and the evidence suggests that the index case had eaten food brought into the hospital . The results also suggest that the incubation period of the organism may be longer than is currently recognized, particularly for person-to-person spread . A Fatal Accident Inquiry was held into the deaths, and the Sheriff's Determination is discussed together with the implications of the results for infection control procedures.

J Appl Physiol, 1994 Mar, 76(3), 1020 - 30
Severe VA/Q mismatch in perfused lungs evoked by sequential challenge with endotoxin and E . coli hemolysin; Walmrath D et al.; Escherichia coli hemolysin (ECH), an important pathogenicity factor in extraintestinal E . coli infections, provokes pulmonary hypertension and microvascular leakage in buffer-perfused rabbit lungs . We investigated gas exchange abnormalities in response to low doses of ECH, lipopolysaccharides (LPS), and sequential and combined application of these bacterial agents by using the multiple inert gas elimination technique . In control lungs and after admixture of 100 ng/ml of LPS, unimodal narrow distribution of perfusion and ventilation to midrange ventilation-perfusion (VA/Q) areas was noted . ECH {0.08 hemolytic units (HU)/ml} caused a moderate increase in pulmonary arterial pressure (< 10 mmHg), progressive lung edema formation (approximately 10 g within 20 min), and a broadening of perfusate and gas flow dispersion . Application of 0.08 HU/ml of ECH in lungs "primed" with 100 ng/ml of LPS in a preceding 125-min perfusion period provoked a large increase in pulmonary arterial pressure (> 50 mmHg within 5 min), rapid edema formation (approximately 10 g within 10 min), and severe VA/Q mismatch with predominance of shunt flow . Vasoconstrictor response and VA/Q mismatch, but not edema formation, were largely inhibited by pretreatment of lungs with acetylsalicylic acid or the thromboxane receptor antagonist BM-13.505 . In addition, "rescue" application of BM-13.505 rapidly reversed pressure rise and shunt flow due to sequential LPS and/or ECH stimulation, whereas edema formation was not affected . We conclude that the marked pulmonary hypertension in response to low doses of ECH in LPS-primed lungs is paralleled by severe gas exchange abnormalities with predominance of shunt flow . Both the vasoconstrictor response and the development of shunt are closely related to toxin-induced thromboxane generation.

Zhonghua Yu Fang Yi Xue Za Zhi, 1994 Mar, 28(2), 84 - 7
{Detection of SOS response inhibitor with E . coli GW1104 and GW1107 and its mechanism}; Qian J et al.; E . coli GW1104 and GW1107 are temperature-sensitive strains carrying a fusion gene umu:: Mud(Ap,lac) with genotypes recA441 (tif-1) and recA441 (tif-1),lexA (Def) (spr), respectively . SOS response can be produced spontaneously at 32 degrees C in E . coli GW1107, and induced at 42 degrees C in both E . coli GW1107 and GW1104 . To detect SOS response inhibitor and to study its mechanism, a quick-test-system was established based on their genetic characteristics of the two strains . It was found some kinds of Chinese herbal medicine, vegetables, and chemicals could inhibit SOS response in the strains to different extent with varied mechanisms . Shell of water chestnut could inhibit temperature-induced SOS response at 42 degrees C in E . coli GW1104, and spontaneously-produced one at 32 degrees C in E.coli GW1107 . Chinese chives can only inhibit temperature-induced SOS response in E . coli GW1104 . It suggested the former's inhibition effect on SOS response occurred at lexA gene or on the pathway of SOS response before lexA gene, and so did the latter's on the pathway after lexA gene.

Biotechniques, 1994 Mar, 16(3), 476 - 7, 480-3
Intra- and extracellular expression of an scFv antibody fragment in E . coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins; Duenas M et al.; We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression . Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA) . Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein . Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter . For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector . The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding . Co-expression of chaperonin-encoding plasmid pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein twofold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding . The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.

Protein Eng, 1994 Mar, 7(3), 405 - 12
The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E . coli; Almo SC et al.; Two refined crystal structures of aspartate aminotransferase from E . coli are reported . The wild type enzyme is in the pyridoxal phosphate (PLP) form and its structure has been determined to 2.4 A resolution, refined to an R-factor of 23.2% . The structure of the Arg292Asp mutant has been determined at 2.8 A resolution, refined to an R-factor of 20.3% . The wild type and mutant crystals are isomorphous and the two structures are very similar, with only minor changes in positions of important active site residues . As residue Arg292 is primarily responsible for the substrate charge specificity in the wild type enzyme, the mutant containing a charge reversal at this position might be expected to catalyze transamination of arginine as efficiently as the wild type enzyme effects transamination of aspartate {Cronin, C.N . and Kirsch, J.F . (1988) Biochemistry, 27, 4572-4579} . This mutant does in fact prefer arginine over aspartate as a substrate, however, the rate of catalysis is much slower than that of the wild type enzyme with its physiological substrate, aspartate . A comparison of these two structures indicates that the poorer catalytic efficiency of R292D, when presented with arginine, is not due to a gross conformational difference, but is rather a consequence of both small side chain and main chain reorientations and the pre-existing active site polar environment, which greatly favors the wild type ion pair interaction.

FEBS Lett, 1994 Feb 28, 340(1-2), 93 - 8
Cloning of the complete coding region for human protein phosphatase inhibitor 2 using the two hybrid system and expression of inhibitor 2 in E . coli; Helps NR et al.; The yeast two hybrid system has been employed to identify cDNAs encoding proteins which interact with the gamma 1 isoform of human protein phosphatase 1 . Here we report the isolation of cDNA encoding human protein phosphatase inhibitor . The deduced human sequence of 205 amino acids shows 92% identity to inhibitor 2 from rabbit . Human inhibitor 2 was expressed in E . coli and purified to homogeneity . The expressed human protein inhibited both native and bacterially expressed PP1, with the same Ki (1 nM) as inhibitor 2 purified from skeletal muscle . A gene or pseudogene for inhibitor 2 may be present near the major histocompatibility complex on chromosome 6.

FEBS Lett, 1994 Feb 14, 339(1-2), 181 - 4
Production of platelet-derived growth factor receptor (PDGFR-beta) in E . coli . Mapping ligand binding domain; Rooney BC et al.; Portions of the extracellular domain of the platelet-derived growth factor receptor beta (PDGFR-beta) were expressed as fusion proteins with a hexa His tag in E . coli . Following purification by Ni chelate chromatography, the recombinant receptors were tested in cross-competition studies with 125I-labelled PDGF-AA and -BB . Although of lower affinity than the native receptor (IC50 values of 10(-8) M) the recombinant molecules retained ligand binding specificity and neutralized the mitogenic effect of PDGF-BB . These data indicate that the ligand binding region lies within the first four immunoglobulin-like domains on PDGFR-beta . This E . coli expression system could be further used as a rapid and economical means to produce mutated receptors and map the ligand binding domain.

Nucleic Acids Res, 1994 Feb 11, 22(3), 308 - 13
A mutation in helicase motif III of E . coli RecG protein abolishes branch migration of Holliday junctions; Sharples GJ et al.; The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP . To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced . The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein . This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases . RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions . However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction . RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor . These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.

Nucleic Acids Res, 1994 Feb 11, 22(3), 279 - 84
Mutations in the peptidyl transferase region of E . coli 23S rRNA affecting translational accuracy; Gregory ST et al.; We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253 . These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA . Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy . Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion . Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo . C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene . The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12 . These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.

Cell, 1994 Feb 11, 76(3), 577 - 88
Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E . coli; Braaten BA et al.; We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E . coli . Two Dam methyltransferase sites, GATC1028 and GATC1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells . In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined . Our results show that methylation of GATC1028 blocks formation of the ON state by inhibiting the binding of Lrp and PapI regulatory proteins to this site . Conversely, methylation of GATC1130 is required for the ON state . Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter . A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Acta Anaesthesiol Scand, 1994 Feb, 38(2), 130 - 5
Effects of metabolic pH-alterations on cerebral blood flow and oxygen uptake following E . coli endotoxin in dogs; Westerlind A et al.; The aim of the present study was to investigate if metabolic pH-alterations have an influence on cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) after an injection of E . coli endotoxin . Following endotoxin in dogs with normal pH a decreased CBF and an increased CMRO2 have earlier been found . Thirteen anaesthetized dogs were subjected to metabolic pH-variations in blood by infusion of hydrochloric acid or sodium bicarbonate . Ten dogs received E . coli endotoxin in a dose of 1 mg.kg-1 bodyweight . CBF, CMRO2 and noradrenaline and adrenaline concentrations in blood and cerebrospinal fluid were measured repeatedly during normoxia and normocarbia . Measurements before endotoxin served as controls, together with three additional animals, where endotoxin was never given . In control measurements pH showed no influence on the variables studied . After endotoxin CBF, CMRO2 and noradrenaline in cerebrospinal fluid increased with decreasing arterial blood pH . The influence exerted by metabolic pH alterations in blood after endotoxin may be explained by hydrogen ions and monoamines passing over a blood-brain barrier (BBB), damaged by endotoxin, into the brain tissue causing vasodilation and neuronal activation.

Nihon Kyobu Shikkan Gakkai Zasshi, 1994 Feb, 32(2), 130 - 7
{Effects of liposome-entrapped E . coli endotoxin on lung pathophysiology in rats}; Takihara H et al.; The effects of repeated intravenous injections of E . coli endotoxin (ETX)-containing liposomes (LP) on the development of emphysematous change in rats were examined . Male Wistar rats, weighting 150-200 g, were divided into 3 groups . Group 1; rats treated with phosphate buffered saline (PBS), Group 2; rats treated with liposome-entrapped PBS, Group 3; rats treated with liposome-entrapped ETX (10 mg/ml) . In each group, 0.5 ml was injected intravenously once a week for 8 consecutive weeks . After 8 weeks, functional and morphometrical analyses of lungs were performed . Functional residual capacity (FRC), total lung capacity (TLC), and static compliance (Cst.) were measured using a pressure plethysmograph for small animals . Mean linear intercept (MLI) and internal surface area (ISA) were also determined . TLC did not differ among the 3 groups . In rats treated with liposome-entrapped ETX, Cst . corrected by body weight (Cst./body weight) was significantly increased compared with that in liposome-entrapped PBS-treated rats . Further more, MLI was larger than that of the other 2 groups and ISA was less than that of the liposome-entrapped PBS-treated rats . These results suggest that liposome-entrapped ETX induces emphysematous changes in rats.

Anticancer Drug Des, 1994 Feb, 9(1), 41 - 50
Towards an understanding of the reactivity of E . coli R2 ribonucleotide reductase: a mechanistic approach to inactivation; Swarts JC et al.; Five categories of reaction of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) are defined from mechanistic studies with hydroxyurea, methylhydroxylamine, hydroxamic acid derivatives, long-lived organic radicals of which methyl viologen MV+ is a good example, hydrazine, catechol and their derivatives . Attention is focused on whether a particular reagent reduces only the tyrosyl radical (Tyr.) giving metR2, or the Tyr . and Fe(III)2 in consecutive steps to give fully reduced R2 . In the case of hydrazine (N2H4), reduction of both the Tyr and Fe(III)2 occurs in a uniphasic process, while with di-imide (N2H2) it has already been demonstrated that the Tyr . is reduced and that Fe(II)Fe(III) semi-metR2 is formed . A further mechanism is observed with catechol and catechol-like derivatives (in this work Didox), in which there is reduction of the Tyr . in the first stage, followed by scavenging of the Fe(III) in the second . The latter offers a more permanent inactivation of R2, meriting more extensive study in the context of cancer drug therapy . Comparative studies on mouse R2 suggest that the Tyr . and Fe(III)2 of mammalian R2 forms may be more exposed, and as compared to E . coli R2 are approximately an order of magnitude more reactive.

Braz J Med Biol Res, 1994 Feb, 27(2), 527 - 33
Molecular dynamics at a cytoplasm/membrane interface of a signal sequence from an E . coli maltoporin; Areas EP et al.; We used a recently developed software that mimics a cytoplasm/membrane environment, with an interface separating two continuous media of different dielectric constants (1) . This software has been designed to allow modelling of different kinds of molecules of biological interest such as proteins and drugs, in each of the isolated continuous media, as well as in their interactions with membrane-like structures, making use of the dielectric discontinuity represented by the interface . In the present study we have applied this program ("THOR") to model a polypeptide sequence corresponding to a 50% active mutant of the signal sequence of the lamB gene product of E . coli, known as maltoporin or lambda receptor (2) . The peptide was first submitted to optimization of its molecular geometry followed by molecular dynamics in water (epsilon = 80) until thermalization was achieved . The conformation evolved from a rather extended random conformation to increasingly folded structures . The presence of the dielectric discontinuity induced the movement of the molecule's center of mass from water towards the interface . The entry of the peptide into the lower dielectric constant medium (epsilon = 2) through the interface was paralleled by a decrease in the total potential energy, indicating the affinity of the peptide for the lipid-mimetic phase.

Circ Shock, 1994 Feb, 42(2), 92 - 103
Retrospective description and experimental reconstitution of three different responses of the baboon to lethal E . coli; Taylor FB Jr et al.; This paper is divided into a retrospective descriptive section in which we report on three distinctly different and spontaneous responses of the baboon to LD100 Eschericia coli observed over the last 6 years . This section is followed by an experimental section in which we reproduce the immediate and delayed responses based on hypothetical mechanisms . In the descriptive section, we arbitrarily divided all the non-survivor animals on which we had sufficient data into three groups based on duration of survival (i.e., 12 hr or less, immediate, 12 to 30 hr, intermediate, and 30 hr or more, delayed) . The natural history and pathophysiology of the 12 hr or less group matched that of capillary leak syndrome with a rapid fall in blood pressure, rise in hematocrit, massive edema, and congestion with leukocyte sequestration in both lung and liver, with only limited adrenal cortical hemorrhage . The 12 to 30 hr group matched the natural history of a consumptive hemorrhagic diatheses with a biophasic blood pressure response, limited change in hematocrit, a severe consumptive coagulopathy, severe adrenal cortical hemorrhage, and a moderate renal cortical tubular necrosis, but limited renal cortical thrombosis . The greater than 30 hr group matched the natural history of a microvascular thrombotic (hemolytic uremic) syndrome with a stable blood pressure, a fall in hematocrit associated with a massive renal cortical thrombosis with a severe medullary, and cortical tubular necrosis . We did not analyze these groups further (i.e., type of intervention etc.) once we found that time of survival correlated with a unique clinical syndrome, because based on these observations, we hypothesized that we could reproduce the immediate capillary leak and pulmonary failure, and the delayed microvascular thrombosis and renal failure syndromes experimentally . We reproduced the immediate (< 12 hr) and delayed (> 30 hr) responses by infusion of either tumor necrosis factor or C4b binding protein with sublethal E . coli . This provides models of the immediate and delayed as well as the intermediate responses to E . coli for study of mechanism and the efficacy of therapeutic interventions.

Nature, 1994 Jan 13, 367(6459), 138 - 46
Three-dimensional structure of the 67K N-terminal fragment of E . coli DNA topoisomerase I; Lima CD et al.; The three-dimensional structure of the 67K amino-terminal fragment of Escherichia coli DNA topoisomerase I has been determined to 2.2 A resolution . The polypeptide folds in an unusual way to give four distinct domains enclosing a hole large enough to accommodate a double-stranded DNA . The active-site tyrosyl residue, which is involved in the transient breakage of a DNA strand and the formation of a covalent enzyme-DNA intermediate, is present at the interface of two domains . The structure suggests a plausible mechanism by which E . coli DNA topoisomerase I and other members of the same DNA topoisomerase subfamily could catalyse the passage of one DNA strand through a transient break in another strand.

Nucleic Acids Res, 1994 Jan 11, 22(1), 79 - 87
Unusual anticodon loop structure found in E.coli lysine tRNA; Watanabe K et al.; Although both tRNA(Lys) and tRNA(Glu) of E . coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations . (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys) . (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu) . (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised . (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu) . These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop . The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu) . The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.

