Escherichia coli

FEBS Lett, 1997 May 12, 408(1), 1 - 4
The E . coli SRP: preferences of a targeting factor; De Gier JW et al.; Research on the targeting of proteins to the cytoplasmic membrane of E . coli has mainly focused on the so-called 'general secretory pathway' (GSP) which involves the Sec-proteins . Recently, evidence has been obtained for an alternative targeting pathway in E . coli which involves the signal recognition particle (SRP) . The constituents of this SRP pathway in E . coli are homologous to those of the well-characterized eukaryotic SRP pathway, which is the main targeting pathway for both proteins translocated across and inserted into the endoplasmic reticulum membrane . However, until recently, no clear function could be assigned to the SRP in E . coli . New studies point to an important role of the E . coli SRP in the assembly of inner membrane proteins.

Carbohydr Res, 1997 May 9, 300(1), 69 - 76
Motional properties of E . coli polysaccharide K5 in aqueous solution analyzed by NMR relaxation measurements; Hricovini M et al.; 13C NMR relaxation measurements at three different magnetic field strengths have been used to analyse the motional properties of a low molecular weight K5 polysaccharide (delta UA-{-->4)-beta-D-GlcNAc(1-->4)-beta-D-GlcA(1-->}n-GlcNAcred) from E . coli . Two-dimensional double INEPT spectra with suppression of cross-correlation effects between dipolar and chemical shift anisotropy relaxation mechanisms were collected in order to determine carbon longitudinal and transverse relaxation times . The values of the overall correlation time and the rate of internal motions were obtained using the model free spectral densities . The data indicate that the overall motion of the molecule is non-isotropic and can be approximated with the symmetric top model with an axial ratio of approximately 22 . The magnitude of the generalized order parameters (S2 approximately 0.8) and the internal motion correlation time (tau e approximately 30 ps) differ from those found for iduronic acid-containing glycosaminoglycans and suggest that the internal motions in K5 polysaccharide are more limited.

Biochem Mol Biol Int, 1997 May, 41(6), 1101 - 8
Characterization of a soluble IL-6 receptor alpha mutant C277D/H280I expressed in E.coli; Duan J et al.; The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal transducer gp130 . To construct the antagonist of IL-6, a DNA segment encoding the soluble human IL-6R alpha (shIL-6R alpha) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction . The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level . The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold . DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells . DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.

J Membr Biol, 1997 May 1, 157(1), 17 - 25
Molecular dissection of the large mechanosensitive ion channel (MscL) of E . coli: mutants with altered channel gating and pressure sensitivity; Hase CC et al.; In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E . coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system . The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions . Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique . The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating . Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity . Similarly, deletion of the internal amphipathic region of the MscL abolished activity . In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic alpha-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.

J Clin Microbiol, 1997 May, 35(5), 1066 - 70
Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E . coli O157:H7 and O157:NM; Fields PI et al.; Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen . Identification of the H7 flagellar antigen is critical for the confirmation of E . coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen . To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E . coli . The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis . Two hundred eighty E . coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed . A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates . Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A . A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern . Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups . The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E . coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E . coli strains.

Neurosci Lett, 1997 Apr 18, 226(1), 53 - 6
Dissociative expression of adenoviral-mediated E . coli LacZ gene between ischemic and reperfused rat brains; Abe K et al.; A replication-defective adenoviral vector containing the E . coli lacZ gene was directly injected into the ischemic or reperfused cerebral cortex of rats . An administration of adenoviral vector showed a slight to moderate expression of the lacZ gene in the cerebral cortex of the middle cerebral artery (MCA) region until 2 days after the MCA occlusion . In contrast, expression of the lacZ gene was not observed, or only minimally so, in the reperfused brain until 2 days after a 90 min of transient MCA occlusion . However, the lacZ expression dramatically increased at 7 days after the reperfusion, then diminished by 21 days . The majority of brain cells that expressed the lacZ gene were neurons and a fraction (5-10%) were astroglial cells . The present study showed that an exogenous gene was transferred and expressed in neural cells of ischemic and reperfused brains in vivo, but the temporal profile of the expression is dissociative.

Hosp Pract (Off Ed), 1997 Apr 15, 32(4), 123 - 6, 129-30, 133 passim
Recognizing E . coli O157:H7 infection; Greenwald DA et al.; This infection first surfaced as a U.S . public health problem in the early 1980s . The problem persists, in the United States and elsewhere . Illness may resemble that in other infectious colitides and should be considered in the differential diagnosis of ischemic colitis . Prompt diagnosis may be made by specific stool culture.

J Biotechnol, 1997 Apr 4, 54(1), 53 - 67
Characterisation of non-porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E . coli extract; O'Brien SM et al.; The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described . Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents . Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E . coli extracts in a single step . Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose . The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale . By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.

J Mol Biol, 1997 Apr 4, 267(3), 476 - 80
Allosteric activation increases the maximum velocity of E . coli phosphofructokinase; Auzat I et al.; Several mutations that cause a decrease of 25 to 65% of the catalytic activity, were introduced at different positions in the phosphofructokinase from Escherichia coli, and the influence of the allosteric activator GDP on these mutants was measured . In the case of the wild-type enzyme, GDP converts the highly cooperative saturation towards fructose-6-phosphate into a hyperbolic saturation with almost no change in the maximum velocity . The mutants Glu148 --> Leu, Leu178 --> Val and Leu178 --> Trp are still cooperative for fructose-6-phosphate, and their cooperativity is also abolished or markedly decreased by GDP . In addition, GDP acts on these mutants as an activator of maximum velocity, and increases their catalytic rate constants by 35 to 65% depending on the mutation . The Leu178 --> Val mutant is even as active as the wild-type enzyme in the presence of GDP . The Thr125 --> Ser mutation decreases the maximum velocity by 60% and also suppresses the cooperativity towards fructose-6-phosphate . Accordingly, the only effect of GDP on the Thr125 --> Ser mutant is on its maximum velocity and not on its affinity for fructose-6-phosphate . However, the maximum velocity of this mutant is not increased by GDP but reduced by 33% . These results show that GDP affects the maximum velocity of these mutants and suggest that the activation by GDP of the wild-type enzyme measured by steady-state kinetics could be partially due to an increase of the maximum velocity, and not exclusively to an increase of the affinity for fructose-6-phosphate.

Mol Biotechnol, 1997 Apr, 7(2), 189 - 95
An efficient random mutagenesis technique using an E . coli mutator strain; Greener A et al.; Random mutagenesis of a cloned gene remains a central method to understand many aspects of the gene products function and structure . Having the ability to introduce a limited number of changes within a gene in a controlled fashion allows one to evaluate single changes and study the effect these variants have on the gene of interest . The in vivo random mutagenesis strategy described in this article, using an E . coli host, is a convenient method to introduce a limited number of mutations in a controlled manner.

J Clin Microbiol, 1997 Apr, 35(4), 892 - 9
Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E . coli serotypes isolated from sheep; Kudva IT et al.; The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E . coli (STEC) strains from sheep are described . One flock was investigated for E . coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons . Variation in the occurrence of E . coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter . E . coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis . Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E . coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E . coli O157:H7 strains, and that the strains shed by individuals changed over time . E . coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces . In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment . The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM . Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity . The most common toxin-eae genotype was positive for stx1, stx2, and eae . A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene . The report demonstrates that sheep transiently shed a variety of STEC strains, including E . coli O157:H7, that have potential as human pathogens.

Insect Biochem Mol Biol, 1997 Apr, 27(4), 337 - 43
Molecular cloning of cDNA for Sarcophaga prolyl endopeptidase and characterization of the recombinant enzyme produced by an E . coli expression system; Ohtsuki S et al.; A cDNA for prolyl endopeptidase (PEP) of Sarcophaga peregrina (flesh fly) was cloned and its sequence determined . The overall amino acid sequence identity between Sarcophaga and mammalian PEPs was 53%, indicating that these enzymes are structurally very similar . Northern blot hybridization revealed that the Sarcophaga PEP gene was activated significantly at the eversion stage of imaginal disc differentiation . We obtained recombinant PEP by expressing the cDNA in Escherichia coli . The recombinant and authentic enzymes showed almost identical characteristics, in terms of substrate specificities and sensitivities to inhibitors.