FEBS Lett, 1994 Jan 3, 337(1), 88 - 92
Structure of partially-activated E . coli heat-labile enterotoxin (LT) at 2.6 A resolution; Merritt EA et al.; Biological toxicity of E . coli heat-labile enterotoxin and the closely related cholera toxin requires that the assembled toxin be activated by proteolytic cleavage of the A subunit and reduction of a disulfide bond internal to the A subunit . The structural role served by this reduction and cleavage is not known, however . We have crystallographically determined the structure of the E . coli heat-labile enterotoxin AB5 hexamer in which the A subunit has been cleaved by trypsin between residues 192 and 195 . The toxin is thus partially activated, in that it has been cleaved but the disulfide bond has not been reduced . The structure of the A subunit in the cleaved toxin is substantially the same as that previously observed for the uncleaved AB5 structure, suggesting that although such cleavage is required for biological activity of the toxin it does not by itself cause a conformational change.

Arch Biochem Biophys, 1994 Jan, 308(1), 37 - 41
Expression of recombinant inhibitor-2 in E . coli and its utilization for the affinity chromatography of protein phosphatase-1; Zhang Z et al.; The coding sequence for rabbit muscle protein phosphatase inhibitor-2 was inserted into the pET3a expression vector . This vector allowed the expression of recombinant inhibitor-2 at levels of ca . 1.5% of the soluble protein . A simple procedure allowed the purification of inhibitor-2 from Escherichia coli lysates . This involved heat-treatment, followed by chromatography on Blue-Sepharose and Q-Sepharose . Recombinant inhibitor-2 inhibited rabbit muscle protein phosphatase-1 with a potency similar to that reported for the wild type protein . The recombinant protein was coupled to CH-Sepharose and this support was found to bind the catalytic subunit of protein phosphatase-1 with high efficiency . A procedure for a single-step affinity purification of recombinant ppase-1 from E . coli lysates was shown to be feasible.

Vopr Virusol, 1994 Jan-Feb, 39(1), 34 - 7
{The isolation and expression in E . coli of a recombinant plasmid containing the 3'-terminal fragment of the cDNA of the influenza A virus nucleoprotein gene}; Stefanovich LE et al.; A recombinant plasmid pN62 determining the synthesis of a hybrid protein consisting of a full-size beta-galactosidase and C-terminus fragment of influenza A virus nucleoprotein was constructed . The complete identity of pN62 insert with the 3'-terminus cDNA fragment of influenza A virus NP-gene and conservation of beta-galactosidase reading frame was confirmed by Maxam-Gilbert sequencing of pN62 . An expression of pN62 plasmid in E . coli JM103 in the presence of IPTG resulted in accumulation of fused protein as poorly soluble inclusion bodies in the bacterial cells . Analysis of E . coli JM103/pN62 bacteria lysates by 7% PAGE revealed that molecular weight of hybrid polypeptide was 18 kDa heavier than normal beta-galactosidase and corresponded to the previously deduced weight of 135 kDa.

Am J Respir Crit Care Med, 1994 Jan, 149(1), 41 - 9
Liver-lung interactions during E . coli endotoxemia . TNF-alpha:leukotriene axis; Matuschak GM et al.; The liver modulates host responses to endotoxemia by production and clearance of tumor necrosis factor alpha (TNF-alpha) and eicosanoid lipoxygenation products . Reductions in liver blood flow (QL) are common during endotoxemia, but it is unknown whether the kinetics of TNF-alpha and leukotrienes (LTs) are thereby altered to amplify lung inflammation . To test this hypothesis, reductions in QL were modeled by an end-to-side portacaval shunt (PCS) in Sprague-Dawley rats . Conscious animals received 2.5 mg/kg of intravenous E . coli lipopolysaccharide (LPS) serotype 055:B5 (PCS + LPS; n = 17) or saline (n = 5) . Responses were compared with those in sham-operated rats (sham + LPS; n = 13) and NSS-challenged control rats (n = 5) . Cardiopulmonary changes, serum TNF-alpha, and formed elements were determined at t = 0, 1.5, 3.5, and 24 h, when organ wet/dry ratios (W/D) were measured with TNF-alpha, LTB4, and polymorphonuclear neutrophils (PMN) in bronchoalveolar lavage fluid (BALF) . In PCS + LPS rats, mortality was 59% and serum TNF-alpha peaked at 1.5 h (2,784 +/- 658 U/ml, mean +/- SEM) coincident with the onset of hypotension . Despite equivalent endotoxemia and liver- and lung-associated TNF-alpha in sham + LPS rats at 1.5 h, peak serum TNF-alpha was 38% less and mortality was 15% (p < 0.05) . Cardiac, hepatic, and cecal W/D were likewise increased in PCS + LPS versus sham + LPS rats, as were BALF PMNs (p < 0.05) . In parallel studies, the disappearance kinetics of infused rTNF-alpha were not altered in nonendotoxemic PCS animals, implicating enhanced lung uptake of LPS and systemic export of TNF-alpha in PCS + LPS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophys Chem, 1994 Jan, 48(3), 383 - 93
On the consensus structure within the E . coli promoters; Nair TM et al.; Using the theoretical model of DNA curvature, we have studied about 112 different E . coli promoters with a view to obtain some common super structures associated with them . Out of the 112 promoters analyzed by theoretical gel electrophoresis permutation about 66 of them have their minima lying between the -10 and the -35 region . The analysis of the bases at the minima reveals strong structural similarities . The differences can account for the varying strengths of the promoters as well as for different degree with which the RNA polymerase binds to these regions . The effects of mutation in each of these 112 promoters and their changes in curvature dispersion have also been evaluated.

Arch Insect Biochem Physiol, 1994, 26(1), 1 - 14
Cellular defense mechanisms in C . capitata: recognition and entrapment of E . coli by hemocytes; Marmaras VJ et al.; The mechanism of recognition of foreignness and entrapment of invaders by the immune system of insects is unknown . In this report using hemocyte monolayer preparations and biochemical analysis we demonstrate the requirements for recognition of E . coli in vitro, their entrapment by hemocytes, and nodule formation . A model system consisting of an isolated hemocyte protein (47 KDa), isolated hemocyte tyrosinase, isolated hemocytes, tyrosine, and E . coli was used to obtain these results . The 47 kDa polypeptide has the ability to form adducts with tyrosine derivatives generated by the action of tyrosinase and to attach to the E . coli surface . The latter process takes place independently of tyrosinase activity . When the E . coli-47KDa protein complex was overlaid on hemocyte monolayers followed by tyrosine and tyrosinase or vice versa, the bacteria were entrapped by hemocytes . The same results were obtained when the monolayers were overlaid with 47 KDa protein, followed by E . coli-47 KDa protein complex and then tyrosine and tyrosinase . The same experimental procedure in test tubes resulted in nodule formation . These results permit us to propose that the most likely explanation for the entrapment of E . coli to hemocytes and the formation of nodules is the production of E . coli-47 KDa complexes, and their crosslinking through a quinone intermediate generated by the action of tyrosinase on hemocytes.

Acta Biochim Pol, 1994, 41(1), 25 - 34
The epidermal growth factor-like domain from tissue plasminogen activator . Cloning in E . coli, purification and ESR studies of its interaction with human blood platelets; Pietrucha T et al.; To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E . coli . The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E . coli . The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC . After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements . Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.

Appl Biochem Biotechnol, 1994 Spring, 45-46, 349 - 66
Effect of oxygen on ethanol production by a recombinant ethanologenic E . coli; Lawford HG et al.; Escherichia coli strain B, bearing the pet plasmid pLO1297, and the wild-type culture, lacking the plasmid, responded to aeration of the complex medium by an approximate three- and fourfold increase in both growth rate and growth yield with glucose and xylose, respectively . At a relatively low oxygen transfer rate (8 mmol O2/L/h), the sugar-to-ethanol conversion efficiency exhibited by the recombinant strain decreased 40% and 30% for glucose and xylose, respectively . At a high aeration efficiency (100 mmol O2/L/h), the ethanol yield with respect to xylose was 0.15 g/g for the recombinant and 0.25 g/g for the culture lacking the plasmid . These observations suggest that oxygen reduces the ethanologenic efficiency of recombinant E . coli by diverting carbon to growth and end products other than ethanol . Previous observations, by others, on the effect of oxygen on ethanologenic recombinant E . coli were made with different strains bearing different plasmids . In addition to the possibility of strain and plasmid specificity, the results of this study suggest that previous conclusions were influenced by the particular environmental conditions imposed on the culture, including poor aeration efficiency and lack of pH control.

Environ Mol Mutagen, 1994, 24(3), 176 - 80
Localization of the xanthine guanine phosphoribosyl transferase gene (gpt) of E . coli in AS52 metaphase cells by fluorescence in situ hybridization; Michaelis KC et al.; The purpose of this study was to localize the xanthine guanine phosphoribosyl transferase gene (gpt) to a specific chromosome to investigate its proposed autosomal location in the AS52 cell line . AS52 cells are hgprt-deficient Chinese hamster ovary (CHO) cells which carry a single functional copy of the E . coli gpt gene . Fluorescence in situ hybridization (FISH) and digoxigenin-labeled probes, as small as 673 bp, were used in an attempt to localize the 456 bp gpt gene to a specific chromosome . Chi-square analysis of 13 metaphases showed significant labeling on autosomal chromosomes 6 or 7, which are indistinguishable without further banding analysis . Furthermore, a majority of the signals were on the q arm, proximal to the centromere . The data collected supports incorporation of the gpt gene into an acrocentric autosome of the AS52 cell line.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1994, 12(2), 107 - 10
{Preliminary study on expression of Leishmania donovani antigen gene in E . coli}; Jing B et al.; We constructed previously the expressing library of Leishmania donovani genomic DNA with lambda gt11 as vector . In this paper, 2 x 10(4) phages were plated on E . coli Y1090r-, and screened with a rabbit antiserum prepared by immunization with Leishmania donovani promastigotes . Bound antibodies were detected using alkaline phosphatase labeled anti-rabbit antibodies . A positive expressing clone was detected and transferred into E . coli Y1098r- to prepare lysate, a 39 kDa polypeptide in E . coli Y1089r-lysate was recognized by anti-Leishmania donovani serum . The result indicated that the 39 kDa polypeptide which was not fused with the major portion of beta-galactosidase existed disconnectedly . This finding remained to be further studied.

Environ Mol Mutagen, 1994, 24(4), 317 - 24
Induction of genotoxic effects by chlorohydroxyfuranones, byproducts of water disinfection, in E . coli K-12 cells recovered from various organs of mice; Fekadu K et al.; The genotoxic effects of three chlorohydroxyfuranones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2{5H}-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2{5H}furanone (CMCF) and 3,4,-dichloro-5-hydroxy-2{5H}furanone (MCA), which are formed as byproducts of water disinfection with chlorine, were investigated in bacterial differential DNA repair assays in vitro and in animal-mediated assays in vivo . As indicators of DNA damage, E . coli K-12 strains were used that differ in their repair capacity (uvrB/recA vs . uvr+/rec+) . Liquid incubation of the compounds without metabolic activation caused a pronounced reduction of the viability of the repair-deficient strain relative to the repair-proficient wild-type strain . The order of potency of genotoxic activity in vitro (dose range 0.004-10 micrograms/ml) was MX > CMCF > MCA . Addition of mouse S-9 mix or bovine serum albumin to the incubation mixtures resulted in an almost complete loss of the activity of all three test compounds . In the animal-mediated assays, mixtures of the indicator bacteria were injected intravenously into mice which were subsequently treated with the test compounds (200 mg/kg b.w.) . Two hours later, the cells were recovered from various organs and the relative survival frequencies determined . Under these conditions, all three compounds caused pronounced genotoxic effects, MX and CMCF being stronger genotoxins than MCA . The strongest effects were consistently found in the gastrointestinal tract, but statistically significant DNA damage was also observed in indicator cells recovered from lungs, liver, spleen and kidneys . In a further experiment, the effects of lower doses of MX (4.3, 13 and 40 mg/kg) were investigated . In these experiments dose-dependent effects were measured in all organs . CMCF and MA caused only marginal effects at 40 mg/kg except in the stomach where approximately a 50% reduction of relative survival frequency was observed with CMCF . The results of these animal-mediated assays indicate that (i) all three CHFs cause genotoxic effects in the living animal, and (ii) the potencies of the three compounds observed under in vivo conditions are not commensurate with their extremely high activities measured in vitro . One possible explanation for the weaker responses observed in the animal-mediated assays might be that CHFs are inactivated by non-specific protein binding.

Prog Clin Biol Res, 1994, 388, 175 - 94
Studies on the inflammatory-coagulant axis in the baboon response to E . coli: regulatory roles of proteins C, S, C4bBP and of inhibitors of tissue factor; Taylor FB Jr; The baboon model of E . coli sepsis illustrates three concepts with respect to the host response and vascular endothelium . First, the endothelium is the primary target . E . coli sepsis is an acute inflammatory disease of the vascular endothelium . Second, the endothelium is not a passive target . Initially it regulates both the inflammatory and coagulopathic aspects of E . coli sepsis through membrane associated regulatory receptor/plasma protein assemblies including protein C/thrombomodulin, activated protein C/protein S, C4bBP/protein S, tissue factor pathway inhibitor/Xa, antithrombin III/glycosaminoglycans . Third, when overridden by inflammatory events, the endothelium can change its anticoagulant phenotype and mount a massive procoagulant fibrinolytic counter-attack on its luminal side through the expression of tissue factor and release of tissue plasminogen activator . Fourth, again when overridden by inflammatory events, the endothelium can change its antioxidant phenotype and produce a "distal" tissue hypoxia on its abluminal side through induction of free radical generation and peroxidation of mitochondrial lipid membranes of those tissues with high metabolic rates . It has become increasingly clear that the so-called anticoagulant systems which act on the proximal factors of the clotting cascade (protein C, TFPI, AT-III, PGI2) also attenuate the amplification of the inflammatory response . Aspects of the mechanism by which this occurs are coming to light . This includes the attenuation of Il-6 response by TFPI and the attenuation of the complement effects by C4bBP/PS . The specifics of these observations in the E . coli sepsis model will be reviewed.

Receptors Channels, 1994, 2(4), 295 - 302
Expression of rat NK-2 (neurokinin A) receptor in E . coli; Grisshammer R et al.; With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein . As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor . Western-blots showed that the fusion protein was associated with the membranes . The agonist {4,5-3H-Leu9}neurokinin A and the NK-2 antagonist {3H}SR48,968 bound to the receptor in a highly specific manner . Saturation binding of the {3H}agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein) . The {3H}antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein) . Competition of {3H}agonist binding with other agonists demonstrated a potency order of: neurokinin A > {Nle10}NKA(4-10) = {beta-Ala8}NKA(4-10) >> substance P >>> senktide Against the {3H}antagonist, agonists were only partially inhibitory . Selective NK-2 antagonists inhibited binding of both {3H}ligands with an identical order of potency: SR48,968 >> R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.

Int Rev Immunol, 1994, 11(2), 103 - 11
Fusion proteins with heterologous T helper epitopes . Recombinant E . coli heat-stable enterotoxin proteins; Lowenadler B et al.; Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation . Chimeric proteins, containing single or multiple copies of the Th epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E . coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation . Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova . Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope . In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position . A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein . T cell recognition of ova in all chimeric peptides, independently of its position, following the same pattern of genetic restriction (i.e . immunodominant in H-2d and nonimmunogenic in H-2k) as in the native ovalbumin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1590 - 4
N E . coli, inducible DNA repair is error-prone with regard to lethality; Ewing D; The umuC gene is a member of the inducible SOS repair network in E . coli . The experiments reported here indicate that the product of the umuC locus can make lethal mistakes during its normal repair function . Such "lethal repair" may be a major factor in establishing a cell's sensitivity to radiation.

Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 478 - 85
Purified human vitamin D receptor overexpressed in E . coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract; Nakajima S et al.; We report here that highly purified human vitamin D receptor (hVDR) derived from E . coli or baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand binding when these preparations alone are analyzed . Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3 binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd = 0.13-0.22 nM) . This observation should facilitate the valid assay of 1,25(OH)2D3 binding activity and kinetics in samples of overexpressed hVDR . Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3 binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.