EMBO J, 1997 Apr 1, 16(7), 1670 - 85
Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E . coli: role of two new phosphoprotein phosphatases PrpA and PrpB; Missiakas D et al.; It is now well established that the sigmaE regulon of Escherichia coli is induced by misfolding of proteins in the periplasm and the outer membrane . htrA belongs to this regulon and encodes a periplasmic protease involved in the degradation of misfolded proteins . htrA transcription is also under the positive control of a two component signal transduction system CpxR CpxA . Closer examination of the putative signal transduction pathway modulating htrA transcription has led us to the identification of two new genes . Biochemical and genetic evidence shows that these two genes encode two phosphoprotein phosphatases, designated PrpA and PrpB . These are the first examples of typical serine/threonine and tyrosine phosphatases described in E . coli . PrpA and PrpB are involved in signaling protein misfolding via the CpxR CpxA transducing system . In addition, both PrpA and PrpB modulate the phosphorylated status of some other phosphoproteins in E . coli . Finally, we show that PrpA is a heat shock protein.

Biochem Mol Biol Int, 1997 Apr, 41(4), 815 - 20
Renaturation and purification of recombinant tissue-type plasminogen activator expressed in E . coli; Hua ZC; Under the control of trp promoter, human tissue-type plasminogen activator was expressed in E . coli in the form of inclusion body . The recombinant t-PA was recovered for renaturation from preparative native PAGE, gel by zinc acetate staining and electroelution . After renaturation in vitro, the recombinant t-PA was purified by benzamidine affinity chromatography and lysine affinity chromatography . The purified t-PA showed homogeneous on silver-stained SDS-PAGE gel, with a specific activity of 240,000 I.U./mg protein.

Cancer Lett, 1997 Mar 19, 114(1-2), 85 - 7
Antimutagenic activity of casein against MNNG in the E . coli DNA repair host-mediated assay; van Boekel MA et al.; The effect of caseinate and soy protein in the diet on the mutagenicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was assessed in-vivo and ex-vivo in the DNA-repair host-mediated assay and liquid suspension assay, respectively . Of the two proteins only casein showed a strong antimutagenic activity over the whole digestive tract, except in the stomach . It is suggested that the molecular structure of a protein determines its protective effect against mutagens: casein lacks secondary and tertiary structure so that amino acids are more readily available for interaction with the mutagen than with the amino acids in soy protein which is a globular protein.

Nat Struct Biol, 1997 Mar, 4(3), 198 - 201
Solution structure of the N-terminal domain of the delta subunit of the E . coli ATPsynthase; Wilkens S et al.; NMR studies of the delta subunit of the Escherichia coli F1F0-ATPsynthase reveal that it consists of an N-terminal six alpha-helix bundle and a less well ordered C terminus . Both domains are part of one of two separate connections between F1 and F0.

Int J Radiat Biol, 1997 Mar, 71(3), 253 - 8
Production of radiation-resistant E . coli strains by daily X-irradiation; Ewing D; Exposure of E . coli AB1157 (a K-12 wild-type strain) to very large, daily X-ray doses has produced mutant strains resistant to both X-rays and UV photons . Results reported here are with KS0(160), the most resistant strain isolated thus far . Relative to its parent, KS0(160) is about 2.3 x more resistant to X-rays and about 2.2 x more resistant to ultraviolet photons (ratios of sensitivities in air) . Two other characteristics of KS0(160) make its responses to X-rays different from those of AB1157: KS0(160) has an oxygen enhancement ratio of only 1.8 compared with 2.7 for its parent; glycerol reduces the sensitivity of AB1157 by about 75% (in air), but the maximum effect in KS0(160) is only a 49% reduction in response . P1 transduction experiments showed that all the acquired resistance in KS0(160) is lost when SOS repair activity is genetically blocked by an inserted lexAl, indicating that the mutation(s) associated with the acquired resistance in KS0(160) are in wild-type genes involved with induced DNA repair (i.e . SOS repair activity).

Vet Microbiol, 1997 Mar, 54(3-4), 329 - 41
Chicken egg yolk antibodies against F18ab fimbriae of Escherichia coli inhibit shedding of F18 positive E . coli by experimentally infected pigs; Imberechts H et al.; F18ab and F18ac are antigenic variants of a colonizing fimbria commonly found on E . coli associated with postweaning diarrhea and edema disease in pigs . Chicken F18ab antibodies were obtained by immunising hens with purified F18ab fimbriae . For their in vitro characterisation antibodies were isolated from diluted egg yolks by ammonium sulfate precipitation . In vitro adhesion tests demonstrated that the chicken F18ab antibodies inhibited attachment of F18ab positive E . coli bacteria to the intestinal mucosa . Just weaned piglets were experimentally infected with an F18ab positive edema disease strain of E . coli, or with an F18ac positive postweaning diarrhea E . coli strain . The animals were infected on the second day of a period during which chicken F18ab antibodies were added to their feed . During the same period, pigs of the control group received commercial eggs in which no F18 antibodies were detected . In both experimental infections the excretion of the F18 positive strain was reduced in pigs that received the F18ab antibodies as compared to the control animals . The F18ab antibodies diminished the cases of diarrhea and death in animals infected with F18ac positive E . coli.

Kidney Int Suppl, 1997 Mar, 58, S91 - 6
Association of verotoxin-producing E . coli and verotoxin with hemolytic uremic syndrome; Arbus GS; It has been over 10 years since we first showed an association {1} between classical hemolytic uremic syndrome (hemolytic anemia, thrombocytopenia, and uremia, following a diarrheal prodome) and certain E . coli {1} capable of producing a toxin, initially called verotoxin (VT) because of its cytopathic effect on Vero cells {2} and later Shiga-like toxin (SLT) because of the toxin's close biological and structural similarity to Shiga toxin . Although we initially reported that 75% of children with idiopathic hemolytic uremic syndrome (HUS) {1} had evidence of a verotoxin-producing E . coli (VTEC) {1}, a later study showed that over a seven year period (1980-86), of 86 children seen with HUS, 91% had a classical presentation and 88% of these had evidence of a VTEC infection {3} . This paper traces the path of the incriminating organisms (VTEC) from the time of ingestion, up to and including internalization of the toxin into a target cell; in vitro experiments demonstrating the effect of toxin on endothelial cells are included . It is hoped that we might gain a clearer insight into factors that might predispose an individual to contracting HUS . Once a better understanding of the pathogenesis of VTEC associated HUS is known, areas for therapeutic intervention might be realized.

J Mol Biol, 1997 Feb 14, 266(1), 15 - 22
Heteronuclear NMR studies of E . coli translation initiation factor IF3 . Evidence that the inter-domain region is disordered in solution; Moreau M et al.; Initiation factor IF3 from Escherichia coli plays a critical role in the selection of the correct initiation codon . This protein is composed of two domains, connected by a lysin-rich hydrophilic linker . The conformation of native IF3 was investigated by heteronuclear NMR spectroscopy . The two domains are independent and show little or no interaction . Heteronuclear relaxation studies of a sample selectively labelled on lysine residues demonstrates that the inter-domain linker is highly flexible, exhibiting increased 15N T2 values and negative 1H{15N} nuclear Overhause effects over a length of at least eight residues . Analysis of the rotational correlation times further shows that the motions of the two domains are most likely uncorrelated . The inter-domain linker thus displays almost totally unrestricted motions . Accordingly, the amide protons in the central region are shown to be in fast exchange with water . Such a high degree of flexibility of the inter-domain linker might be required for IF3 domains to interact with distant regions of the ribosome.

FEBS Lett, 1997 Feb 3, 402(2-3), 209 - 12
Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E . coli; Vishnuvardhan D et al.; The serine protease domain of HCV comprising amino acids 1027-1218 (deltaNS3) was expressed in E . coli with a His tag at its N-terminal end . The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (approximately 3 mg/l of cultured cells) . The deltaNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with kcat/Km = 160 x 10(-3) min(-1) microM(-1) in the presence of NS4A peptide . Our deltaNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far . The deltaNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203-2506 amino acids) that was synthesized by in vitro transcription and translation system.

Biochemistry (Mosc), 1997 Feb, 62(2), 184 - 90
Interaction of the E . coli alkaline phosphatase precursor with model phospholipid membranes; Mikhaleva NI et al.; An interaction of a precursor of the E . coli secreted alkaline phosphatase (prePhoA) containing a signal peptide and model bilayer membranes prepared from the lipids of E . coli has been studied . The interaction was evaluated by monitoring of the state of the lipid phase by fluorescence spectroscopy . The role of the signal peptide in this process was evaluated by a comparative study of the interaction of the mature alkaline phosphatase which does not contain the signal peptide with the model membranes . The precursor was shown to be inserted into the lipid bilayer since the fluorescence anisotropy of a hydrophobic probe, diphenylhexatriene, was enhanced . The intensity of the process is determined by the presence of the signal peptide and depends on the pH of the medium . The data indicate that the mature polypeptide chain of the enzyme also has affinity for the membrane.

Bioorg Khim, 1997 Feb, 23(2), 104 - 9
{Irreversible specific inhibition of E . coli inorganic pyrophosphatase with amines}; Burobin AV et al.; An unusually high reactivity of the carboxyl groups of the active site of E . coli inorganic pyrophosphatase towards amines was shown . Amino acid esters and other amines are specific irreversible inhibitors of the enzyme . The reaction involves the formation of an enzyme-inhibitor complex followed by the chemical modification of dicarboxylic amino acid residues . It is assumed that the binding of the positively charged inhibitor occurs at the binding site of cations-activators.

Gene Ther, 1997 Feb, 4(2), 101 - 10
Selective cell ablation in transgenic mice expression E . coli nitroreductase; Clark AJ et al.; The gene encoding E . coli nitroreductase (NTR) was expressed in the luminal cells of the mammary gland of transgenic mice using the ovine beta-lactoglobulin promoter . Treatment of NTR expressing animals with the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) resulted in a rapid and selective killing of this population of cells whereas the closely associated myoepithelial cells were unaffected . NTR-mediated inducible cell ablation offers a number of advantages over the use of HSV1-tk for the selective killing of cells in vivo.

Cell Mol Biol (Noisy-le-grand), 1997 Feb, 43(1), 47 - 58
Human coproporphyrinogen oxidase . Biochemical characterization of recombinant normal and R231W mutated enzymes expressed in E . coli as soluble, catalytically active homodimers; Martasek P et al.; To obtain recombinant human coproporphyrinogen oxidase (CPX), a cDNA for the coding region of mature human CPX has been expressed in E . coli . CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage just preceding first amino acid of the CPX protein . The human CPX was found to be in the soluble fraction . This previously unobtainable human heme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration . Recombinant human CPX exhibits homogeneous behavior during high performance liquid chromatography (HPLC) and the N-terminal sequence, confirmed by protein sequencing, revealed a single polypeptide chain . In its active form, human CPX is a homodimer . According to the hydrodynamic properties derived from analytical ultracentrifugation, dimeric CPX has a nearly globular shape . Additionally, naturally occurring Arg to Trp (R231W)-mutated CPX has been also expressed in E . coli and further characterized . The mutated enzyme has a Km value of 0.55 microM as compared to 0.30 microM for the wild type . The catalytic efficiency (specificity constant, kcat/Km) of the mutated CPX was four fold lower than wild-type enzyme . The activity measurement of the mutated enzyme showed higher thermal sensitivity as compared with wild type CPX . The measured pI for mutated CPX is 5.65, compared to 6.40 for wild type . The pH optima for the mutated and wild-type protein are 6.6 and 6.8, respectively . The R231W mutation of CPX does not affect dimer formation and both normal and mutated CPX exhibit identical sedimentation properties . The thermal denaturation of both wild type and mutant CPX was found to be irreversible . The mutated CPX contained a significant amount of tightly bound porphyrin coproporphyrin . No metal association was found either in wild type or in mutated CPX . The availability of the recombinant human CPX will aid in structural and mechanistic studies.

Biophys Chem, 1997 Jan 31, 63(2-3), 107 - 18
Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E . coli: ligand-dependent quenching of enzyme intrinsic fluorescence; Kierdaszuk B et al.; Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E . coli purine nucleoside phosphorylase (PNP) with phosphate (Pi; a substrate) and a close non-substrate analogue (sulfate; SA) . The quenching of enzyme fluorescence by Pi was bimodal, best described by two modified Stem-Volmer equations fitted independently for "low" (below 0.5 mM Pi) and "high" (above 0.5 mM Pi) ligand concentrations . At Pi > 0.5 mM, binding is characterized by a fortyfold higher dissociation constant (Kd2 = 1.12 +/- 0.10 mM), i.e . by a lower affinity for phosphate, with a sevenfold lower quenching constant and 1.6-fold higher accessibility . By contrast, the binding of SA, and the resultant fluorescence quenching, was unimodal, with Kd = 1.36 +/- 0.07 mM, comparable to the Kd for Pi at "high" Pi, with a total binding capacity of one sulfate or phosphate group per enzyme subunit . SA proved to be a competitive inhibitor of phosphorolysis with Ki = 1.2 +/- 0.2 mM vs . Pi, hence similar to its Kd . SA at a concentration of 5 mM did not affect the Pi affinity at Pi < 0.5 mM, but led to a reduced affinity and twofold higher Pi binding capacities at Pi > 0.5 mM . The resultant fluorescence quenching by Pi decreased at 5 mM SA, with lower Stern-Volmer constant (KSV) and fractional accessibility (fa) values . Increasing concentrations of Pi reduced the enzyme affinity for SA, characterized by a higher Kd . The Hill model showed negative cooperative binding of Pi in the absence and presence of 5 mM SA with Hill coefficients h = 0.60 +/- 0.01 and h = 0.83 +/- 0.07, respectively . SA exhibited non-cooperative binding in the absence of Pi (h = 1.08 +/- 0.01) and negative cooperative binding in the presence of Pi (h < 1) . PNP fluorescence decays were best fitted to a sum of two exponentials, with an average lifetime of 2.40 +/- 0.14 ns unchanged on interaction with quenching ligands, and pointing to static quenching . The overall results are relevant to the properties of PNP from various sources, in particular to the design of potent bisubstrate analogue inhibitors.

Cell, 1997 Jan 24, 88(2), 187 - 96
The E . coli signal recognition particle is required for the insertion of a subset of inner membrane proteins; Ulbrandt ND et al.; E . coli homologs of the signal recognition particle (SRP) and its receptor are essential for viability, but their role in protein export is unclear . To elucidate their function, we devised a genome-wide screen to identify genes that encode SRP substrates . Inhibition of the SRP pathway sharply blocked the membrane insertion of several polytopic inner membrane proteins (IMPs) that were predicted to be SRP substrates, but had a smaller effect on the insertion of other IMPs and no significant effect on preprotein translocation . Our results suggest that whereas most E . coli preproteins and some IMPs can utilize SRP-independent targeting pathways effectively, the structural features of a subset of IMPs have required the conservation of an SRP-based targeting machinery.