FEBS Lett, 1993 Dec 13, 335(3), 358 - 60
Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E . coli groES; Rospert S et al.; The mitochondrial chaperonin system consists of chaperonin 60 (also termed hsp60), which is homologous to E . coli groEL, and chaperonin 10, which is homologous to E . coli groES . In yeast, chaperonin 60 function has been shown to be essential for viability . We report here that the same is true for chaperonin 10 . We have cloned, sequenced and disrupted the nuclear chaperonin 10 gene CPN10 from Saccharomyces cerevisiae . This gene encodes a protein of 11,372 Da that is imported into the mitochondrial matrix without detectable cleavage . Haploid cells lacking a functional copy of CPN10 fail to grow at temperatures between 23 and 37 degrees C.

Nucleic Acids Res, 1993 Dec 11, 21(24), 5748 - 53
Conformational changes in E . coli RNA polymerase during promoter recognition; Brodolin KL et al.; We analysed complexes formed during recognition of the lacUV5 promoter by E . coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent . Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21 . The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions . In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes . In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.

J Chemother, 1993 Dec, 5(6), 422 - 9
In vitro and in vivo results of brodimoprim and analogues alone and in combination against E . coli and mycobacteria; Seydel JK; New diaminodiphenylsulfone inhibitors of dihydropteroate synthase are described with increased inhibitory activity against mycobacteria and plasmodia, whereas their side effect of methemoglobin formation could be suppressed . The optimization of diaminobenzylpyrimidines, inhibitors of dihydrofolate reductase, led to derivatives with increased inhibitory effect against mycobacteria, especially M . leprae and plasmodia . Some of these derivatives show autosynergism . Finally the combination of brodimoprim (BDP) and dapsone (DDS) was developed for the treatment of leprosy . First clinical trials in Paraguay and Ethiopia show that combinations of BDP/DDS and BDP/DDS plus rifampicin were highly effective and may become an alternative multi-drug therapy for the treatment of leprosy . The tolerance of the regimens used was generally good.

Prostaglandins Leukot Essent Fatty Acids, 1993 Dec, 49(6), 955 - 8
Comparison of the potency of various serotypes of E . coli lipopolysaccharides in stimulating PGI2 production and suppressing ACE activity in cultured human umbilical vein endothelial cells; Watanabe K et al.; We investigated the potency of various serotypes of lipopolysaccharides (LPS) by examining LPS-induced stimulation of PGI2 production and suppression of ACE activity in cultured human umbilical vein endothelial cells (HUVEC) . HUVEC which had been incubated with E . coli 055:B5 and 0111:B4 for 24 h produced more prostacyclin (PGI2) in response to thrombin than HUVEC incubated with E . coli 026:B6 . Also, angiotensin converting enzyme activity (ACE) in cell lysates of HUVEC incubated for 24 h with 055:B5 or 0111:B4 was suppressed significantly compared to control HUVEC or HUVEC incubated with 026:B6 . From these experimental results, E . coli 055:B5 and 0111:B4 appear to be more potent than 026:B6 . It is concluded that this difference in potency among various serotypes of LPS should be taken into account when experiments are designed to examine the effect of LPS on endothelial cell function.

Trans R Soc Trop Med Hyg, 1993 Dec, 87 Suppl 3, 49 - 53
Diarrhoeal disease: current concepts and future challenges . Enteroadherent Escherichia coli: a heterogeneous group of E . coli implicated as diarrhoeal pathogens; Savarino SJ; Despite a great expansion in our knowledge of the causative agents of infectious diarrhoea over the past 20 years, a significant proportion of diarrhoeal cases remains undiagnosed . Enteroadherent Escherichia coli are a relatively recently identified group of enteric bacteria which have been implicated as diarrhoeal pathogens . These organisms, defined by their ability to adhere to human epithelial-derived tissue culture cells, have been closely studied over the past 10 years and appear to be quite heterogeneous . This review summarizes our current understanding of enteroadherent E . coli and the recognized subgroups . At least 3 distinct tissue culture cell adherence patterns have been recognized: localized adherence, characteristic of enteropathogenic E . coli, diffuse adherence, and aggregative adherence . Studies examining the epidemiological and pathogenic significance of the latter 2 groups, so-called diffusely adhering E . coli and enteroaggregative E . coli, are discussed in detail.

FEBS Lett, 1993 Nov 29, 335(1), 47 - 50
Adaptability of nonnatural aromatic amino acids to the active center of the E . coli ribosomal A site; Hohsaka T et al.; 3'-N-Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E . coli in vitro protein synthesizing system was evaluated . The analogs with L-2-naphthylalanine, L-p-biphenylalanine, L-2-anthrylalanine and trans-L-p-phenylazophenylalanine were found to inhibit the protein synthesis with high efficiency . The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E . coli ribosomal A site . A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed.

Biochim Biophys Acta, 1993 Nov 28, 1158(3), 287 - 92
The induction of lipid peroxidation by E . coli lipopolysaccharide on rat hepatocytes as an important factor in the etiology of endotoxic liver damage; Portoles MT et al.; Oxygen-derived radicals have been suggested to produce tissue injury during endotoxic shock by initiating lipid peroxidation . In order to investigate the induction of lipid peroxidation by Escherichia coli 0111:B4 lipopolysaccharide (LPS) on hepatocytes, malondialdehyde (MDA) and superoxide dismutase (SOD) activity have been evaluated in vivo and in vitro using two experimental models: rat liver after the establishment of endotoxic reversible shock, and cultured hepatocytes after treatment with LPS . Liver MDA levels were increased in vivo during the acute-phase of endotoxic shock, decreasing below control values in the recovery phase . An inverse pattern was obtained when SOD activity was measured, consistent with an active system of cellular protection . Similar results were obtained in vitro after treatment of cultured hepatocytes with LPS (50 micrograms/ml), thus indicating that a direct LPS cytotoxic effect on hepatocytes exits during the endotoxic process . The direct LPS interaction induced alterations in Ca2+ permeability of hepatocyte plasma membrane as detected by flow cytometry using the fluorescent probe Indo-1.

Nucleic Acids Res, 1993 Nov 25, 21(23), 5485 - 8
NMR evidence for the RNA stem-loop structure involved in the transcription attenuation of E . coli trp operon; Ramesh V; High field 1H-NMR studies of a synthetic 21-mer RNA fragment, corresponding to residues +114 to +134 within the trp leader mRNA transcript, have been carried out . Seven well resolved imino proton resonances corresponding to six C-G and one A-U hydrogen bonded base pairs, together with their characteristic NOE patterns can be identified in the NMR spectrum . This experimental result provides direct evidence for the postulated stem-loop secondary structure, 3:4, which has been reported to act as a transcription termination signal for RNA polymerase.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5192 - 7
In vivo cloning of PCR products in E . coli; Oliner JD et al.; This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities . PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector . PCR products and vector DNA are then simply co-transfected into E . coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation . The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).

Protein Eng, 1993 Nov, 6(8), 893 - 900
Replacing the (beta alpha)-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme; Mainfroid V et al.; In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein, assuming that the pseudosymmetrical beta-barrel can be divided into eight successive loop/beta-strand/loop/alpha-helix motifs . We replaced the eighth (beta alpha)-unit of E.coli TIM with the corresponding chicken (beta alpha)-unit . The substitution, involving the replacement of 10 of the 23 residues of this (beta alpha)-unit, was evaluated first by modelling, then experimentally . Modelling by homology suggests how the amino acid replacements might be accommodated in the hybrid E.coli/chicken TIM (ETIM8CHI) . Both natural and hybrid recombinant TIMs, overproduced in E.coli, were purified to homogeneity and characterized as to their stability and kinetics . Our kinetic studies show that the modification performed here leads to an active enzyme . The stability studies indicate that the stability of ETIM8CHI is comparable to that of the wild type TIM.

Sci China B, 1993 Nov, 36(11), 1361 - 6
Cloning, sequencing and expression in E . coli of interferon-omega 1 gene; Li MF et al.; Human interferon omega 1 (huIFN-omega 1) gene was isolated and cloned from chromosome DNA derived from a Chinese fetal liver via polymerase chain reaction (PCR) . By determining its nucleotide sequence we proved that the 88th codon should be GGA, coding for Gly . After engineering the original IFN-omega 1 gene clone to a form that may be expressed as a nonfused protein, we also took the IFN-omega 1 gene under the control of the PRPL promoter with an expression vector pBV220 in E . coli . The antivirus activity of the recombinant IFN-omega 1 is about 6.5 x 10(7) units/L CULTURE (OD600 = 0.75) . Since IFN-omega 1 not only has antivirus activity but also shows considerably high homology with animal trophoblast proteins which have been proved antiluteolysins as a maternal recognition signal for pregnancy, we believe that study on it will be practically and theoretically significant.

Int J Radiat Biol, 1993 Nov, 64(5), 485 - 95
Radiosensitization and radioprotection of E . coli by alcohols; Worm KH et al.; The survival of E . coli K12 strain AB1157 and the isogenic repair-deficient mutant E . coli AB2480 (recA13, uvrA6) was measured after gamma-irradiation in the presence of various alcohols as well as after incubation and subsequent removal of the alcohols before irradiation . Irradiation in the presence of alcohols leads to the already known protection effect, which has been attributed to OH radical scavenging . However, it was not possible to explain the protection solely in terms of the reactivity of the OH radical with the various alcohols, because addition of some of the alcohols did not yield the expected survival values . It was found that incubation with and subsequent removal of various alcohols before irradiation led to radiosensitization . The degree of radiosensitization increases with the hydrophobicity of the alcohol . In the case of glycerol no radiosensitization was observed . We can conclude that alcohols protect predominantly by OH radical scavenging . The comparatively small protection of cell survival by the more hydrophobic alcohols can be attributed to the sensitizing effect of these alcohols.

Eur J Clin Chem Clin Biochem, 1993 Nov, 31(11), 777 - 85
Determination of the catalytic activity of phospholipase A2: E . coli-based assay compared to a photometric micelle assay; Aufenanger J et al.; Phospholipase A2 activity in human sera was determined of the basis of the E . coli assay and compared to a photometric micelle assay . The E . coli assay is based on the hydrolysis of phospholipids from {1-14C}oleic acid-labelled E . coli biomembranes . In the photometric assay the phospholipase A2 acts on mixed phospholipid micelles . The amount of fatty acid produced is quantitated in a subsequent photometric assay by coupling in the reaction to the coenzyme A metabolism . The E . coli membranes are essentially resistant to other lipases in human sera, i.e . lipoprotein lipases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A2 of human serum . The photometric assay, though, is susceptible to other lipases in human serum . The ratio of {1-14C}oleic acid to released total fatty acids served as the basis for the calculation of the true enzymatic activity . The assay closely correlated with the photometric assay based on mixed micelles in the higher ranges of phospholipase A2 activity, but not in the normal range . The sensitivity is higher by at least two powers of 10 . The human serum phospholipase A2 strongly preferred E . coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine . In conclusion, the modified phospholipase A2 assay based on E . coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A2 activity in human sera . The photometric assay suffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation.

Cell, 1993 Oct 22, 75(2), 351 - 61
Two related recombinases are required for site-specific recombination at dif and cer in E . coli K12; Blakely G et al.; The stable inheritance of ColE1-related plasmids and the normal partition of the E . coli chromosome require the function of the Xer site-specific recombination system . We show that in addition to the XerC recombinase, whose function has already been implicated in this system, a second chromosomally encoded recombinase, XerD, is required . The XerC and XerD proteins show 37% identity and bind to separate halves of the recombination site . Both proteins act catalytically in the recombination reaction . Recombination site asymmetry and the requirement of two recombinases ensure that only correctly aligned sites are recombined . We predict that normal partition of most circular chromosomes requires the participation of site-specific recombination to convert any multimers (arising by homologous recombination) to monomers.

Biochim Biophys Acta, 1993 Oct 20, 1182(3), 264 - 74
Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E . coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation; Bross P et al.; The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation . Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein . (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting . In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme . Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly . Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate . Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers . A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.

Nucleic Acids Res, 1993 Oct 11, 21(20), 4788 - 95
Drosophila Rrp1 complements E . coli xth nfo mutants: protection against both oxidative and alkylation-induced DNA damage; Gu L et al.; Drosophila Rrp1 protein has four tightly associated enzymatic activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease and apurinic/apyrimidinic (AP) endonuclease . The carboxy-terminal region of Rrp1 is homologous to Escherichia coli exonuclease III and several eukaryotic AP endonucleases . All members of this protein family cleave abasic sites . Rrp1 protein was expressed under the control of the E . coli RNA polymerase tac promoter (pRrp1-tac) in two repair deficient E . coli strains (BW528 and LG101) lacking both exonuclease III (xth) and endonuclease IV (nfo) . Rrp1 confers resistance to killing by oxidative, antitumor and alkylating agents that damage DNA (hydrogen peroxide, t-butylhydroperoxide, bleomycin, methyl methanesulfonate, and mitomycin C) . Complementation of the repair deficiency by Rrp1 provides up to a two log increase in survival and requires the C-terminal nuclease region of Rrp1, but not its N-terminal region . The AP endonuclease activity in extracts from the repair deficient strain LG101 is increased up to 12-fold when the strain contains pRrp1-tac . These results indicate that pRrp1-tac directs the synthesis of active enzyme, and that the nuclease activities of Rrp1 are likely to be the cause of the increased resistance to DNA damage of the mutant cells.

Nucleic Acids Res, 1993 Oct 11, 21(20), 4690 - 5
Binding of nucleic acids to E . coli RNase HI observed by NMR and CD spectroscopy; Oda Y et al.; To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR . Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated . The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme . The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme . From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes . The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.

Carbohydr Res, 1993 Oct 4, 248, 241 - 50
Structural comparison of the O4-specific polysaccharides from E . coli O4:K6 and E . coli O4:K52; Jann B et al.; Two distinct forms of the O4 antigen (LPS) from E . coli were analysed by 1H and 13C NMR spectroscopy . Both consisted of D-glucose, L-rhamnose, 2-acetamido-2,6-dideoxy-L-galactose (L-FucNAc), and 2-acetamido-2-deoxy-D-glucose . Their structures were found to be {formula: see text} . In the O4-specific polysaccharide from E . coli O4:K3, O4:K6, and O4:K12, X is alpha-D-Glcp . In the O4 specific polysaccharide from E . coli O4:K52, the rhamnose residue is not substituted (X = H).

J Biochem (Tokyo), 1993 Oct, 114(4), 453 - 6
Characterization of wild type and mutant chicken gizzard alpha calponin expressed in E . coli; Gong BJ et al.; Calponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction . Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, alpha and beta, the expression of the alpha-isoform being uniformly more abundant in various smooth muscle tissues {Takahashi, K . & Nadal-Ginard, B . (1991) J . Biol . Chem . 266, 13284-13288} . For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of alpha calponin . The amino acid composition and N-terminal sequence of the recombinant alpha calponin were found to be identical to those deduced from its nucleotide sequence . Recombinant alpha calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity . A mutant alpha calponin with a replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.

J Trop Pediatr, 1993 Oct, 39(5), 304 - 6
A study of the importance of the enterotoxigenic E . coli in children with acute diarrhoea in Recife, Brazil; Sarinho SW et al.; Even today acute diarrhoea is a major cause of morbidity and mortality in young children . It is a serious public health problem in less-developed countries, primarily in households whose income is low . As such, it is one of the main causes of death, affecting the undernourished in particular . The potential enteropathogenic agents are distributed universally . However, there is a major difference in the prevalence, according to the areas and the characteristics of the population groups which were studied . In Brazil, since the 1970s, our attention has been drawn to the importance of the classic enteropathogenic strains of E . coli (EPEC) in the aetiology of diarrhoeal diseases, primarily those present in weaned infants in inner city neighbourhoods . On the other hand, Guerrant and co-workers, and Queiroz and co-workers found significant percentages of isolated colonies of enterotoxigenic coli (ETEC) in the stool material of children with symptoms of acute diarrhoea . In rural areas of less-developed countries the incidence of episodes of ETEC provoking diarrhoea is estimated to be between five and ten per person/year . Due to the social and medical gravity of acute diarrhoea, and to the lack of studies dealing with the aetiopathogenic role of the strains of ETEC in our area, we decided to analyse the frequency of isolation of ETEC in children with and without diarrhoea who were attending the out-patient department of the Instituto Materno Infantil de Pernambuco/IMIP.

Circ Shock, 1993 Oct, 41(2), 103 - 12
Effect of dexamethasone on the onset and persistence of vascular hyporeactivity induced by E . coli lipopolysaccharide in rats; Paya D et al.; The effects of dexamethasone (DEX) were studied on early and delayed hyporesponsiveness to noradrenaline (NA) induced by Escherichia coli lipopolysaccharide (LPS) in pentobarbitone-anesthetized rats, and in aortic rings, which were either removed from LPS-treated rats or exposed to LPS in vitro . In all three preparations, the LPS-impaired responses to NA were restored by N omega-nitro-L-arginine methyl ester . In addition, delayed depression of NA-induced aortic contractions was enhanced by L-arginine (1 mM) . In control conditions, DEX had no effect on responses to NA . When administered before LPS, or before hyporeactivity was fully developed, DEX (5-10 mg/kg or 10 microM) entirely prevented both the early decline in responses to NA or its progression, and the delayed impaired aortic contraction induced by LPS . However, DEX did not prevent the transient drop in mean arterial blood pressure (which was maximal at 20 min after the onset of LPS infusion) observed before the full development of impaired reactivity to NA (reached after 55 min) . Neither did DEX modify the responses to NA, in vivo or in vitro, once LPS-induced hyporesponsiveness was fully established . These results indicate that DEX inhibits both the onset of impaired responsiveness to NA, which probably involves the early stimulation of the constitutive nitric oxide (NO) synthase, and persistent vascular hyporeactivity resulting from the delayed induction of NO-synthase by LPS . In addition, they show that DEX has no effect on hyporeactivity to NA once fully established.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1371 - 8
Expression of an EF-1 alpha-like rat cDNA, S1, in E . coli and production of a rabbit polyclonal antiserum to the recombinant protein; Liu CH et al.; A previously identified rat cDNA (S1) that shares 78% nucleotide homology and a predicted 92% amino acid homology with human EF-1 alpha was constructed into PGEX-2T and inducibly expressed in E . coli as a fusion protein . The purified recombinant S1 protein (rpS1) was used to raise rabbit antiserum which recognized rpS1 but not EF-1 alpha on immunoblots . A polyclonal antiserum to EF-1 alpha failed to react with the rpS1 . Our results indicate that rpS1 protein can be used to produce antibodies which distinguish pS1 from EF-1 alpha despite their extensive amino acid sequence homology.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4467 - 75
Contributions of discrete tRNA(Ser) domains to aminoacylation by E.coli seryl-tRNA synthetase: a kinetic analysis using model RNA substrates; Sampson JR et al.; The aminoacylation kinetics of T7 transcripts representing defined regions of Escherichia coli serine tRNAs were determined using purified E.coli seryl-tRNA synthetase (SerRS) and the kinetic values were used to estimate the relative contribution of various tRNA(Ser) domains to recognition by SerRS . The analysis revealed that the extra stem/loop structure, characteristic of type II tRNAs such as tRNA(Ser), is the domain which makes the largest contribution to kcat/Km of aminoacylation . Moreover, Km of aminoacylation was increased by a factor of about 1000 when the extra stem/loop was changed to the consensus sequence of type I tRNA extra loops indicating that the stem structure contributes significantly to the binding of tRNA(Ser) to SerRS . A model RNA, which represents only the tRNA(Ser) coaxial acceptor-T psi C stem/loop domain, was also specifically aminoacylated by SerRS having a kcat/Km about 1000-fold greater than background levels . A significant portion of the contribution of this domain to aminoacylation is attributable to the acceptor stem sequence making the acceptor stem the second most important domain for recognition by SerRS . Finally, kcat/Km was essentially unchanged when the entire anticodon stem/loop of tRNA(Ser) was deleted indicating that neither the anticodon nucleotides nor the surrounding stem/loop structure are important for recognition by SerRS.

FEBS Lett, 1993 Sep 6, 330(1), 90 - 4
Cooperative homodimeric hemoglobin from Scapharca inaequivalvis . cDNA cloning and expression of the fully functional protein in E . coli; Gambacurta A et al.; The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc, Scapharca inaequivalvis, has been accomplished in E . coli from its cDNA . The latter was isolated by PCR amplification of total RNA and sequenced . The cDNA-derived sequence differed by a single amino acid when compared to that previously obtained from purified protein . Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins . The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.

J Biochem (Tokyo), 1993 Sep, 114(3), 438 - 44
Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E . coli; Fujita-Becker S et al.; Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli . The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex . The three subunits expressed and purified are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC . beta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region . TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively . Each subunit was separately expressed in E . coli, purified by column chromatography including HPLC, and reassembled to form troponin complex . The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S1 ATPase rate, as well as the superprecipitation, was calcium-sensitive . Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials.

Virus Genes, 1993 Sep, 7(3), 229 - 40
Subcloning of HIV-2 nef genes in E . coli and immunological reactivity of expressed fusion proteins; Ulrich R et al.; The nef gene located in the 3' region of the HIV-2 genome encodes an N-terminally myristylated protein of 27-35 kD, likely to be involved in the regulation of viral transcription . The nef genes of HIV-2 isolates GH-1, ROD, ST, BEN, and D194.17 were inserted into E . coli pEX vectors and expression of Nef beta-galactosidase fusion proteins was detected in stained gels . All fusion proteins specifically reacted with a rabbit serum raised against bacterially expressed Nef from HIV-2D194.17 . Sera from monkeys inoculated with HIV-2BEN or SIVMAC251 recognized the Nef proteins of only certain HIV-2 isolates . No cross-reactivity of these sera with HIV-1 Nef and of a rabbit anti-HIV-1-Nef serum with the described HIV-2 Nef fusion proteins was observed.

Biochem Mol Biol Int, 1993 Sep, 31(1), 161 - 8
Studies on phosphorylated transcriptional regulator (NarL) for E . coli nar operon by 31P-NMR spectroscopy; Takahashi K et al.; The sequential transphosphorylation from autophosphorylated nitrate-sensing protein (NarX) to the transcriptional regulator protein (NarL), both operating in signal transduction to control the expression of the respiratory nitrate reductase (nar) operon in E . coli, was demonstrated with an in vitro reconstructed system to function similarly to other bacterial two-component regulatory systems . Over-expression system established by means of the pT7 promoter/polymerase provided both NarX and NarL proteins to reconstruct the in vitro transphosphorylation system . The phosphorylated NarL was detected, and the unstable phosphorylated group was directly assigned to acyl phosphate in the in vitro system by 31P-NMR spectroscopy.

J AOAC Int, 1993 Sep-Oct, 76(5), 988 - 1005
Substrate supporting disc method for confirmed detection of total coliforms and E . coli in all foods: collaborative study; Feldsine PT et al.; The ColiComplete substrate supporting disc (SSD) method for simultaneous confirmed total coliform count and Escherichia coli determination in all foods was compared with AOAC most probable number (MPN) methods, 966.23 and 966.24 . Twenty-nine laboratories participated in this collaborative study in which 6 food types were analyzed . Four food types, raw ground beef, pork sausage, raw liquid milk, and nut meats, were naturally contaminated with coliform bacteria . Two foods, dry egg and fresh frozen vegetables, were seeded with coliforms . Three food types, ground beef, raw liquid milk, and pork sausage, were naturally contaminated with E . coli . Although pork sausage was naturally contaminated, the level was very low (<10/50 g); therefore, additional E . coli were inoculated into 1 lot of this food type . Three food types, nut meats, dry egg, and fresh frozen vegetables, were inoculated with E . coli . For naturally contaminated samples, duplicate determinations were made on 3 separate lots for each food type . For inoculated samples, low, medium, and high contamination levels plus uninoculated control samples were examined in duplicate . Data were analyzed separately for total coliform bacteria and for E . coli . Mean log MPN counts were determined by the SSD method and the appropriate AOAC MPN method . Results were then analyzed for repeatability, reproducibility, and mean log MPN statistical equivalence . Results were statistically equivalent for all total coliform levels in all food types except frozen vegetable and raw nut meat uninoculated control samples and 1 lot of pork sausage where the SSD method produced statistically significant greater numbers . For the E . coli determinations, results were statistically equivalent across all samples and all levels for each food type . The SSD method has been adopted first action by AOAC International for confirmed detection of total coliforms and E . coli in all foods.

Biull Eksp Biol Med, 1993 Sep, 116(9), 306 - 7
{The role of the chromosomal Thr-Leu segment of E . coli K-12 in the expression of the fin-V transfer inhibition system of the F-like plasmid pAP53}; Buianova NI et al.; The role E . coli K-12 cell chromosome genetic region (tis-region) in expression of fin V-transfer inhibition system of F-like plasmid pAP53 was shown . The results obtained testify the linkage of tis region and Thr-Leu chromosomal segment.

Biull Eksp Biol Med, 1993 Sep, 116(9), 304 - 6
{The genetic regulatory systems of cointegrative plasmid transfer in E . coli K-12 cells}; Miandina GI et al.; On the basis of transfer factor pAP42 and nonconjugative plasmids pRSF2124 and pUB781, the cointegrative plasmids pAP42/pRSF2124 and pAP42/pUB781 were constructed . Complex systems of plasmid transfer inhibition (funU and finV) were detected in the structure of cointegrative plasmids.

Cell, 1993 Aug 27, 74(4), 713 - 22
Strand switching of a replicative DNA helicase promoted by the E . coli primosome; Allen GC Jr et al.; The E . coli primosome assembles at an origin on a single-stranded DNA, like that of phi X174, to promote replication of that template . Upon conversion to the duplex form, the primosome can generate a rolling circle product from this template . Rolling circle synthesis implies the transfer of the DnaB helicase from its initial loading site on the viral strand to a displaced complementary strand . Isolated primosomes promote only unit-length synthesis; supplementation with PriC, DnaC, and DnaT is necessary to reconstitute rolling circle synthesis . Rolling circle replication is sensitive to salts, whereas primosome assembly and unit-length synthesis are not . Thus, the primosome promotes two distinct reactions: assembly for first-round synthesis and strand switching for rolling circle synthesis.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4019 - 23
Identification and characterization of E.coli ribosomal binding sites by free energy computation; Schurr T et al.; Sequences upstream from translational initiation sites of different E.coli genes show various degrees of complementarity to the Shine-Dalgarno (SD) sequence at the 3' end of the 16S rRNA . We propose a quantitative measure for the SD region on the mRNA, that reflects its degree of complementarity to the rRNA . This measure is based on the stability of the rRNA-mRNA duplex as established by free energy computations . The free energy calculations are based on the same principles that are used for folding a single RNA molecule, and are executed by similar algorithms . Bulges and internal loops in the rRNA and mRNA are allowed . The mRNA string with maximum free energy gain upon binding to the rRNA is selected as the most favorable SD sequence of a gene . The free energy value that represents the SD region provides a quantitative measure that can be used for comparing SD sequences of different genes . The distribution of this measure in more than 1000 E.coli genes is presented and discussed.

FEBS Lett, 1993 Aug 2, 327(3), 279 - 83
Cyanide-reactive sites in cytochrome bd complex from E . coli; Krasnoselskaya I et al.; Cyanide reacts with cytochrome bd from E . coli in an 'aerobically oxidized' state (mainly, an oxygenated complex b558(3+) b595(3+) d(2+)-O2), bringing about (i) decomposition of the heme d2+ oxycomplex (decay of the 648 nm absorption band) and (ii) extensive red shift in the Soret region accompanied by minor changes in the visible range assigned to ferric heme b595 . MCD spectra show that the Soret red shift is associated with heme b595(3+) high-to low-spin transition . This is the first unambiguous demonstration that heme b595 can bind exogenous ligands . No reaction of cyanide with b558 is observed . In about 70% of the enzyme which forms the cyano complex, the spin-state transition of b595 decay of heme d oxycomplex match each other kinetically (keff ca . 0.002 s-1 at 50 mM KCN, pH 8.1, 25 degrees C) . This points to an interaction between the two hemes . The concerted binding of cyanide to d3+ and b595(3+), perhaps as a bridging ligand, is probably rate-limited by d2+ oxycomplex autoxidation . In the remaining 30% of the isolated bd, there is a rapid phase of cyanide-induced b595 spin-state transition which can be tentatively assigned to that proportion of the enzyme in which heme d is initially in the ferric rather than ferrous-oxy form.

J Protein Chem, 1993 Aug, 12(4), 489 - 97
Recombinant human IL-6 expressed in E . coli undergoes selective N-terminal degradation: evidence that the protein consists of a stable core and a nonessential flexible N-terminal; Proudfoot AE et al.; A synthetic gene for human interleukin-6 has been expressed in E . coli . The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay . The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15 . The truncated proteins retain full biological activity . The degradation results in the loss of sharp amide resonances in the 1H-NMR spectrum, and little change to the ultraviolet CD spectrum . Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment . The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.

Nihon Kyobu Shikkan Gakkai Zasshi, 1993 Aug, 31(8), 924 - 31
{Measurement of myeloperoxidase and thiobarbituric acid-reactive material in plasma and bronchoalveolar lavage in E . coli-induced acute lung injury}; Hasegawa N et al.; Myeloperoxidase (MPO), which is exclusively contained in neutrophils, is released on their activation . Therefore, MPO may possibly be used as a parameter of neutrophil activation . Thiobarbituric acid-reactive material (TBARM) reflects lipid peroxidation and is a parameter of oxygen radical-mediated cell membrane damage . Using our guinea pig model of septic lung injury we measured MPO and TBARM in the setting of acute lung injury . The two experimental groups were saline controls (n = 8) and an E . coli septic group to which 2 x 10(9) live E . coli were administered intravenously (n = 8) . Lung damage was assessed by measuring wet to dry lung weight ratio (W/D) and lung tissue to plasma accumulation of 125I-albumin (AL: albumin leakage) . We measured MPO and TBARM in plasma and BAL fluid . Increased W/D and AL were observed in the E . coli group suggesting the development of acute lung injury . In the E . coli group, plasma MPO increased and MPO in BAL fluid was significantly increased as compared with the saline control group . There was no difference in plasma TBARM between the two groups, while TBARM in BAL fluid of the E . coli group was greater than in that of controls . Although BAL fluid TBARM correlated with both W/D and AL, there was no relation between BAL fluid MPO and either of these parameters . We conclude that TBARM in BAL fluid may be useful for assessing E . coli-induced acute lung injury in guinea pigs.

Biochem Mol Biol Int, 1993 Aug, 30(5), 969 - 81
The collision of cotranscribing E . coli RNA polymerases studied in vitro; Wang FJ et al.; The collision of cotranscribing E . coli ternary complexes was brought about in vitro by forcing the first "leading" complex to stall before first template C, due to the absence of CTP, and then permitting initiation of a second "following" complex . Following collision, the transcript of the leading complex was increased in length by as much as 7 nt., despite the absence of CTP, but did not dissociate . Upon addition of CTP, the leading complex aborted at exactly the positions requiring incorporation of the next Cs', the following complex continued transcription . The observations point to the importance of linking appropriate promoter efficiency with transcriptional pausing times, and the role of transcriptional collisions in termination events.

APMIS, 1993 Aug, 101(8), 582 - 6
Reduction of E . coli adherence to rubber slices treated with phospholipids; Yu JL et al.; The present study aimed at modifying the surface of biliary drain material to reduce bacterial adherence . The adherence of cells of seven E . coli strains to rubber slices treated with phosphatidylcholin (PC) or phosphatidylinositol (PI) and the adherence of cells of E . coli strain NG7C to PC- or PI-treated rubber slices implanted in the common bile duct in rats were studied in vitro . The rubber slices were incubated with 1 x 10(7) cfu radiolabeled E . coli cells/ml at 37 degrees C for 60 min and then drained and washed thrice in 2 ml PBS, and adherent E . coli cells were quantified by radioactivity counting . The results show that both PC and PI absorbed on the surface of slices reduced the adherence of E . coli cells in at least two ways, i.e . by changing surface properties in vitro and by reducing deposition of host-derived molecules on phospholipid-treated surfaces in vivo . The results may be of use for modification of the biomaterial surface in the clinical situation.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3359 - 64
An E . coli RuvC mutant defective in cleavage of synthetic Holliday junctions; Sharples GJ et al.; Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products . The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins . Each of the three mutations is a single base-pair substitution . ruvC51 converts glycine-15 to an aspartic acid residue . The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity . However, it has lost the ability to resolve these structures by symmetrical cleavage . A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain . The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon . Neither of these genes produces a stable product . The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3521 - 7
Gel retardation analysis of E . coli M1 RNA-tRNA complexes; Hardt WD et al.; We have analyzed complexes between tRNA and E . coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels . The RNA subunit of E . coli RNase P formed a specific complex with mature tRNA molecules . A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA . Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA . A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function . Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion . Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist . Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding . Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation . At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed . This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.