Cell, 1997 Jan 24, 88(2), 175 - 85
Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E . coli; Hale CA et al.; FtsZ is a soluble, tubulin-like GTPase that forms a membrane-associated ring at the division site of bacterial cells . While this ring is thought to drive cell constriction, it is not well understood how it is assembled or how it affects cell wall invagination . Here we report that FtsZ binds directly to a novel integral inner membrane protein in E . coli that we call ZipA . We present genetic and morphological evidence indicating that this interaction is required for cell division, and show that a fluorescent ZipA-Gfp fusion protein is located in a ring structure at the division site, both before and during cell wall invagination . ZipA is an essential component of the division machinery, and, by binding to both FtsZ and the cytoplasmic membrane, is likely to be directly involved in the assembly and/or function of the FtsZ ring.

Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 306 - 10
Expression of Sulfolobus solfataricus trpE and trpG genes in E . coli; Tutino ML et al.; The genes trpE and trpG of the hyperthermophilic archaeon Sulfolobus solfataricus, encoding the components I and II of anthranilate synthase, were cloned and co-expressed in Escherichia coli . The properties of the recombinant protein were determined and compared to those of the wild type complex . Gel filtration chromatography revealed an alpha2beta2 composition . The heteromeric enzyme is fully active above 85 degrees C and can be considered to be an "extremozyme" according to Adams et al.{1} . Sulfolobus solfataricus anthranilate synthase is subject to feedback inhibition by L-tryptophan even if it lacks the co-operativity that has been observed for all the other tetrameric anthranilate synthases.

Adv Exp Med Biol, 1997, 419, 175 - 80
Expression and comparative analysis of recombinant rat and mouse RT6 T cell mono(ADP-ribosyl)transferases in E . coli; Karsten S et al.; Recombinant RT6 proteins of rat and mouse were analyzed for NAD-metabolizing, i.e . mono(ADP-ribosyl)transferase, NAD-glycohydrolase (NADase) and ADP-ribosyl cyclase activities . The results reveal surprising intra- as well as inter-species differences in enzyme activities . While mouse Rt6 proteins were found to be strong arginine-specific transferases, but comparatively weak NADases, the opposite held true for rat RT6, for which transferase activity could only be detected in the form of arginine-specific auto-ADP-ribosylation, displayed by RT6.2 but not by RT6.1 . NADase activity of rat RT6 was not accompanied by production of cyclic ADPR (cADPR) . Rat RT6 gained potent arginine-specific transferase activity by exchange of a single amino acid for the corresponding residue of the mouse proteins.

Adv Exp Med Biol, 1997, 412, 357 - 61
Adhesion of K88ab fimbriated E . coli in piglet small intestines in relation with iron transport molecules; Grange P et al.; Enteropathogenic K88 fimbricated E . coli colonize the piglet small intestine . In swine, it has been previously established that some pigs lack intestinal receptors for K88 lectins and that these animals are resistant to infections by K88-positive E . coli . The receptor is inherited as a simple mendelian character . The interactions established between the glycoconjugate receptors of pig brush borders and K88 lectins are mediated by O- and N-linked glycoproteins which differ between adhesive and non-adhesive piglets . In this study the adhesion of E . coli K88+ in crossbred F2 (LW x MS) x (LW x MS) populations . By using in vitro brush border test, we observed modulation of the adhesion of K88 fimbriae and distinguished high and low affinity receptors . Furthermore, we correlated the attachment with glycoprotein pattern of epithelial cells and mucus . Epithelial cells and mucus contained several glycopeptides (from 42 to 74 kDa) recognized by K88ab fimbriae . The 74 kDa glycoprotein was characteristic of adhesive phenotype and was a mucosal transferrin (iTf) . It appeared that iTf was more abundant in adhesive intestines than in non-adhesive ones, suggesting that susceptibility/resistance phenotype could be related to iron metabolism in the intestinal tract . Furthermore, we visualized the intestinal transferrin receptors on the brush border membrane of epithelial cells, probably implicated in iron absorption.

Adv Exp Med Biol, 1997, 412, 193 - 200
Evaluation of DNA "fingerprinting" for predicting the potential of E . coli O157:H7 isolates to cause hemolytic uremic syndrome (HUS); McAdoo KK et al.; Escherichia coli O157:H7 has been recognized since 1982 as a serious human pathogen spread by contaminated food and water . Pulsed-field gel electrophoresis has proven useful for identification of specific isolates/strains of this organism . Hemolytic uremic syndrome (HUS), generally occurring in children or the aged, is the most severe sequela associated with E . coli O157:H7 infection . The presently described work was designed to compare the genomic profile of isolates known to have caused HUS with those having had no such involvement . We asked the question: "Can we develop the means to recognize an 'HUS-prone' E . coli isolate and thereby alert medical personnel to the increased risk?" Twenty-two HUS-related and 10 HUS-unrelated E . coli O157:H7 samples were chosen for genomic analysis . Isolates were cultured overnight prior to being embedded in agarose gel plugs . Plugs were digested, using Xbal restriction endonuclease, and subjected to pulsed-field gel electrophoresis (PFGE) for 20 hours . Gels were stained with ethidium bromide, photographed under ultraviolet light, and Southern blotted . Radiolabeled toxin gene probes were used for hybridization assays . The two classes of isolates were compared by optical imaging software . A computer-generated dendrogram, based on restriction profiles, offered strong initial evidence that the HUS sequela may be produced by a particularly virulent and identifiable clone . The predictive value of this finding appears to be substantial.

Biochemistry (Mosc), 1997 Jan, 62(1), 66 - 70
Purification and some properties of Thermotoga neapolitana thermostable xylanase B expressed in E . coli cells; Velikodvorskaya TV et al.; A xylanase was purified from the recombinant strain E . coli TG1 carrying the pTT32 vector with a fragment of the Thermotoga neapolitana chromosomal DNA . The enzyme was purified 419-fold with 36% yield after heating at 70 degrees C and pH 4.5 and subsequent ion-exchange chromatography . By polyacrylamide gel electrophoresis in the presence of SDS, the molecular weight of the apparently homogeneous protein is 39 kD . By isoelectric focusing, the protein is of a single form with pI = 5.9 . The optimal pH for hydrolysis is 5.5, and the optimal temperature is 90 degrees C . The xylanase is stable to heating at 70 degrees C for 4 h . The enzyme is inactivated by 50% at 80, 90, and 100 degrees C after 227, 162, and 30 min, respectively . Enzyme activity was tested using xylans and glucans as substrates . By thin-layer chromatography of the xylan hydrolysis products, the enzyme was classified as an endoxylanase.

Chem Biol, 1997 Jan, 4(1), 17 - 24
Ability of Streptomyces spp . acyl carrier proteins and coenzyme A analogs to serve as substrates in vitro for E . coli holo-ACP synthase; Gehring AM et al.; INTRODUCTION: The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes . The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS . ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP . When ACPs of Streptomyces type II aromatic PKS are overproduced in E . coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form . RESULTS: We demonstrate that E . coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin . The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate . Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS . CONCLUSIONS: E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties . Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.

Biotechniques, 1997 Jan, 22(1), 112 - 8
Relationship between opacity of transformed E . coli colonies and over-expression of the recombinant transcript; Barik S; Following transformation with recombinant plasmid clones, E . coli BL21(DE3) often produced extremely opaque colonies on a standard semisolid agar plate, as compared with the translucent colonies produced by normal, untransformed bacteria A standard BL21(DE3) culture consisted of two kinds of cells one kind produced translucent colonies and the other produced opaque colonies upon transformation by recombinant plasmids . The translucent-generating phenotype often switched to the opaque-generating phenotype, which was irreversible . Opacity in the BL21(DE3) background was correlated to a higher preinduced level of T7 RNA polymerase, presumably through a stable and inheritable genetic change . In all E . coli strains tested, a robust transcription of the recombinant gene from the plasmid clone was found to be an essential prerequisite for every high opacity; translation of the RNA was not required . The degree of opacity was also determined by the nature of the insert in a given strain background . Colony, opacity generally, but not invariably, correlated with a smaller colony size on semisolid agar and a reduced growth rate in liquid culture.

Shock, 1997 Jan, 7(1), 65 - 9
Adrenocorticotropin correlates strongly with endotoxemia after intravenous but not after intraperitoneal inoculations of E . coli; Carlson DE; To determine the influence of the site of infection on circulating endotoxin and hormonal release, male rats were prepared with arterial catheters and with either intravenous (i.v.) or intraperitoneal (i.p.) catheters under pentobarbital . Four days later, they were injected either i.v . or i.p . with Escherichia coli suspended in saline . Plasma was assayed for adrenocorticotropin (ACTH), corticosterone, and endotoxin activity . After approximately 10(9) colony-forming units (CFU) of E . coli, plasma endotoxin in i.v . rats (496 +/- 96 EU/mL) differed from that in i.p . rats (12.6 +/- 3.6 EU/mL, p < .01) . However, ACTH and corticosterone increased to the same extent in both groups . After approximately 10(7) CFU, plasma endotoxin in i.v . rats (9.15 +/- 2.09 EU/mL) was greater than in i.p . rats (2.56 +/- .42 EU/mL, p < .05), and ACTH and corticosterone increased more at 1 h in i.v . rats than in i.p . rats (p < .01) . Additional rats given approximately 0.3 x 10(9) CFU i.p . had plasma endotoxin that did not differ from values measured after either approximately 10(9) CFU i.p . or approximately 10(7) CFU i.v . However, the ACTH responses in these three groups differed from one another (p < .01) . ACTH was more strongly correlated to plasma endotoxin in i.v . rats (r = .915) than in i.p . rats (r = .528, p < .01 for difference from i.v . group) . The weak relationship between plasma endotoxin and ACTH after i.p . inoculations suggests that peritoneal infections activate important pathways that are independent of the circulation.

J Clin Microbiol, 1997 Jan, 35(1), 223 - 7
Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E . coli strains isolated from piglets and calves with diarrhea; Yamamoto T et al.; Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E . coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization . The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin . One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene . In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene . The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid . The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E . coli strains, respectively . The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.

Biochim Biophys Acta, 1996 Dec 5, 1298(2), 241 - 9
Reaction of E . coli catalase HPII with cyanide as ligand and as inhibitor; Maj M et al.; Cyanide forms an inhibitory complex with the haem d-containing E . coli catalase HPII, spectrally similar to the cyanide complex of beef liver enzyme but with absorption bands shifted 90 nm towards the red end of the spectrum . Both the Kd and Ki values are approximately 7 microM in the wild-type enzyme . The cyanide reaction is slow, with a bimolecular 'on' constant approx . 2000 x smaller than that of eukaryotic enzyme, and an 'off' constant diminished by a similar amount . Catalases with a mutated distal histidine (H128) fail to bind cyanide at cyanide concentrations below 50 mM . Catalases with a mutated distal asparagine (N201) show only small changes in cyanide affinity from the wild type . The major fraction of HPII N201A has a Kd approximately 40 microM, and a minor fraction has a lower cyanide affinity; the major fraction of HPII N201Q has a Kd approximately 15 microM . The Kd and Ki for HPII N201D is approximately 8 microM, essentially identical with that of the wild type but N201D appears to bind cyanide somewhat more rapidly than does wild-type enzyme . The HPII mutant N201H can be obtained in both haem d and protohaem forms; it exhibits two types of cyanide binding behaviour . In its protohaem form it binds cyanide poorly (Kd > or = 0.25 mM) . After peroxide treatment converts t into haem d or a closely related species it binds cyanide with a much higher affinity (Kd approximately 15 microM) . Cyanide binding to HPII requires a distal histidine to provide hydrogen-bonding stability, but not a distal asparagine . Rates of cyanide binding and release are controlled by haem group accessibility through the channel leading to the outside . In HPII N201H channel opening may depend upon oxidation of the haem from the starting protohaem to the final haem d form.

FEBS Lett, 1996 Dec 2, 398(2-3), 274 - 8
Six-fold rotational symmetry of ClpQ, the E . coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY; Kessel M et al.; ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome . In E . coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX . ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 350,000, respectively, estimated by gel filtration . Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP . Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings . The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring . The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.

Mutat Res, 1996 Dec 2, 364(3), 193 - 207
A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E . coli and T4; Purmal AA et al.; Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V . These N-glycosylases are known to have associated AP lyase activities . In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites . Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities . Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates . For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)) . Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V . DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively . Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.

Bioorg Khim, 1996 Dec, 22(12), 941 - 3
{Mutation ssF29 in the gene for E . coli ribosomal protein S1, suppressing a defect in transmembrane protein transport results from insertion of the IS10R element}; Artamonova VS et al.; The nature of the ssyF29 mutation causing the synthesis of a truncated form of the ribosomal protein S1 and its location in the rpsA gene were determined . The ssyF mutation was found to result from insertion of the IS10(R) element which causes the termination of translation of the corresponding mRNA at the first insertion nucleotide and the production of the S1 protein which is truncated at the C-terminus and composed of 464 amino acid residues (instead of 557 residues in the wild-type protein) . The mutant rpsA gene (ssyF) encodes no additional amino acid residues as compared with the wild-type rpsA gene.

Microbiol Res, 1996 Dec, 151(4), 379 - 85
Transfer of attaching and effacing from a strain of enteropathogenic Escherichia coli to E . coli K-12; O'Gorman LE et al.; The ability to cause attaching and effacing (AE) lesions in intestinal epithelial cells is an essential virulence trait of enteropathogenic E . coli (EPEC) that requires several chromosomal genes acting in concert with one another . In this study, we show that the ability to cause AE lesions can be transferred by conjugal mating from a high frequency recombinant (Hfr) derivative of a rabbit EPEC strain, E . coli RDEC-1, to a strain of E . coli K-12 . Although the recipient acquired a considerable amount of donor DNA during the transfer process, it expressed the AE phenotype phenotype only weakly . The findings suggest the AE is a multigene phenomenon, the genes for which may not reside on a single region of the bacterial chromosome.

Horm Metab Res, 1996 Dec, 28(12), 694 - 7
Purification of milligram quantities of human leptin from recombinant E . coli; Fawzi AB et al.; Leptin, the product of the obese (ob) gene, is a 16 kilodalton protein secreted from adipose tissue . Restoration of leptin to obese ob/ob mice leads to normalization of body weight . The effect of leptin in larger animals has not been explored, in part because of limited supplies of leptin . To date, the potency and yield of recombinant leptin purified from a variety of eukaryotic sources or from E . coli has been highly variable . While purification of leptin from E . coli inclusion bodies has afforded the greatest yield of protein, its potency is at least an order of magnitude lower than that of leptin secreted from E . coli or eukaryotic cells . The mechanistic basis of this difference in potency is not clear at present . The ability to purify significant quantities of highly active leptin will be crucial for the evaluation of leptin structure, as well as its function in additional animal models of obesity . We now report a facile protocol for the preparation of recombinant leptin using an E . coli expression system . 75-85 milligrams of leptin with a purity of greater than 97 % was prepared from a liter of recombinant E . coil . The procedure can be performed in less than 48 h and requires no chromatography . Intraperitoneal injection of 0.1 mg/kg renatured leptin into ob/ob mice results in a significant reduction in food consumption . The potency of this material is similar to the most potent recombinant leptin described to date . The ability to rapidly prepare large quantities of high specific activity material will hasten the definition of leptin's role in non-rodent models of obesity.

J Biomol NMR, 1996 Dec, 8(4), 429 - 44
1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E.coli rho protein; Briercheck DM et al.; Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E . coli . NMR spectra of the fragment containing residues 1-116 of rho protein (Rho116) show that a region of this protein is unfolded in solution . Addition of (dC)10 to this fragment stabilizes the folded form of the protein . The fragment comprising residues 1-130 of rho protein (Rho130) is found to be stably folded, both in absence and presence of nucleic acid . NMR studies of the complex of Rho130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho130 are similar to those of intact rho protein; therefore, Rho130 is a suitable model of the RNA-binding domain of Rho protein . NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggests that Rho130 forms oligomers in the presence of longer oligonucleotides . 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY . The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.