Biochem Biophys Res Commun, 1993 Jul 15, 194(1), 274 - 9
Mouse osteopontin expressed in E . coli exhibits autophosphorylating activity of tyrosine residues; Ashkar S et al.; Osteopontin is a secreted glycosylated phosphoprotein found in various normal and transformed tissues . Mouse osteopontin expressed in bacteria has been found to autophosphorylate in vitro using ATP or GTP as phosphoryl donors . The reaction does not occur using inorganic orthophosphate as the donor . Only tyrosine residues are phosphorylated . Neither serine nor threonine residues, both of which are found phosphorylated in osteopontin extracted from bone, is autophosphorylated in vitro . The autophosphorylation of tyrosine residues by a secreted protein such as osteopontin may provide additional insight into its biological functions.

FEBS Lett, 1993 Jul 12, 326(1-3), 101 - 4
Human cathepsin E produced in E . coli; Hill J et al.; A cDNA for procathepsin E was generated from human gastric adenocarcinoma (AGS) cells, amplified by PCR and inserted into the T7 dependent vector pET 22b for expression in E . coli . Purification of the resultant product was accomplished simply, without the need to resort to column chromatography . The recombinant protein displayed comparable properties to those of its naturally occurring counterpart . The yield of homogeneous active enzyme obtained was approximately 3 mg per 40 g of cells . This is sufficient to permit crystallisation and structural analysis to begin and a mutagenesis programme to examine structure/activity relationships now to be undertaken.

Nucleic Acids Res, 1993 Jul 11, 21(14), 3205 - 9
Antipairing and strand transferase activities of E . coli helicase II (UvrD); Morel P et al.; The product of the uvrD gene of Escherichia coli, UvrD (helicase II), is known to be involved in methyl-directed mismatch repair, transposon excision and uvrABC excision repair . In conjugational crosses, various uvrD mutants have been reported to result in higher, lower or unaffected recombination frequencies . In an attempt to clarify the role of UvrD in recombination, we have studied in vitro its effects on two key reactions driven by RecA, homologous pairing and strand exchange . We show here that UvrD efficiently prevents or reverses RecA-mediated homologous pairing . Unexpectedly, we also found that it can stimulate RecA-driven branch migration and even catalyze strand exchange in the absence of RecA . A possible in vivo role for these antagonistic activities is discussed.

FEBS Lett, 1993 Jul 5, 325(3), 299 - 302
Cytochrome bo from E . coli does not exhibit the same proton transfer characteristics as the bovine cytochrome c oxidase during oxygen reduction; Hallen S et al.; The reaction where fully reduced cytochrome bo from E . coli partially reduces dioxygen has been characterized with respect to the kinetics of the associated proton uptake, and with respect to the pH- and D2O-sensitivity of the electron transfer reactions . A monophasic proton uptake with a rate constant of about 8 x 10(3) s-1 and a stoichiometry of 0.8 H+/bo were recorded, using the indicator dye, Cresol red, at pH 8.2 . The electron transfer reactions were independent of pH in the range 6.0-9.5 and were not affected by exchanging H2O to D2O as solvent . Comparison of these results with those obtained in an earlier investigation of the bovine cytochrome c oxidase {(1992) Biochemistry 31, 11853-11859}, indicates differences between the two oxidases with respect to the role of protons in oxygen reduction and/or the mechanism of proton uptake from the medium.

Fertil Steril, 1993 Jul, 60(1), 154 - 8
Adherence of Escherichia coli to sperm: a mannose mediated phenomenon leading to agglutination of sperm and E . coli; Wolff H et al.; OBJECTIVE: To investigate the mechanism of adherence between Escherichia coli and sperm . DESIGN: Experimental study performed with donor sperm and male genital tract-derived E . coli . SETTING: Andrology unit of a university hospital . PATIENTS: None . INTERVENTIONS: Monitoring of sperm-E . coli agglutination; addition of sugars to block adherence; electron microscopy . MAIN OUTCOME MEASURE: Sperm-E . coli agglutination . RESULTS: Escherichia coli readily adhered to and agglutinated sperm . The phenomenon was observed at E . coli to sperm ratios as low as 1:20; maximum sperm agglutination involving approximately 90% of spermatozoa was seen with ratios of 1:5 or higher . By transmission electron microscopy, E . coli adherence was observed both on sperm heads and tails . Heteroagglutination could be blocked by D-mannose and alpha-methyl-mannopyranoside but not by other sugars . Preincubation of sperm or E . coli with mannose resulted in block of agglutination, indicating mannose-binding structures both on sperm and E . coli . CONCLUSIONS: Adherence of E . coli to sperm is mediated by mannose and mannose-binding structures present on both cell types . Agglutination of sperm by E . coli may be relevant in male and female infertility.

Neurochem Res, 1993 Jul, 18(7), 795 - 800
The E . coli envY gene encodes a high affinity opioid binding site; Cabon F et al.; In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template . Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated . An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites . The insert was sequenced and comparison with data banks showed a 100% homology with the E . coli envY gene . We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E . coli DNA . A search for opioid binding sites on E . coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates . On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands . These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides . In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties . In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites . Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

Biofizika, 1993 Jul-Aug, 38(4), 678 - 83
{H+-K+-exchange and formation of H2 in E . coli mutants with defects in the H+-ATPase complex and potassium transport}; Bagramian KA et al.; It was shown that 2H(+)-K(+)-exchange through H(+)-K(+)-pump composed of H(+)-ATPase complex F0F1 and Trk-system of potassium accumulation, H(+)-K(+)-exchange through H(+)-K(+)-antiport, composed of H(+)-channel F0 and of system with Trk-defect, and H2-production in E . coli grown in anaerobic conditions, change in mutants with defects of F0F1 and potassium transport . In unc-mutant of E . coli AN 936 with defects of c-subunit F0 (M(r) 8,4 kDa) H(+)-K(+)-exchange and H2-production disappeared . Mutants of E . coli TK 509 (2240) with defect of Trk-system not able to perform 2H(+)-K(+)-exchange, performed, however H(+)-K(+)-exchange and H2-production which can be blocked of N, N'-dicyclohexylcarbodiimide (DCCD) . In mutant of E . coli Tk 509 (2242) with defects of KdpA-protein of Kdp-system of potassium accumulation, Trk A and Trk D proteins of corresponding systems of potassium accumulation, the H(+)-K(+)-exchange disappeared, while H(+)-formation and H2-production, blocked by DCCD, external osmotic pressure and absence of potassium in the medium, persisted . It is assumed that 2H(+)-K(+)-exchange, H(+)-K(+)-exchange and H2-production are connected, and the membrane-bound format-hydrogenlyase oxidizing format to H2 and CO2, directly take part in interaction with F0F1 and Trk, as well as F0 and defective Trk-system in formation of supercomplexs . It is assumed that the format-hydrogen-lyase can interact with F0F1 without the Trk-system.

Carcinogenesis, 1993 Jul, 14(7), 1475 - 8
3-Amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) inhibits the removal of both cyclobutane dimers and (6-4) photoproducts from the DNA of ultraviolet-irradiated E . coli; Mori T et al.; Heterocyclic amines have been isolated from cooked foods and found to be mutagens and carcinogens . Among them, 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) were also found to enhance UV-induced mutation frequencies in Escherichia coli at the concentrations where they were neither toxic nor mutagenic by themselves . Using an immunological method recently developed to detect UV-induced DNA damage, we investigated the inhibitory effect of Trp-P-1 on the removal of both cyclobutane dimers and (6-4)photoproducts from the DNA of UV-irradiated E.coli . Cells repaired 60% of the initial cyclobutane dimers within 30 min and 75% at 120 min after UV-irradiation . Furthermore, the same cells repaired 90% of the initial (6-4)photoproducts within 30 min . On the other hand, Trp-P-1 clearly showed inhibition of repair of both photolesions in a concentration-dependent manner . The levels of repair inhibition by Trp-P-1 were almost the same between cyclobutane dimers and (6-4)photoproducts . These results suggested that the enhancing effect of Trp-P-1 on UV-induced mutagenesis in E.coli stemmed from the inhibition of the removal of photolesions from the DNA.

Carcinogenesis, 1993 Jul, 14(7), 1251 - 4
Mutations induced by saturated aqueous nitric oxide in the pSP189 supF gene in human Ad293 and E . coli MBM7070 cells; Routledge MN et al.; Nitric oxide is an important bioregulatory agent that may also be an endogenous and exogenous human mutagen . In order to study mutations generated following exposure of a shuttle vector-borne target gene to nitric oxide, mutations were induced in the supF gene of the pSP189 shuttle vector by treatment with nitric oxide in aerobic buffered solution followed by replication of the plasmid in either human Ad293 or Escherichia coli MBM7070 cells . The induced mutation frequency, which increased with nitric oxide dose, was 44-fold greater than the spontaneous background in human cells and > 15-fold greater than background in the bacterial cells when a total of 100 mmol of nitric oxide was oxidatively absorbed/I of pH 7.4 buffer containing the plasmid . The majority of point mutations analysed (61 and 75% for human and E . coli cells respectively) were AT-->GC transitions with GC-->AT transitions (29 and 23%) being the next most prevalent . The overall frequencies of the various point mutations seen in the supF gene were similar in the two cell types, although the distribution of hotspots showed differences . The results are consistent with a mutational mechanism initiated by deamination of DNA bases.

EMBO J, 1993 Jul, 12(7), 2959 - 67
Selection for active E . coli tRNA(Phe) variants from a randomized library using two proteins; Peterson ET et al.; In vitro selection was used to isolate active Escherichia coli tRNA(Phe) variants from randomized libraries . Functional tRNAs were first selected by multiple rounds of binding to Escherichia coli phenylalanyl-tRNA synthetase . These variants were then aminoacylated and selected for affinity to elongation factor-Tu . By randomizing potential recognition nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E . coli phenylalanyl-tRNA synthetase (FRS), was confirmed . However, the sequences of several active variants imply that the wild-type tertiary interactions G10-C25-U45 and A26-G44 are not required for recognition, as previously suggested . Selection of functional tRNAs from a second library randomized at positions normally involved in conserved tertiary interactions revealed new combinations of nucleotides at these positions, suggesting the presence of novel tertiary interactions . In both libraries, active sequences containing deletions were isolated . Taken together, it is clear that FRS is active with substrates having an unexpectedly broad sequence diversity . Finally, the potency of this method is illustrated by the identification of a second class of variants that was isolated by virtue of the presence of an impurity in the FRS preparation.

FEBS Lett, 1993 Jun 21, 324(3), 253 - 7
Expression in E . coli and purification of a chimeric p22-NS3 recombinant antigen of hepatitis C virus (HCV); Osborne S et al.; A recombinant antigen (p22-NS3), possessing putative HCV nucleocapsid protein (p22) and non-structural protein 3 (NS3) epitopes, was heavily expressed in E . coli and purified . The p22-NS3 purified recombinant antigen strongly reacts with sera containing human antibodies directed against p22 and NS3 providing a starting point for the design of an HCV single all-encompassing antigen for a blood screening assay.

J Biochem (Tokyo), 1993 Jun, 113(6), 747 - 53
Kinetic study of human beta-1,4-galactosyltransferase expressed in E . coli; Nakazawa K et al.; Recombinant beta-1,4-galactosyltransferase which synthesizes the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains was obtained as a soluble form from Escherichia coli by transfection of the human cDNA lacking the transmembrane segment . Kinetic study revealed that the soluble transferase has the same apparent Km values toward sugar nucleotide and sugar acceptors as those of mouse membrane-bound beta-1,4-galactosyltransferase previously characterized {Nakazawa et al . (1991) Eur . J . Biochem . 196, 363-368} . However, the Vmax value of this transferase was low when compared to that of the mammalian transferase, probably due to the instability of the transferase caused by the lack of protein glycosylation . The soluble transferase was purified from the E . coli lysates almost to homogeneity by chromatography on DEAE-Sepharose and alpha-lactalbumin-Sepharose columns . Using this purified transferase, the acceptor specificity of the transferase has been studied . The results showed that the transferase has apparent Km values of 170, 190, and 830 microM for agalacto-poly-N-acetyllactosamine, lacto-N-triose II, and lacto-N-triaosylceramide, respectively, but has apparently no activity toward glucosylceramide . These results suggest that the beta-1,4-galactosyltransferase may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose, and probably lacto-N-neotetraosylceramide in addition to the formation of the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains and lactose.

Virus Genes, 1993 Jun, 7(2), 151 - 6
Expression of the "helper component" protein of potato virus Y (PVY) in E . coli: possible involvement of a third protease; Stram Y et al.; Transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (HC) that also exhibits protease activity . HC was expressed in E . coli from two types of clones: a full-length cDNA clone of PVY and two 5' end clones containing the first three cistrons (3.6-3.7 kbp) . The clones derived from the 5' end of PVY expressed HC of the size of the mature component . Other proteins reacting with antibodies to HC were also observed, and their sizes corresponded with those of expected intermediates resulting from partial protease cleavage of the three-cistron polyprotein . On the other hand, the only detectable HC-related product of the full-length clone was a mature-size HC . The presence of a third PVY protease among the first three cistrons is therefore suggested.

Onderstepoort J Vet Res, 1993 Jun, 60(2), 153 - 4
The use of the GENETRAK Escherichia coli probe kit for the detection of three atypical E . coli isolates; Davis AJ et al.; A commercially available E . coli probe kit was used to test 1 lactose negative E . coli isolate and 2 hydrogen sulphide-producing E . coli isolates . The isolates were confirmed as E . coli by means of the API system . The GENETRAK E . coli DNA probe kit reacted positively with the lactose negative isolate, but negatively with the hydrogen sulphide-producing isolate.

Bratisl Lek Listy, 1993 Jun, 94(6), 297 - 301
{Verotoxin as an important factor in the virulence of E . coli strains}; Puzova H et al.; Verotoxin as a factor of virulence of E . coli strains is involved in the pathogenesis of hemorrhagic colitis, hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura . The most frequent producers of verocytotoxin are E . coli O157:H7 and O157:H-, yet further 57 E . coli serotypes producing verotoxin have been described . The occurrence rate of verocytotoxigenic strains of E . coli is increasing worldwide and should be a matter of concern also in our conditions . (Ref . 56.)

Biull Eksp Biol Med, 1993 Jun, 115(6), 655 - 6
{The genetic regulation of the conjugative properties of plasmid complex pAP18 in E . coli cells}; Grishina EV et al.; Natural plasmid complex pAP18 discovered in E . coli cells consists of F-like plasmid pAP18-1 (Tc, ColV) and N-like plasmid pAP18-2 (Sm) which differ in their genetic transfer . Plasmid pAP18-1 is able to inhibit transfer of plasmid pAP18-2 but not vice versa . These systems are active both in cells of serologically typed and untypable E . coli strains.

Mutat Res, 1993 Jun, 294(1), 77 - 87
Resistance to cisplatin in an E . coli B/r NalR mutant; Bouayadi K et al.; The effects of various mutations in DNA-repair processes have been reported to either enhance or decrease bacterial sensitivity to cis-diamminedichloroplatinum(II) (cis-DDP) . In the search for other mutations affecting bacterial sensitivity to this antitumor compound, we tested the E . coli B/r BS80 mutant, which is resistant to nalidixic acid (NalR) . This mutation maps in the topoisomerase II gene (gyrA subunit) and leads to cross-resistance to cis-DDP . The mechanism underlying the resistance phenotype was only partly due to decreased DNA platination . BS80 was cross-resistant to mitomycin C and, to a lesser extent, to UV light, while it was normally sensitive to MNNG . The mechanisms involved in cis-DDP and mitomycin C resistance were independent of uvrA (excision repair) and recA (SOS repair and recombination) gene expression . In contrast, UV resistance was dependent upon recA gene expression . Both the reversion to NalS in BS80 and the transduction of NalR in the parental wild type (F26) did not modify cis-DDP toxicity; in addition, platinated plasmids equally survived in BS80 and F26 strains . Hence, it is possible that selection of the NalR phenotype induced other mutation(s) than gyrA responsible for cis-DDP, mitomycin C and UV resistance and/or that lesions with a different toxic potential were introduced by cis-DDP into the BS80 and F26 chromosomes.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 413 - 9
A gel-concentration-independent retardation detected in two fragments of the rrnB P1 promoter of E . coli using transverse polyacrylamide pore gradient gel electrophoresis; Wheeler D; Two fragments of the E . coli rrnB ribosomal RNA P1 promoter Upstream Activation Region exhibit a constant gel retardation, over a polyacrylamide gel concentration range of 3% to 10% . Gel retardation is usually seen to increase with polyacrylamide gel concentration as in the case of the 219 base pair Crithidia fasciculata kinetoplast DNA fragment which represents the classic case of sequence directed curvature . Computer modeling of the Upstream Activation Region fragments suggests that their unusual electrophoretic behavior can be accounted for on the basis of a "screw" model proposed by Drak and Crothers (1991, Proc . Natl . Acad . Sci . USA 88,3074-3078).

FEBS Lett, 1993 May 24, 323(1-2), 83 - 8
E . coli expression and characterization of a mutant troponin I with the three cysteine residues substituted; Kluwe L et al.; A TnI cDNA was cloned from rabbit fast skeletal muscle, and site-directed mutagenesis was applied to replace all the three cysteine residues, Cys-48 and Cys-64 by Ala and Cys-133 by Ser . The mutant and wild-type TnI were expressed in E . coli and purified to homogeneity . No significant functional differences were observed between the mutant and the authentic TnI in terms of the interactions with TnT and TnC, and the ability of the reassembled Tn complex to regulate the acto-S1 ATPase activity in a calcium-dependent manner . These findings suggest that none of the cysteine residues in TnI are essential for the function of this protein and can be replaced to obtain a non-oxidizable mutant TnI which is much easier to handle and suitable as an alternative to the authentic TnI for various purposes, such as crystallization of TnI and the whole Tn, and 1H NMR studies.

J Inorg Biochem, 1993 May 15, 50(3), 173 - 80
Effects of various salts on the steady-state enzymatic activity of E . coli alkaline phosphatase; Poe RW et al.; Seventeen salts were tested at various concentrations for their effects on E . coli alkaline phosphatase steady-state activity . Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects . 1 . All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca . 100% at moderate (0.05-0.3 M) concentrations . 2 . Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M . The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations . These do not provide the secondary stimulatory effect of cations such as Na+ or Li+ . 3 . Anions associated with greater "salting in" effectiveness such as thiocyanate also reduce activity at ca . 1 M concentrations . These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation . There is little effect on the fluorescence or fluorescence-polarization spectrum of the enzyme so there is no general effect of 1 M salts on the conformation of the protein . The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax . Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.

Bioessays, 1993 May, 15(5), 355 - 8
RuvA, RuvB and RuvC proteins: cleaning-up after recombinational repairs in E . coli; Kuzminov A; After the completion of RecA protein-mediated recombinational repair of daughter-strand gaps in E . coli, participating chromosomes are held together by Holliday junctions . Until recently, it was not known how the cell disengages the connected chromosomes . Accumulating genetic data suggested that the product of the ruv locus participates in recombinational repair and acts after the formation of Holliday junctions . Molecular characterization of the locus revealed that there are three genes--ruvA, ruvB and ruvC; mutations in any one of the genes confer the same phenotype . Recently, the RuvC protein was found to be a Holliday junction resolvase . At first glance, the resolving activity of RuvC alone would appear to be sufficient for the separation of recombining chromosomes . However, in vitro studies show that the filament of RecA protein is unable to dissociate from the products of the recombination reaction . Thus, in vivo, even if the Holliday junctions are resolved by RuvC, RecA filament must be holding two DNA duplexes together . New findings about enzymatic activities of RuvA and RuvB proteins foster the hope that the machinery for removing the RecA filament from DNA has been found.

Mikrobiol Zh, 1993 May-Jun, 55(3), 7 - 11
{The count and distribution of saprophytic bacteria and bacteria of the E . coli group in the water of Odessa Bay and the adjacent waters}; Teplinskaia NG et al.; The quantity and distribution of heterotrophic and enteric bacteria as indicators of the human activity influence on the marine ecosystem have been studied in water thickness of the Odessa Bay . It has been established that in the late autumn period the average quantity of heterotrophic bacteria in Bay waters is 6500 cells/ml and the number of the enteric ones--2400 cells/l . The spatial distribution of the heterotrophic bacterioplankton was mosaic and a tendency to decrease their quantity while moving away from the shore was not observed . This indicated the absence of the shore flow influence on the distribution of heterotrophic bacteria . On the contrary, the number of enteric bacteria significantly decreased with an increase of the distance from the shore, but on the greater part of the Bay they were absent at all . As to the vertical distribution of the heterotrophic bacteria quantity their maximum was in the water layer (0.5) below the surface, whereas the distribution of enteric bacteria did not correspond to this rule . Proceeding from the number of heterotrophic bacteria in the late autumn period waters of the Odessa Bay can be characterized as mesoeutrophic and according to the value of coli-index--as mesosaprobic ones.

Trends Genet, 1993 May, 9(5), 173 - 7
Nucleotide excision repair I: from E . coli to yeast; Hoeijmakers JH; Genetic information is constantly deteriorating, mainly as a consequence of the action of numerous genotoxic agents . In order to cope with this fundamental problem, all living organisms have acquired a complex network of DNA repair systems to safeguard their genetic integrity . Nucleotide excision repair (NER), one of the most important of these, is a complex multi-enzyme reaction that removes a remarkably wide range of lesions . This is the first of a series of two reviews on this repair process . Part I focuses on the main characteristics of the NER pathway in E . coli and yeast . Part II, to appear in the next issue of TIG, deals with NER in mammals and compares it with the process in yeast.

Acta Anaesthesiol Scand, 1993 May, 37(4), 334 - 42
Effects of high dose E . coli lipopolysaccharide on rabbit lung function, and of subsequent addition of chemotactic granulocyte activators to their isolated, blood-perfused lungs; Opdahl H et al.; E . coli LPS was infused (1 microgram/kg to 5 mg/kg over 30 min) to spontaneously breathing rabbits, and their arterial blood pressure (ABP), blood leukocyte count and blood gases were observed for 2.5-3.5 h . Pulmonary vascular and airway function were subsequently evaluated in vitro by comparing weight changes, fluid filtration rates, pulmonary vascular resistance (PVR) and airway pressures in their isolated, blood-perfused lungs with those in lungs from untreated rabbits . Lung preparations from both groups of animals were then exposed to autologous zymosan-activated plasma (ZAP) or n-formyl-methionyl-leucyl-phenylalanine (FMLP) and perfused for 2 more hours . LPS addition to isolated rabbit leukocytes increased cell aggregation; cell chemiluminescence after activation with FMLP was also enhanced . Infusion of 1-5 mg/kg LPS decreased the count of all types of leukocytes and caused a metabolic acidosis (BE < -8 mmol), but no decrease in ABP . PAO2-Pao2 increased by about 2.0 kPa . No vascular permeability increase was detected in the lungs of these animals during subsequent in vitro perfusion . Addition of ZAP or FMLP during perfusion markedly increased the PVR in lungs from LPS animals, but did not induce major microvascular leakage . No significant differences in edema between lungs from LPS-treated and control animals were found by microscopy.

Comp Biochem Physiol Comp Physiol, 1993 May, 105(1), 123 - 8
High-level expression of biologically active chicken prolactin in E . coli; Ohkubo T et al.; 1 . A large quantity of chicken prolactin (cPRL) was produced by manipulating the cPRL cDNA clone and an expression vector pKK223-3 . To augment the production of the hormonal protein in E . coli, in addition to the potent tac promoter, a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron sequence was inserted into adjacent 5'-region of the coding region . 2 . In sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, recombinant cPRL protein exhibited a molecular mass of 23 kDa . 3 . The recombinant cPRL showed equivalent binding kinetics to an antiserum raised against turkey PRL . Also, this product increased the weight of pigeon crop sac mucosa to a degree comparable to that induced by turkey PRL . 4 . These results indicate that this recombinant cPRL has immunological and biological activities identical to those of authentic avian PRL.

Mutat Res, 1993 May, 299(3-4), 157 - 63
Mutagenesis induced by single UV photoproducts in E . coli and yeast; Lawrence CW et al.; Data from experiments with single-stranded vectors that carry a site-specific cyclobutane dimer, pyrimidine (6-4) pyrimidone adduct, or abasic lesion, replicated in either E . coli or, in some cases, bakers' yeast, Saccharomyces cerevisiae, are used to examine two questions: (i) what factors are responsible for the lesion's mutagenicity? and (ii) what are the relative contributions of different photoproducts to the spectrum of UV-induced mutations? With respect to the first question, we suggest that the structure of the mutagen-modified template itself largely determines the kinds of mutations induced, but the relative frequencies of these mutations, the error frequency, and the bypass frequency are strongly dependent on the particular organism studied . With respect to the second question, we suggest that cyclobutane dimers may be responsible for most of the mutations in slowly replicating genomes because of the deamination of cytosine, and that the T-T, and to a lesser extent the T-C, (6-4) adducts play a greater role in the UV mutagenesis of quickly replicating viruses, such as M13 and lambda phage.

FEBS Lett, 1993 Apr 26, 321(2-3), 241 - 6
Domain of E . coli translational initiation factor IF2 homologous to lambda cI repressor and displaying DNA binding activity; Vachon G et al.; The carboxy-terminal region of translational initiation factor IF2 is a common region to the three active forms of the factor (alpha, beta and gamma) but its function is still unknown . We report here that this region of IF2 carries at least one domain which is homologous to the N-terminal and middle part of the cI repressor of lambda phage . The IF2 homologous domain harbors functionally important features of the lambda repressor, e.g . the helix-turn-helix motif and some of the residues essential for the structure of the hydrophobic core of the repressor . This homologous domain of IF2 was fused to the beta-galactosidase protein . The hybrid protein, as well as IF2 itself, shows a consistent DNA binding activity in nitrocellulose filtration assays but does not display the specificity of the cI repressor for the PR operator . The implication of this domain in the transcriptional activity of IF2, reported by others, is discussed.

FEBS Lett, 1993 Apr 26, 321(2-3), 163 - 8
Ligand binding properties of human cellular retinoic acid binding protein II expressed in E . coli as a glutathione-S-transferase fusion protein; Redfern CP et al.; To test the hypothesis that 9-cis-retinoic acid is a ligand for cellular retinoic acid binding protein II (CRABP II), human CRABP II was expressed as a glutathione-S-transferase fusion protein (GST-CRABP II) and a single affinity purification step used to extract it from bacterial lysates . GST-CRABP II bound all trans-retinoic acid with high affinity (Kd 14.2 +/- 6.5 nM), but 9-cis-retinoic acid bound poorly . These studies suggest that 9-cis-retinoic acid is not a ligand for CRABP II . Their ease of purification makes GST-CRABP fusion proteins useful tools for ligand binding studies with different retinoids.

Nucleic Acids Res, 1993 Apr 11, 21(7), 1507 - 16
Compilation of E . coli mRNA promoter sequences; Lisser S et al.; An updated compilation of 300 E . coli mRNA promoter sequences is presented . For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally . We comment on the reliability of the sequence databanks and analyze the conservation of known promoter features in the current compilation . This database is available by E-mail.

Cell, 1993 Apr 9, 73(1), 87 - 96
The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E . coli RecBCD enzyme; Dixon DA et al.; The RecBCD enzyme is a multifunctional enzyme that is essential for homologous recombination in E . coli . In vitro, the RecBCD enzyme degrades linear double-stranded DNA nonspecifically during the process of unwinding the double-stranded DNA . Here we demonstrate that this DNA degradation is asymmetric, with the strand that is 3' terminal at the entry site of RecBCD enzyme being degraded much more vigorously than the 5' terminal strand . Furthermore, interaction with the recombination hotspot chi causes an attenuation of the nuclease activity but not of the helicase activity and is accompanied by a pause of RecBCD enzyme at the chi site . These results demonstrate that chi is a unique regulatory element that acts by controlling the degradative function of RecBCD enzyme and, thereby, enhancing its recombination function.

J Immunol Methods, 1993 Apr 2, 160(2), 207 - 14
Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E . coli strain; Akita EM et al.; The importance of eggs as a source of specific antibodies is well recognized . Egg yolk contains 8-20 mg of immunoglobulins (IgY) per ml . However, the major problem in isolation is removal of lipids which are present in high concentrations . A method had been developed by employing water dilution to separate the yolk plasma proteins from the granules and lipids . Further purification of IgY from plasma proteins was achieved by a protocol involving salt precipitation and ultrafiltration . The water dilution method (WD) was compared with three other methods, namely, polyethylene glycol (PEG), dextran sulphate (DS) and xanthan gum (Xan) in terms of yield, purity, ease of use, potential scaling up and immunoactivity of IgY . The WD method gave the highest yield, followed by DS, Xan and PEG methods in that order . 9.8 mg IgY/ml egg yolk was routinely obtained from the WD method compared to 4.9 mg IgY/ml egg yolk with the popular PEG method with purities of 94% and 89% respectively . Purification methods had no adverse effect on the immunoactivities of IgY . WD was also found superior in terms of ease of use and large scale production of IgY . WD method therefore provides a simple, rapid and efficient means of purifying IgY with high activity.

Biokhimiia, 1993 Apr, 58(4), 613 - 9
{Mechanism of discrimination of tRNA(Phe) from E . coli by yeast phenylalanine-tRNA-synthetase}; Khvorova AM et al.; Some peculiarities of aminoacylation of homologous and heterologous tRNA by yeast phenylalanyl-tRNA synthetase have been studied . It was found that the enzyme is inactivated during E . coli tRNA aminoacylation due to the formation of a pyrophosphorylated form of the enzyme . Inorganic pyrophosphatase splits off the bound pyrophosphate and reactivates the enzyme; a similar effect is produced by homologous tRNA . The differences in the reaction mechanism observed with E . coli tRNA in comparison with yeast tRNA seem to be due to the sole difference in the nucleotide sequences, i . e., substitution of G-U at position 20 . A putative mechanism of the enzyme inactivation during aminoacylation of heterologous tRNA is discussed.

Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 3093 - 7
Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E . coli heat-stable toxin; Savarino SJ et al.; Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children . Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1) . We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue . DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100) . Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity . The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels . A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity . These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene . EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E . coli and with guanylin, a mammalian analog of STa . Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues . Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Kansenshogaku Zasshi, 1993 Apr, 67(4), 342 - 8
{Morphological studies on liver in septic state by mice with E . coli injection into the abdominal cavity}; Kodama H et al.; The purpose of this study is to elucidate morphological changes in septicemic mice induced by an injection of E . coli into the abdominal cavity and to discuss the relationship between the clinical findings and morphological ones . The liver of septicemic mice was morphologically demonstrated, a) Kupffer cells entrapped bacteria and erythrocytes, and large lysosomes were found in Kupffer cells, b) hepatocytes with low dense cytosol increased, and in the hepatocytes, many conglomerations of lysosome and cytolysome, and degeneration of rER, sER and mitochondria were identified, c) intercellular bile canaliculus became narrow . The conclusion is as follows: 1) the increase of the serum level of T . bili is caused by the increase of the phagocyte of erythrocytes by Kupffer cells and by the dysfunction of bile transformation and transportation in hepatocytes, 2) the increase of the serum level of GOT, GPT and LDH is caused by the degeneration of mitochondria in hepatocytes, 3) the decrease of the capacity to synthesize protein and lipid is due to the degeneration of cell organelles in hepatocytes.