Gene Ther, 1996 Dec, 3(12), 1143 - 50
Investigation of alternative prodrugs for use with E . coli nitroreductase in 'suicide gene' approaches to cancer therapy; Bailey SM et al.; The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system . We have examined the efficacy of an alternative approach utilising the E . coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs . V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells . In general other prodrugs which were found by HPLC to act as substrates for purified E . coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute . In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold) . The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.

J Bioenerg Biomembr, 1996 Dec, 28(6), 483 - 94
Calcium binding to the subunit c of E . coli ATP-synthase and possible functional implications in energy coupling; Zakharov SD et al.; The 8-kDa subunit c of the E . coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes . The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes . The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment . The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E . coli mutants, Asp61-->Asn and Asp61-->Gly . The Ca++ binding is pH dependent in both the E . coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0 . A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells . Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment . An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment . The data are consistent with the notion that Ca++ bound to the periplasimic side of the E . coli F0 proton channel can block H+ entry into the channel . A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation . The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.

Structure, 1996 Nov 15, 4(11), 1317 - 24
Binding of the anticancer drug ZD1694 to E . coli thymidylate synthase: assessing specificity and affinity; Rutenber EE et al.; BACKGROUND: Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by 5, 10-methylenetetrahydrofolate (CH2H4folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate) . The essential role of TS in the cell life cycle makes it an attractive target for the development of substrate and cofactor-based inhibitors that may find efficacy as anticancer and antiproliferative drugs . Antifolates that compete specifically with the binding of CH2H4 folate include the cofactor analog CB3717 (10-propargyl-5,8-dideazafolate) . However, the development of potent cofactor analog inhibitors of TS, such as CB3717, as drugs has been slowed by their toxicity, which often becomes apparent as hepatic and renal toxicity mediated by the specific chemistry of the compound . Attempts to abolish toxicity in human patients while preserving potency against the target enzyme, have led to the development of ZD1694, which has already shown significant activity against colorectal tumours . RESULTS: The three dimensional crystallographic structure of ZD1694 in complex with dUMP and Escherichia coli TS has been determined to a resolution of 2.2 . This was used to evaluate the specific structural determinants of ZD1694 potency and to correlate structure/activity relationships between it and the closely related ligand, CB3717 . ZD1694 binds to TS in the same manner as CB3717 and H2 folate, but a methyl group on its quinazoline ring, its thiophene ring and the methyl group at N10 are compensated for by plastic accommodation of the enzyme active site coupled with specific rearrangement in the solvent structure . A specific hydrogen bond between the protein and the inhibitor CB3717 is absent in the case of ZD1694 whose monoglutamate tail is reoriented and more well ordered . CONCLUSIONS: The binding mode of ZD1694 to thymidylate synthase has been determined at atomic resolution . ZD1694 forms a ternary complex with dUMP and participates in the multi-step TS reaction through the covalent bond formation between dUMP and Cys146 thereby competing with CH2H4 folate at the active site . Analysis of this inhibitor ternary complex structure and comparison with that of CB3717 reveals that the enzyme accommodates the differences between the two inhibitors with small shifts in the positions of key active site residues and by repositioning an active site water molecule, thereby preserving a general binding mode of these inhibitors.

Adv Dent Res, 1996 Nov, 10(2), 187 - 93; discussion 194
High expression of human amelogenin in E . coli; Deutsch D et al.; A human cDNA, encoding for the 175-amino-acid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E . coli . The expressed protein was characterized by SDS-PAGE Western blotting, and N-terminal amino acid sequencing.

Genes Cells, 1996 Nov, 1(11), 1017 - 30
Specific nicking at the 3' ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E . coli protein ACP; Maekawa T et al.; BACKGROUND: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition . The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini-Tn3 as the donor and another plasmid as the target . Transposase has the ability to bind to the 38-bp terminal inverted repeats (IRs) of Tn3 . The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood . RESULTS: We found that nicking occurred efficiently in the cell-free system at each of the 3' ends of the IRs of mini-Tn3 in the closed circular or linear donor molecules . The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP-regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction . By using the nicking assay employed here, transposase was purified almost to homogeneity . Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 M NaCl . The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor . The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein . CONCLUSIONS: Nicking occurred efficiently at the 3' ends of mini-Tn3 in the reaction mixture containing transposase and ACP . ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form . ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3' ends of Tn3.

Bioorg Med Chem, 1996 Nov, 4(11), 1809 - 17
PapG adhesin from E . coli J96 recognizes the same saccharide epitope when present on whole bacteria and as isolated protein; Nilsson U et al.; Purified PapG adhesin from the genetically well-defined uropathogenic Escherichia coli strain J96, as well as whole bacteria, were bound to microtiter plates that carried covalently bound globotetraose and galabiose . The binding was inhibited by soluble saccharide derivatives corresponding to the glycolipids, including all di-, tri-, tetra-, and pentasaccharide fragments of the Forssman antigen and all monodeoxy analogues of galabiose . Analysis of the inhibition pattern showed no significant difference between purified adhesin and whole bacteria . The glucose unit at the reducing end of the natural saccharides was detrimental to PapG binding since deletion of the glucose unit increased the inhibitory power 10-20 fold . The five hydroxyl groups HO-6, -2', -3', -4', -6' of the galabiose unit were shown to be important for PapG binding, presumably via intermolecular hydrogen bonds.

Proteins, 1996 Nov, 26(3), 314 - 22
An extended sampling of the configurational space of HPr from E . coli; de Groot BL et al.; Recently, we developed a method (Amadei et al., J . Biomol . Str . Dyn . 13: 615-626; de Groot et al., J . Biomol . Str . Dyn . 13: 741-751, 1996) to obtain an extended sampling of the configurational space of proteins, using an adapted form of molecular dynamics (MD) simulations, based on the essential dynamics (ED) (Amadei et al., Proteins 17:412-425, 1993) method . In the present study, this ED sampling technique is applied to the histidine-containing phosphocarrier protein HPr from Escherichia coli . We find a cluster of conformations that is an order of magnitude larger than that found for a usual MD simulation of comparable length . The structures in this cluster are geometrically and energetically comparable to NMR structures . Moreover, on average, this large cluster satisfies nearly all NMR-derived distance restraints.

Am J Physiol, 1996 Nov, 271(5 Pt 1), G841 - 8
Products of enteropathogenic E . coli inhibit lymphokine production by gastrointestinal lymphocytes; Klapproth JM et al.; Previously we have shown that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated peripheral blood mononuclear cells (PBMCs) . The aim of the present study was to determine whether products of EPEC alter lymphokine expression by gastrointestinal mucosal lymphocytes . Lysates from EPEC clones inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, IL-5, and interferon-gamma (IFN-gamma) but not IL-8 mRNA expression by lamina propria mononuclear cells isolated from surgically resected colon specimens . Inhibitory lysates did not significantly change CD25 expression on either CD4, CD8, or CD45R0 lymphocytes by flow cytometry . Bacterial supernatants of EPEC inhibited IL-2 and IL-5 protein secretion by mitogen-stimulated PBMCs . EPEC lysates inhibited IL-2 mRNA expression induced by lysates of nonpathogenic E . coli . In conclusion, EPEC contains a novel gene(s) that encodes factors that selectively inhibit IL-2, IL-4, IL-5, and IFN-gamma expression by mucosal mononuclear cells without affecting CD25 or IL-8 expression . Thus enteric bacteria can produce factors that may regulate the function of the gastrointestinal mucosal immune system.