Mutat Res, 1993 Apr, 286(2), 253 - 65
Participation of the SOS system in producing deletions in E . coli plasmids; Balbinder E et al.; The participation of the SOS response in the deletion of palindromic and non-palindromic inserts of about 66 and 100 bp cloned within the EcoR1 site of the chloramphenicol acetyl transferase (cat) gene of plasmid pBR325 was tested after introducing the derived plasmids into strains containing different combinations of lexA, recA and umuC alleles and the auxotrophic mutation trpE65 . This allowed for a comparison of deletion frequency in the plasmids, measured as the reversion of chloramphenicol sensitivity to resistance (Cms-->Cmr), to point-mutation frequency measured from the reversion of trpE65 to tryptophan independence (Trp(-)-->Trp+) . We found that the spontaneous deletion frequency of palindromic inserts was increased by the overproduction of activated RecA* and UmuC+ in lexA (Def) backgrounds but the deletion of the non-palindromic inserts was unaltered . Overproduction of RecA+ had no significant effect on deletion incidence but it did increase Trp(-)-->Trp+ reversions . The SOS stimulation of palindrome deletions paralleled the SOS mutator effect of certain recA and umuC alleles on Trp(-)-->Trp+ reversions, suggesting that some form of SOS processing was responsible for the observed increases . The results further suggest that the SOS effect on deletions depends on the distinction between palindromy vs . non-palindromy, rather than on the sizes or sequences of the inserts or those of the terminal homologies bracketing them.

FEBS Lett, 1993 Mar 22, 319(3), 257 - 60
Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E . coli transformed cells are identical; Hervas M et al.; Native plastocyanin from Synechocystis 6803 has been isolated and purified to electrophoretic homogeneity . The corresponding gene (petE) has been cloned and expressed in E . coli, thus leading to a protein completely identical to plastocyanin purified from the cyanobacterial cells . The petE gene product is correctly processed in E . coli as deduced from the N-terminal amino acid sequences . These results, along with the identical physicochemical and kinetic properties of the two protein preparations, confirm that expression of petE in E . coli is an adequate tool to address the study of Synechocystis plastocyanin by site-directed mutagenesis.

Nucleic Acids Res, 1993 Mar 11, 21(5), 1097 - 101
Efficient cleavage of pre-tRNAs by E . coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site; Kleineidam RG et al.; RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs . It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction . To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions . We have shown that these hydroxyls are important but not essential for the processing of these substrates by E . coli RNase P RNA (M1 RNA) . The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site . Additional effects of modifications in neighbouring positions were smaller . Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.

FEBS Lett, 1993 Mar 8, 318(3), 322 - 4
Purification of recombinant hepatitis delta antigen expressed in E . coli cells; Calogero R et al.; Recombinant DNA technology enables the massive production of recombinant hepatitis delta antigen (recHDAg) retaining immunological properties and transport functions . However, purification procedures of the recombinant delta antigen have, to date, not been described in the literature . We present a purification procedure allowing one to obtain highly purified recHDAg from bacterial cells expressing the hepatitis delta antigen.

Biokhimiia, 1993 Mar, 58(3), 376 - 84
{Change in adenine nucleotide pool in E . coli 1257 bacterial cells under the action of a low intensity He-Ne laser}; Romanova NA et al.; Evidence is presented of co-immobilized bioluminescent reagents which include firefly luciferase, pyruvate kinase and adenylate kinase used for an adenylate assay in bacterial cells . The changes in the ATP, ADP and AMP content induced by irradiation with a low-power He-Ne-laser depend on the laser power and irradiation dose . The data obtained suggest that laser irradiation causes the activation of adenine nucleotide synthesis de novo.

J Bacteriol, 1993 Mar, 175(6), 1590 - 5
Escherichia coli ferredoxin NADP+ reductase: activation of E . coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein; Bianchi V et al.; A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli . This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E . coli fractions . The gene for one of these, previously named dA1, was cloned and sequenced . We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation . In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E . coli . The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases . We therefore conclude that dA1 is identical with E . coli ferredoxin (flavodoxin) NADP+ reductase . Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion . The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M . Morimyo, J . Bacteriol . 170:2136-2142, 1988) . We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.

Infect Immun, 1993 Mar, 61(3), 1152 - 6
Attaching-effacing lesions and intracellular penetration in HeLa cells and human duodenal mucosa by two Escherichia coli strains not belonging to the classical enteropathogenic E . coli serogroups; Pedroso MZ et al.; In the present study, we compared two strains of serotypes O88:H25 and O145:H45 with an enteropathogenic Escherichia coli (EPEC) adherence factor-positive (EAF+) strain of the classic enteropathogenic E . coli serotype O111ab:H2 for their association with HeLa cells and with biopsies of human duodenal mucosa . Both strains not belonging to the classic EPEC serotype showed virulence properties similar to those of the serotype O111ab:H2 strain, i.e., the production of attaching-effacing lesions and intracellular penetration in both systems . These virulence properties associated with the relatively high frequency at which the two serotypes had been detected in infant diarrhea in Sao Paulo, Brazil (T . A . T . Gomes, M . A . M . Vieira, I . K . Wachsmuth, P . A . Blake, and L . R . Trabulsi, J . Infect . Dis . 160:131-135, 1989) allowed us to suggest that strains of serotypes O88:H25 and O145:H45 should be included in the EAF+ EPEC category.

J Appl Physiol, 1993 Mar, 74(3), 1027 - 38
Intratracheal E . coli lipopolysaccharide induces platelet-dependent bronchial hyperreactivity; Vincent D et al.; Bronchial hyperresponsiveness (BHR) characterizes asthma and accompanies respiratory infections . Because endotoxin {lipopolysaccharide (LPS)} induces either hyper- or hyporesponsiveness of the guinea pig airways and protects against bronchopulmonary anaphylaxis in sensitized guinea pigs, we compared the effects of the intratracheal administration of Escherichia coli LPS on bronchopulmonary responsiveness to intravenous serotonin or acetylcholine in sensitized and nonsensitized guinea pigs . LPS (1 mg) induced BHR within 1-2 h, with a threefold increase in the bronchial response after serotonin challenge in both groups (n = 6; P < 0.005) and a marked influx of neutrophils into the perivascular and peribronchial connective tissue and the bronchoalveolar lavage fluid . This BHR was not leukocyte dependent, since it was still observed in animals depleted of circulating leukocytes with vinblastine and was not modified by antineutrophil serum, unless platelet counts were < 100,000/mm3 . This suggested that LPS-induced BHR involves platelets, and indeed antiplatelet serum, which depleted platelets, or prostacyclin, which inhibited platelets, was effective in suppressing BHR . Neither aspirin, mepyramine, nor the platelet-activating factor antagonist WEB 2170, administered before LPS instillation, prevented BHR, whereas the association of methysergide, mepyramine, and aspirin was effective, without modifying platelet and leukocyte counts . This association has been shown to prevent the release of ATP by ex vivo platelets . Our results suggest that platelets or a platelet-derived product mediates LPS-induced BHR.

Biochem Mol Biol Int, 1993 Mar, 29(4), 621 - 33
Plant mitochondrial RNase P and E . coli RNase P have different substrate specificities; Marchfelder A et al.; Substrate specificity requirements of the plant mitochondrial RNase P were investigated with different natural and mutated substrates . Heterologous precursors with intact tRNAs from plant and fungal mitochondria, from bacteria, archaebacteria and of eukaryotic origins were processed faithfully, albeit with different efficiencies . Alteration of the acceptor stem length did not disturb correct processing, while activity is completely inhibited by deletion of the pseudouridine loop . Such and other minimal substrates processed by the E . coli RNase P are not recognized as substrates by the plant mitochondrial enzyme, suggesting different requirements for substrate recognition.

Mutat Res, 1993 Mar, 293(3), 187 - 95
Increased resistance of the Chinese hamster mutant irs1 cells to monofunctional alkylating agents by transfection of the E . coli or mammalian N3-methyladenine-DNA-glycosylase genes; Habraken Y et al.; Irs1 cells are mutants of the Chinese hamster cell line V79-4, and exhibit cross-sensitivity to various DNA-damaging agents, especially to the alkylating compounds methyl methanesulfonate and ethyl methanesulfonate . To test whether this sensitivity was due to the persistence of alkylated residues in DNA, we have transfected irs1 cells with the pMSG expression vector containing two coding sequences for enzymes of different origin, either the E . coli AlkA gene, coding for 3-methyladenine-DNA-glycosylase II, or rat APDG cDNA, encoding alkylpurine-DNA-glycosylase . The two coding sequences for the repair enzymes were ligated in the pMSG vector, under the control of the MMTV-LTR promoter, which is responsive to glucocorticoid regulation . The presence of the AlkA gene or of the APDG cDNA in the transfected cells was detected by Southern blot analysis and the transcription of these foreign sequences was checked by Northern hybridization of the cellular RNA . The transfected irs1 cells treated with {3H}dimethylsulfate removed the 3-methyladenine residues more efficiently from their DNA than the control cells . Irs1 cells harboring the AlkA or the APDG gene become about 2- and 3-fold more resistant to the toxic effect of methyl methanesulfonate, respectively . However, a 3-fold resistance to ethyl methanesulfonate was only observed in irs1 cells harboring the mammalian APDG cDNA.

Biochem Biophys Res Commun, 1993 Feb 26, 191(1), 126 - 33
In vitro phosphorylation of mouse osteopontin expressed in E . coli; Ashkar S et al.; To understand the role of post-translational modifications on the structure and function of osteopontin, a secreted glycosylated phosphoprotein, we expressed mouse osteopontin in E . coli as a fusion protein with glutathione-S-transferase (GST) . The purified fusion protein was cleaved by factor Xa generating GST (26 kDa) and recombinant osteopontin (60 kDa) . The fusion protein was phosphorylated in vitro by cytosolic, microsomal, and casein kinase II fractions from mouse kidney homogenates . The fusion protein and recombinant osteopontin were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase . The suitability of the fusion and recombinant proteins as model substrates for the study of the function(s) and post-translational modifications of osteopontin is discussed.

Nucleic Acids Res, 1993 Feb 25, 21(4), 879 - 85
Identification of the nucleotide sequence recognized by the cAMP-CRP dependent CytR repressor protein in the deoP2 promoter in E . coli; Rasmussen PB et al.; In E . coli repression of transcription initiation by the CytR protein relies on CytR-DNA interactions as well as on interactions between CytR and the cAMP-CRP activator complex . To identify the nucleotide sequence recognized by CytR, mutants of the deoP2 promoter with a reduced regulatory response to CytR have been isolated . Five single bp mutation derivatives of deoP2 with a 2-5-fold decrease in CytR regulation have been characterized . In vitro, the only effect of the mutations was a decrease in the binding affinity of CytR, and a clear correlation was observed between the reduction in CytR regulation in vivo and the reduction in CytR binding in vitro . The mutations all reside in a sequence element that contains an imperfect direct as well as an imperfect inverted repeat . As the active form of CytR, most likely, is an oligomer with two-fold rotational symmetry, CytR probably interacts with the inverted repeat . Degenerate versions of the inverted repeat are present in all CytR binding sites characterized so far, however, the distance between the half-sites varies.

Nucleic Acids Res, 1993 Feb 25, 21(4), 887 - 96
Site-directed cross-linking studies on the E . coli tRNA-ribosome complex: determination of sites labelled with an aromatic azide attached to the variable loop or aminoacyl group of tRNA; Mitchell P et al.; tRNA(Phe) from E . coli, modified with the photoreactive label N-(p-azidobenzoyl)-glycine (ABG) either at the naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine (acp3U47) or the alpha-amino group of Phe-tRNA(Phe), was bound nonenzymatically to 70S ribosomes in the presence of poly (U) or short synthetic mRNA molecules prepared by T7 transcription . The noncovalent complexes were subjected to a mild ultraviolet irradiation treatment and the sites of photo-incorporation were analysed . When the photo-affinity label was attached to the aminoacyl group cross-linking was observed from both A- and P-site bound tRNA and involved exclusively the 50S subunit . In both cases the major target of cross-linking was a single site in 23S RNA, localized to position A-2439 . A lower yield of cross-linking to L27 from both P- and A-sites was also observed . In contrast, cross-linking from the acp3U47 derivative was specific for P-site bound tRNA and involved mainly (but not exclusively) the 50S subunit . In this case rRNA and ribosomal protein were labelled in approximately equal yields, the sites of cross-linking involving A-2309 in 23S RNA and L33 . These results are discussed in the light of our present knowledge concerning the structural arrangement of the tRNA-ribosome complex.

FEBS Lett, 1993 Feb 22, 318(1), 7 - 10
Different sec-requirements for signal peptide cleavage and protein translocation in a model E . coli protein; Nilsson IM et al.; We describe a secretory E . coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non-functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located approximately 30 residues downstream of the signal peptide . When this segment is deleted, normal SecA-dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA-independent . These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.

FEBS Lett, 1993 Feb 15, 317(3), 189 - 94
Variable and constant regions in the C-terminus of vinculin and metavinculin . Cloning and expression of fragments in E . coli; Strasser P et al.; Metavinculin differs from vinculin in having an additional insert of 68 to 79 amino acids in length in the C-terminal half of the molecule . Cross-species comparison of metavinculin sequences from pig, man, chicken and frog reveals a division of the insert into two parts: the first variable and the second highly conserved . The longest insert, 79 amino acids, was found in Xenopus laevis . Three different C-terminal constructs of vinculin and metavinculin over-expressed in E . coli could be purified by column chromatography . Two-dimensional gel electrophoresis and peptide analysis revealed pI values between 8.35 and 10.25 for the recombinant proteins . Biochemical and structural features of the metavinculin-specific sequence and the conserved vinculin/metavinculin carboxy-terminus are discussed.

Cell, 1993 Feb 12, 72(3), 459 - 66
Transcript cleavage factors from E . coli; Borukhov S et al.; Two transcription elongation factors (GreA and GreB) related in primary sequence were isolated from E . coli . Each factor induced cleavage of the nascent transcript in artificially halted elongation complexes followed by the loss of the 3' proximal fragment and resumption of elongation from the new 3' terminus . GreA induced cleavages 2 or 3 nt behind the terminus while GreB released longer oligonucleotides up to 9 nt in length . The pattern of cleavages characteristically changed as the transcription complex advanced, supporting the "inchworm" model of RNA polymerase propagation . In addition to attacking artificially halted complexes, both factors antagonized the action of natural elongation-arresting sites that occasionally trap the advancing complex . GreB rescued the arrested complexes via the transcript cleavage and restart pathway while GreA acted by an unknown mechanism, preventing the arrest only if added before the polymerase reached the arresting site.

J Immunol Methods, 1993 Feb 3, 158(2), 243 - 9
In vitro gene expression for the localization of antigenic determinants: application to the E . coli tryptophan synthase beta 2 subunit; Friguet B et al.; Gene expression of the beta subunit of E . coli tryptophan synthase in an E . coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains . Using specific immunoadsorption of {35S}Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E . coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides . The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay . This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies . The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches . Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.

Zentralbl Hyg Umweltmed, 1993 Feb, 193(5), 481 - 93
{Biological safety investigation of the production of human insulin using genetically altered E . coli K-12 cells . 2 . Plasmid transfer by mobilization and cointegrate formation under environmental conditions}; Mieschendahl M et al.; Plasmid transfer by mobilization and cointegrate formation was tested with expression plasmids for human insulin in laboratory media and in river water samples . Plasmid pLIZP4 which carries a nic/bom sequence could only be mobilized after cloning of mobilization genes . Plasmid mobilization could be detected in laboratory media only, not in river water samples . The Nic/Bom plasmid pSW3 could be transferred by cointegrate formation in laboratory media at 30 degrees C and 37 degrees C, not at room temperature or in river water . The results allow the conclusion that no transfer of the plasmids pLIZP4 and pSW3 will take place by mobilization or cointegrate formation under environmental conditions.

EMBO J, 1993 Feb, 12(2), 683 - 91
Sec dependent and sec independent assembly of E . coli inner membrane proteins: the topological rules depend on chain length; Andersson H et al.; Translocation of proteins across the inner membrane of Escherichia coli normally requires the participation of the sec machinery . A number of proteins are known, however, where translocation can proceed unhindered even when the function of either SecA or SecY, central components of the sec machinery, is blocked . We now show that there is a linear correlation between the length of a translocated region and its degree of dependence on SecA and SecY for lengths between 25 and 55 residues . We also find that positively charged residues have distinctly different topological effects during SecA dependent and SecA independent membrane protein insertion, and that a short cytoplasmic segment in Lep can be converted to a translocated segment (with a concomitant inversion of the original topology of the whole molecule) by increasing its length into the SecA/Y dependent realm.