Artif Cells Blood Substit Immobil Biotechnol, 1996 Nov, 24(6), 567 - 77
The chemical modification of E . coli L-asparaginase by N,O-carboxymethyl chitosan; Qian G et al.; E . coli L-asparaginase was modified with N,O-carboxymethyl chitosan in the presence of normal product L-aspartic acid, which protected the active site of the enzyme . The modified enzyme remained high catalytic activity, showed greater stability against trypsin and alpha-chymotrypsin, but lost its activity more rapidly at high temperature (> 45 degrees C) than did the native enzyme . When tested in vivo, the plasma half-life of the modified enzyme (t1/2 = 40 hr) was over 33 times longer than that of the native enzyme (t1/2 = 1.6 hr) . The results showed that the modified L-asparaginase may be much more useful than did the native enzyme for clinical treatments of tumors.

J Mol Biol, 1996 Nov 1, 263(3), 411 - 22
ATP hydrolysis stimulates binding and release of single stranded DNA from alternating subunits of the dimeric E . coli Rep helicase: implications for ATP-driven helicase translocation; Bjornson KP et al.; DNA helicases are motor proteins that unwind duplex DNA during DNA replication, recombination and repair in reactions that are coupled to ATP binding and hydrolysis . In the process of unwinding duplex DNA processively, DNA helicases must also translocate along the DNA filament . To probe the mechanism of ATP-driven translocation by the dimeric E . coli Rep helicase along single stranded (ss) DNA, we examined the effects of ATP on the dissociation kinetics of ssDNA from the Rep dimer . Stopped-flow experiments show that the dissociation rate of a fluorescent ss oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ssDNA binding to the other subunit, and that the rate of this ssDNA exchange reaction is further stimulated approximately 60-fold upon ATP hydrolysis . This ssDNA exchange process occurs via an intermediate in which ssDNA is transiently bound to both subunits of the Rep dimer . These results suggest a rolling or subunit switching mechanism for processive ATP-driven translocation of the dimeric Rep helicase along ssDNA . Such a mechanism requires the extreme negative cooperativity for DNA binding to the second subunit of the Rep dimer, which insures that the doubly DNA-ligated Rep (P2S2) dimer is formed only transiently and relaxes back to the singly ligated Rep (P2S) dimer . The fact that other oligomeric DNA helicases share many functional features with the dimeric Rep helicase suggests that similar mechanisms for translocation and DNA unwinding may apply to other dimeric as well as hexameric DNA helicases.

EMBO J, 1996 Nov 1, 15(21), 5976 - 87
The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions; Romby P et al.; Threonyl-tRNA synthetase from Escherichia coli represses the translation of its own mRNA by binding to the operator region located upstream from the ribosome binding site . The operator contains two stemloop structures which interact specifically with the homodimeric enzyme . Here, we provide in vitro and in vivo evidence that these two stem-loop structures are recognized by the enzyme in an analogous way and mimic the anticodon arm of E.coli tRNA(Thr) . Determination of the stoichiometry of the different RNA-threonyl-tRNA synthetase complexes reveals that two tRNA(Thr) molecules bind to the enzyme whereas only one thrS operator interacts with the homodimeric enzyme . A model is presented in which the two anticodon-like domains of the operator bind symmetrically to the two tRNA(Thr) anticodon recognition sites (one per subunit) of the dimeric threonyl-tRNA synthetase . Although symmetrical operator-repressor interactions in transcriptional control are widespread, this report stresses the importance of such interactions in translational regulation of gene expression.

Biochem Biophys Res Commun, 1996 Nov 1, 228(1), 165 - 70
Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E . coli; Raza H et al.; Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form . The protein does not form inclusion bodies and is recovered in the membrane fraction . The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E . coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts . It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E . coli inner membrane . This shows that microsomal glutathione transferase has an inside-out orientation in E . coli spheroplasts as compared to liver microsomes . This fact enables us to make topology experiments that were previously not possible . Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus . Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.

J Bacteriol, 1996 Nov, 178(21), 6374 - 7
Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E . coli K-12; Garcia-Martinez J et al.; Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection . Four phylogenetically informative variable sites were identified . The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported . Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

Ann N Y Acad Sci, 1996 Oct 25, 797, 26 - 31
Enteropathogenic E . coli exploitation of host epithelial cells; Finlay BB et al.; Enteropathogenic E . coli (EPEC) is a leading cause of neonatal diarrhea worldwide . These organisms adhere to the intestinal cell surface, causing rearrangement in the epithelial cell surface and underlying cytoskeleton, resulting in a structure termed an attaching/effacing (A/E) lesion . A/E lesion formation is thought necessary for EPEC-mediated disease . EPEC secretes several proteins that trigger signal transduction, intimate adherence, and cytoskeletal rearrangements in epithelial cells . Additionally, it produces intimin, an outer membrane product that mediates intimate adherence . Together these various bacterial molecules contribute to the intimate relationship that is formed by EPEC with host epithelial cells which results in A/E lesion formation and diarrhea.

Biochim Biophys Acta, 1996 Oct 17, 1297(2), 200 - 6
Overexpression in E . coli of the complete petH gene product from Anabaena: purification and properties of a 49 kDa ferredoxin-NADP+ reductase; Martinez-Julvez M et al.; The complete petH gene product from Anabaena PCC 7119 has been overexpressed in E . coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP+ reductase with its substrates . The intact 49 kDa FNR can be easily purified in a two-step procedure using batch extraction with DEAE-cellulose followed by Cibacron blue-Sepharose chromatography of the proteins unbound to DEAE . Isoelectric focusing of FNR shows several forms, with the major band at pH 6.26 . The presence of the N-terminal extension increases the K(m) of FNR for NADPH by 4-fold and by 16.4-fold in the reduction reactions of DCPIP and cytochrome c . However, the K(m) for ferredoxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR than with the 36 kDa protein . This indicates that the presence of the third domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension . Comparable rate constants for both enzymes, were obtained for the photoreduction of NADP+ using photosynthetic membranes and also using rapid kinetic techniques . Slightly different ionic strength dependences of the rate constants were obtained, nevertheless, for both forms of the enzyme . These are a consequence of the structural differences that the proteins show at the N-terminal and of their effect on the interaction with ferredoxin.

Cell, 1996 Oct 4, 87(1), 127 - 36
Crystal structure of a sigma 70 subunit fragment from E . coli RNA polymerase; Malhotra A et al.; The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E . coli RNA polymerase is described . Residues involved in core RNA polymerase binding lie on one face of the structure . On the opposite face, aligned along one helix, are exposed residues that interact with the -10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting . The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

Vet Microbiol, 1996 Oct, 52(3-4), 249 - 57
Experimental inoculation of foals and pigs with an enterotoxigenic E . coli isolated from a foal; Holland RE et al.; Hemolytic E . coli strain 807-13, O149:NM:K88(STb+, LT+), was isolated from the feces of a neonatal diarrheic foal . E . coli 807-13 was examined for adhesion to brush border membranes (BBM) from foals, adult horses and pigs, and its pathogenicity was assessed in neonatal foals and pigs . E . coli 807-13 did not adhere to equine BBM but adhered to pig BBM . It did not cause diarrhea nor did it colonize the intestinal epithelium of 3 colostrum-deprived and 3 suckled foals challenged at 24 h of age . Acute ulcerative gastritis and acute suppurative gastritis were observed in 2 colostrum-deprived challenged foals, and acute neutrophilic enteritis was observed in 1 colostrum-deprived and in 1 suckled challenged foal . No similar histopathologic lesions were detected in the control foals . Both gnotobiotic and suckled pigs developed diarrhea after challenge exposure to E . coli 807-13 and the intestinal epithelium of the pigs was colonized . Histopathologic evidence of gastritis and enteritis among the foals indicated some complicity of E . coli 807-13 in foal enteric disease.

Curr Opin Genet Dev, 1996 Oct, 6(5), 526 - 30
Activation and repression of E . coli promoters; Gralla JD; There is no organism in which transcription initiation is better understood than Escherichia coli . Recent studies using genetics, biochemistry and structure analysis have revealed how RNA polymerase interactions at promoters are regulated . Prominent examples include the recruitment of polymerase by activators touching its alpha and sigma subunits; which subunit is touched depends on which activator is used and where it binds the DNA . The less-common cases centering on enhancer-dependent transcription use an entirely different mechanism, involving either DNA looping or tracking.