Biull Eksp Biol Med, 1993 Feb, 115(2), 187 - 90
{Features of expression of the genetic region of plasmid pAP42, controlling the synthesis of "sex" pili and surface exclusion system in various E . coli cells}; Sokolova SL et al.; The results of investigation of serologically--typed and nontyped E . coli cells carrying F-like derepressed plasmid pAP42 testified to the influence of genome of some bacterial hosts on the expression of plasmid genetic region determining "sex" (plasmid--specific) pili synthesis and surface exclusion system (Sfx--system) . Similar changes in bacterial cells phenotype can also result from the mutational changes of this plasmid.

J Biotechnol, 1993 Feb, 27(3), 307 - 16
Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E . coli signal peptidase; Barthelemy I et al.; We have developed a system for expressing human interleukin 6 into the periplasmic space of Escherichia coli . The method is based on the expression of the hIL6 gene under the control of the regulatory signals of plasmid pINIII-OMPA3, i.e., lpp-lac promoter and the E . coli OMPA ribosome binding site and leader sequence . Since microheterogeneity is known to occur in the amino end of the cytokine, we tested different 'natural' versions of the protein, and we found that the secretion process was only efficient when the N-terminal amino acid was not proline . In flask experiments this procedure yields about 8-10 mg of biologically active hIL6 per liter.

Biotechnology (N Y), 1993 Feb, 11(2), 187 - 93
A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E . coli cytoplasm; LaVallie ER et al.; We have developed a versatile Escherichia coli expression system based on the use of E . coli thioredoxin (trxA) as a gene fusion partner . The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins . Although many of these cytokines previously have been produced in E . coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active . In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E . coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels . Two additional properties of E . coli thioredoxin, its ability to be specifically released from the E . coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps . We also find that the active-site loop of E . coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E . coli cytoplasm.

J Membr Biol, 1993 Feb, 131(3), 151 - 60
Activities of a mechanosensitive ion channel in an E . coli mutant lacking the major lipoprotein; Kubalski A et al.; The activity of the mechanosensitive (MS) ion channels in membrane patches, excised from E . coli spheroplasts, was analyzed using the patch-clamp technique . Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wild-type parent were examined . The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used . These channels showed a stretch sensitivity indicated by the 1/Sp (the suction for an e-fold increase in channel open probability) of 4.9 mm Hg suction . The MS-channel activities of lpp included a prominent substrate and showed a weaker mechanosensitivity with an 1/Sp of 10.0 mm Hg . Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in the lpp membranes . After lysolecithin addition, the lpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve . We discuss one interpretation of these results, in which the major lipoprotein services as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.

EMBO J, 1993 Feb, 12(2), 387 - 96
E . coli DNA polymerase I as a reverse transcriptase; Ricchetti M et al.; The ability of Escherichia coli DNA polymerase I to retrotranscribe an RNA template was examined under steady-state conditions, using a primer extension assay which allows determination of kinetic constants on well-defined heterogeneous sequences . Equilibrium and rate constants for the initial binding step of the enzyme to two homologous DNA and RNA templates do not show striking differences . In both cases, under steady-state conditions, processivity limits the maximal velocity of the translocation process . The lower catalytic efficiency of the enzyme when it operates on RNA is then reflected by a 100-fold greater apparent average Michaelis constant for the deoxynucleotide substrates . We conclude that E.coli DNA polymerase I effectively transcribes both templates, its performances being limited in both cases by its intrinsically low processivity . Furthermore, DNA polymerase I is a strikingly accurate enzyme when operating on RNA . Magnesium has to be substituted by manganese so that a pattern of errors could be detected . This great accuracy results from a combination of factors . The 3' to 5' exonuclease activity is still operating but in a non-discriminative manner . Elongation of a mismatched primer terminus is markedly impaired . The forward polymerization rate of incorporation of an incorrect deoxynucleotide must be extremely low, when Mg2+ is present . In summary E.coli DNA polymerase I preserves its main characteristics when retrotranscribing RNA.

Nucleic Acids Res, 1993 Jan 25, 21(2), 303 - 9
Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E . coli K-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine; Hanck T et al.; The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl methionine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCA/TGG . Sequence-specific, covalent crosslinking of the enzyme to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is demonstrated . This reaction is abolished if serine replaces the cysteine at residue #177 of the enzyme . These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophilic attack by the methyl donor SAM . The enzyme is capable of self-methylation in a DNA-independent reaction requiring SAM and the presence of cysteine at position #177.

Nucleic Acids Res, 1993 Jan 25, 21(2), 319 - 26
E . coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter; Kolb A et al.; A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter . When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter) . We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C) . First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP . By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only . Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40 . The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation . At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.

Gene, 1993 Jan 15, 123(1), 9 - 15
Inactivation of the Escherichia coli B41 (O101:K99/F41) rfb gene encoding an 80-kDa polypeptide results in the synthesis of an antigenically altered lipopolysaccharide in E . coli K-12; Cheah KC et al.; The genetic organisation of the rfb region from Escherichia coli B41 (O101:K99/F41) which determines the biosynthesis of the O101 O-antigen of the lipopolysaccharide (LPS) has been examined in E . coli K-12 . Maxicell analysis of the plasmid-encoded proteins facilitated the construction of a physical map of the rfb region, consisting of six proteins, designated A (87 kDa), B (80 kDa), C (49 kDa), D (38 kDa), E (36.5 kDa) and F (27 kDa) . Proteins E and F are not required for O-antigen biosynthesis . The introduction of frameshift mutations within the region encoding protein B resulted in the synthesis of an antigenically altered LPS which is shorter than the wild-type LPS, as assessed by reaction to antisera in colony and Western immunoblots, and by silver staining of LPS separated on sodium dodecyl sulfate-polyacrylamide-gel electrophoresis . The results demonstrate that protein B has a novel role in O-antigen biosynthesis associated with both the control of LPS chain length and antigenic structure . The nucleotide sequence of the rfb gene encoding protein B has been determined, confirming it to be a 697-amino acid protein of 78.9 kDa predicted to be located in the cytoplasmic membrane.

Cell, 1993 Jan 15, 72(1), 121 - 30
Frameshifting in the expression of the E . coli trpR gene occurs by the bypassing of a segment of its coding sequence; Benhar I et al.; The E . coli trpR gene encodes the 108 amino acid long trp repressor . We have previously shown that a +1 frameshifting event occurs during the expression of trpR . Here we show that the transition from the 0 to the +1 frame of trpR occurs by the bypassing of a 55 nt long segment of the trpR+1-lacZ mRNA . This bypassing event is not pretranslational, and it probably takes place during translation . Two adjacent elements are required: a specific sequence of trpR, which must be preceded by a nonspecific 5' end longer than 10 translatable codons . Unique to trpR-lacZ bypassing is that the 55 nt long region must be translated in frame 0 to enable bypassing into the +1 frame . Translational bypassing as a newly discovered mechanism of gene expression is discussed, and the possible existence of translational introns is suggested.

Cell, 1993 Jan 15, 72(1), 113 - 20
A 27 kd protein of E . coli promotes antitermination of replication in vitro at a sequence-specific replication terminus; Natarajan S et al.; We have discovered a 27 kd protein of E . coli that binds to a terminator site (tau)-terminator protein (ter) complex and abrogates the replication fork-arresting activity of ter protein in vitro . The 27 kd protein also neutralizes the contrahelicase activity of ter protein, allowing dnaB helicase to unwind DNA past a tau-ter complex . The stimulatory activity of low levels of ter protein on helicase II is also abolished by the 27 kd protein . The binding of the 27 kd protein to a tau-ter complex does not appear to dissociate the ter protein from the DNA . Although the in vivo function of the 27 kd protein is unknown at this time, it has the major attributes of a novel replication antiterminator in vitro.

Thromb Haemost, 1993 Jan 11, 69(1), 12 - 5
Evidence of a hypercoagulable state in patients with acute lymphoblastic leukemia treated with low dose of E . coli L-asparaginase: a GIMEMA study; Leone G et al.; Blood coagulation abnormalities induced by administration of E . coli L-asparaginase were investigated in 25 patients with acute lymphoblastic leukemia treated according to the GIMEMA ALL 0288 trial . Dosage of L-asparaginase was relatively low (6,000 U/m2/day for 7 days total dose 42,000 U/m2) as compared to the conventional dosages (120,000-140,000 U/m2 over 10-14 days) . A significant decrease in fibronogen, plasminogen, alpha2-antiplasmin and antithrombin III was observed from day IV of L-asparaginase and it was maximum on day VIII, with return to the baseline levels on day XV . Protein C levels had only a borderline reduction, while no modification of protein S or factor VII was observed . Two of the patients investigated developed thrombosis . The presence of a prothrombotic state induced even by this low dosage of E . coli L-asparaginase was suggested by a significant increase of sensitive markers of hypercoagulability such as fibrinopeptide A, thrombin-antithrombin complexes, and prothrombin fragment F1 + 2.

J Mol Biol, 1993 Jan 5, 229(1), 239 - 42
Purification, crystallization and space group determination of DNA repair enzyme exonuclease III from E . coli; Kuo CF et al.; Escherichia coli exonuclease III possesses multiple catalytic activities: (1) a nucleotidyl hydrolase activity cutting 5' to apurinic/apyrimidinic sites and urea residues in DNA; (2) a 3' to 5' exonuclease activity specific for double-stranded DNA; (3) a RNase H activity preferentially degrading the RNA strand of a DNA.RNA hybrid and (4) an activity that can remove a number of 3' termini from duplex DNA including 3' phosphates, 3' phosphoglycolate residues, 3' phosphoglycolaldehyde residues and 3' trans-4-hydroxy-2-pentenal-5-phosphate residues . These multiple activities make exonuclease III a major enzyme in the base excision repair pathway for DNA damage . We have purified exonuclease III and grown crystals by the vapor diffusion method using polyethylene glycol 4000 as the precipitant . Buffers were found to have profound effects on crystallization with high concentrations of imidazole/malate buffer (0.4 M to 1.0 M) yielding larger crystals with less twinning . The crystals belong to the space group P3(1)21 or its enantiomorph P3(2)21 with unit cell dimensions of a = b = 107.8 A, c = 42.2 A, alpha = beta = 90 degrees, gamma = 120 degrees, have one 31 kDa monomer per asymmetric unit and diffract to 1.6 A . These crystals are stable to X-rays and suitable for high resolution structure determination.

Arch Virol, 1993, 130(1-2), 93 - 107
Structure and expression in E . coli of the gene coding for protein p10 of African swine fever virus; Munoz M et al.; The gene encoding protein p10, a structural protein of African swine fever (ASF) virus, has been mapped, sequenced and expressed in E . coli . Protein p10 was purified from dissociated virus by reverse-phase HPLC, and its NH2-terminal end identified by automated Edman degradation . To map the gene encoding protein p10, a mixture of 20-mer oligonucleotides based upon a part of the amino acid sequence was hybridized to cloned ASF virus restriction fragments . This allowed the localization of the gene in fragment Eco RI K of the ASF virus genome . The nucleotide sequence obtained from this region revealed an open reading frame encoding 78 amino acids, with a high content of Ser and Lys residues . Several of the Ser residues are found in Ser-rich regions, which are also found in some nucleic acid-binding proteins . The gene coding for protein p10 has been inserted in an expression vector which contains the promoter for T7 RNA polymerase . The recombinant plasmid was used to produce the ASF virus protein in E . coli . The bacterially produced p10 protein shows a strong DNA binding activity with similar affinity for both double-stranded and single-stranded DNA.

Mol Biol (Mosk), 1993 Jan-Feb, 27(1), 72 - 80
{Effective expression of genes for interleukin-2 and its mutant analogs in E . coli cells}; Seregin SV et al.; Recombinant plasmids were constructed for the efficient expression in E . coli cells of the human interleukin-2 (HIL-2) gene and two its mutant analogues obtained by of chemical-enzymic synthesis and polymerase chain reaction (deletion of 14 C-terminal amino acids and a change of the codon for Trp121 to Phe) . The recombinant HIL-2 but not the mutant analogues were shown to be biologically active . Both analogues obtained were weak antagonists to HIL-2.

Growth Factors, 1993, 8(2), 119 - 34
Midkine (MK), a retinoic acid (RA)-inducible gene product, produced in E . coli acts on neuronal and HL60 leukemia cells; Maruta H et al.; We have shown previously that (i) retinoic acid (RA), an anti-neoplastic agent, activates the midkine (MK) gene in mammalian embryonic carcinoma cells, and that (ii) the MK of 118 amino acids, purified from L cells, induces neurite outgrowth of mammalian embryonic brain cells . In this paper, we describe an unconventional strategy for the purification of a fully active MK from E . coli with a high yield . The MK was overproduced in E . coli as a glutathione S-transferase (GST) fusion protein . The MK fusion protein extracted from the bacterial inclusion bodies with guanidine-HCl was renatured, refolded slowly and cleaved by thrombin at the site where the GST links to the MK . The purified free MK, like RA, induced neurite outgrowth from central neurons of the mouse spinal cord, and suppressed the growth of human HL60 leukemia cells in vitro . Unlike RA, however, the MK did not induce granulocytic differentiation of HL60 cells . Furthermore, the MK supported the survival of an NGF-insensitive sensory neuron subpopulation(s) from chicken embryo dorsal root ganglion . Thus, the actions of the MK and leukemia inhibitory factor (LIF) are surprisingly similar . There is no sequence similarity between MK and LIF, however, and unlike MK, LIF production does not appear to be RA-inducible.

Vaccine, 1993, 11(2), 159 - 67
Preclinical evaluation of microencapsulated CFA/II oral vaccine against enterotoxigenic E . coli; Reid RH et al.; Colonization Factor Antigen (CFA/II) from enterotoxigenic Escherichia coli (ETEC) prepared under good manufacturing practices (GMP) was successfully incorporated into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer microspheres (BPM) under GMP and found to be safe and immunogenic when administered intraduodenally to rabbits . Following vaccination, Peyer's patch cells responded by lymphocyte proliferation to in vitro challenge with CFA/II . Also, B cells secreting specific anti-CFA/II antibodies were found in spleens following vaccination . No pathological changes were found following total necropsies of ten rabbits vaccinated with CFA/II BPM . Sixty-three per cent of the CFA/II BPM were between 5 and 10 microns diameter by volume particle size distribution; 1.17% protein content; 2.15% moisture; < 0.01% acetonitrile; 1.6% heptane; 22 non-pathogenic bacteria and three fungi per 1 mg protein dose; and passed the general safety test . We conclude that the CFA/II BPM oral vaccine is immunogenic and safe to begin a Phase I clinical safety study following Investigational New Drug approval.

EMBO J, 1993 Jan, 12(1), 17 - 22
Dissociation of synthetic Holliday junctions by E . coli RecG protein; Lloyd RG et al.; The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation . The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes . RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage . In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry . We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB . Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC . Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB . We suggest that RecG and RuvAB provide alternative activities than can promote branch migration of Holliday junctions in recombination and DNA repair.

DNA Seq, 1993, 3(5), 327 - 32
Five open reading frames upstream of the dnaK gene of E . coli; James R et al.; The nucleotide sequence of 3.17 kb of DNA upstream of the dnaK gene of E . coli was determined . Analysis of the sequence indicated the presence of five open reading frames (ORFs), two of which are coded on one strand and three on the other . ORFs 3 and 4 appear to constitute an operon . One of the ORFs (htgA) is preceded by a -10 promoter sequence which is identical to that of the dnaK sigma 32 P1 promoter, and thus suggests that htgA could be a heat-shock gene . ORF4 and htgA overlap each other on opposite strands of DNA, this would offer some interesting regulatory possibilities.

Biosystems, 1993, 29(1), 37 - 48
Frequency and conditions of spontaneous plasmid transfer from E . coli to cultured mammalian cells; Heitmann D et al.; After exposing mammalian cell cultures to E . coli carrying the complete cDNA of poliovirus 1 in a plasmid, formation of polioviruses ensued even when no eukaryotic transcription signals were contained in the plasmid . The age of the bacteria greatly influenced the frequency of gene transfer, which was low if the bacteria were harvested in the exponential stage, reaching a maximum at the stationary phase and declining afterwards . Since the gene transfer was inhibited by cytochalasin B, phagocytosis of the bacteria could be a first step for the transfer; however, other mechanisms could not be ruled out.

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