Genomics, 1996 Oct 1, 37(1), 77 - 86
DNA sequence recognition by hybridization to short oligomers: experimental verification of the method on the E . coli genome; Milosavljevic A et al.; A newly developed method for sequence recognition by hybridization to short oligomers is verified for the first time in genome-scale experiments . The experiments involved hybridization of 15,328 randomly selected 2-kb genomic clones of Escherichia coli with 997 short oligomer probes to detect complementary oligomers within the clones . Lists of oligomers detected within individual clones were compiled into a database . The database was then searched using known E . coli sequences as queries . The goal was to recognize the clones that are identical or similar to the query sequences . A total of 76 putative recognitions were tested in two separate but complementary recognition experiments . The results indicate high specificity of recognition . Current and prospective applications of this novel method are discussed.

J Biomol Struct Dyn, 1996 Oct, 14(2), 153 - 61
A geometrical approach in folding a pseudoknot motif within the E . coli 16S-RNA; Tung CS; Except for tRNA, the tertiary structure of RNA molecules are very little known . The many possibilities in the arrangement of different helices in space and the flexibility in the single-stranded loops that connect the helical regions make the modeling of the tertiary structure of RNA molecule a very complex task . Here, we introduce an approach to fold RNA tertiary structure based only on the information of the secondary structure and the stereochemistry of the molecule . This approach was used to construct an atomic structure of a pseudoknot (bases 500-545) in the E . coli 16S RNA . The resulting structure is a closely packed molecule that is consistent with the predicted secondary structure and stereochemically feasible . This new approach is very general and easily adaptable . Experimental data (e.g., NMR, fluorescence energy transfer, etc.), as they become available, can be incorporated directly into the approach to improve the accuracy of the modeled structure.

EMBO J, 1996 Oct 1, 15(19), 5397 - 407
High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand; Guerin M et al.; We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter . The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center . Furthermore, our analyses reveal protein contacts with the non-transcribed strand within the arrested ternary complex . Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non-transcribed strand is an important regulatory element.

EMBO J, 1996 Oct 1, 15(19), 5202 - 8
In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force; Bassilana M et al.; To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB) . MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease . Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+ . Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly . In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation . Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins . These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.

Br J Haematol, 1996 Oct, 95(1), 123 - 6
Changes in coagulation and fibrinolysis in childhood ALL: a two-step dose reduction of one E . coli asparaginase preparation; Nowak-Gottl U et al.; The influence of two different E . coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes . Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL . Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two-step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10,000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring . Compared with Bayer ASP 10,000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and alpha 2-antiplasmin, along with significantly enhanced thrombin generation . Improvement occurred in children treated with 2500 IU/m2 Medac ASP; alpha 2-antiplasmin and D-dimer no longer differed from the Bayer group . Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 268 - 72
Expression of mouse carbonic anhydrase VII in E . coli and demonstration of its CO2 hydrase activity; Lakkis MM et al.; The alpha-carbonic anhydrase (alpha-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X) . Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2 hydrase activity of its product has not as yet been demonstrated . In this study, we have cloned the mouse CA VII cDNA in an E . coli, glutathione-S-transferase (GST) expression vector . The CO2 hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.

Genes Cells, 1996 Sep, 1(9), 819 - 27
Molecular anatomy of the beta' subunit of the E . coli RNA polymerase: identification of regions involved in polymerase assembly; Luo J et al.; BACKGROUND: The E . coli RNA polymerase is a multisubunit enzyme, which is present in two different forms: the catalytic competent core enzyme (alpha2beta beta') and the promoter selective holoenzyme (alpha2beta beta' sigma) . Correct assembly of individual subunits into core or holoenzyme is essential for the function of this enzyme . RESULTS: Mutant beta' proteins truncated near the centre or at the C-terminus were able to form stable core enzyme-like complexes under reconstitution conditions . Mutant beta' proteins lacking the region between amino acids 201-477 failed to form holoenzyme complexes while retaining the ability to form core enzyme complexes . Furthermore, free beta' subunit interacted with free sigma subunit to form a stable beta' sigma subassembly . Removal of amino acids 201-477 from the beta' subunit strongly interfered with this interaction . CONCLUSION: Our results suggest that the N-terminal region of the beta' subunit is involved in the assembly of core enzyme . The region between amino acids 201 and 477 on beta' may be directly or indirectly involved in the interaction between the beta' subunit and the sigma subunit.

J Virol Methods, 1996 Sep, 61(1-2), 47 - 58
Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E . coli and the baculovirus expression systems; Khromykh AA et al.; All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST) . High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E . coli . The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg) . NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells . All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E . coli cultures transformed with the expression plasmid and induced with IPTG . Only delta NS3 protein was recovered intact by removing GST from the fusion protein by digestion with Factor Xa protease . Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient cleavage or in degradation of the proteins . No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-delta NS3, GST-NS4A, and GST-NS4B was eluted off the GS beads by adding glutathione . Thus, KUN purified recombinant proteins, either in eluted form or while immobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions.

Bioessays, 1996 Sep, 18(9), 767 - 72
recA mutants of E . coli K12: a personal turning point; Clark AJ; A first year graduate student, Ann Dee Margulies, changed my research career in 1962 by challenging me to direct her in the isolation of recombination-deficient mutants of Escherichia coli K-12 . She succeeded in isolating two mutants, which conjugated with donor strains and received the donor DNA, but could not recombine that DNA with their own chromosomes . Ann Dee showed that both mutants were much more sensitive to UV radiation than was the wild type . Furthermore, she showed that one of these mutants carried a single mutation affecting both recombination and resistance . This work, published in 1965, was the first demonstration of the recA gene of E . coli . Subsequent work led to the discovery of many more recombination genes, the phenomenon of post replication-recombination repair, the invention of the SOS hypothesis and the discovery of genes encoding proteins with similar primary structure and function in all major groups of organisms . This article honors the memory of Ann Dee.

Infect Immun, 1996 Sep, 64(9), 3694 - 702
Characterization of a novel hemagglutinin of diarrhea-associated Escherichia coli that has characteristics of diffusely adhering E . coli and enteroaggregative E . coli; Yamamoto T et al.; Escherichia coli 73-1 (serotype O73:H33) and 5-2 (serotype O89:H-) isolated from patients with diarrhea adhered to tissue culture cells (HeLa and HEp-2) as well as coverslips (plastic and glass) in a diffuse pattern . Adherence of strain 73-1 was mediated by a 110-kbp plasmid designated pEDA1 and correlated with D-mannose-resistant hemagglutinin (MRHA) detected with bovine, sheep, or human erythrocytes . The MRHA region was duplicated on pEDA1 and mediated the production of the 57-kDa outer membrane protein whose N-terminal amino acid sequence was hydrophobic . In accordance with MRHA and adherence, the 57-kDa outer membrane protein was observed best at 37 degrees C and to a lesser extent at 25 degrees C . In human intestine, adherence to mucus and colonic epithelium was obvious . No detectable pili were observed . The enteroaggregative E . coli heat-stable enterotoxin 1 (EAST1) gene, whose nucleotide sequence was 99.1% homologous to that of enteroaggregative E . coli, was present adjacent to the MRHA region on pEDA1 . Strain 5-2 also exhibited MRHA activities and adherence and had sequences corresponding to those of the MRHA region and EAST1 gene . The data suggest that strain 73-1 (and strain 5-2), which has characteristics of both diffusely adhering E . coli and enteroaggregative E . coli, possesses a novel hemagglutinin associated with diffuse adherence.

Biochem Biophys Res Commun, 1996 Aug 23, 225(3), 705 - 11
Purification and characterization of phospholipase C-beta 1 mutants expressed in E . coli; Meij JT et al.; In the M1-muscarinic receptor-Gq-phospholipase C-beta 1 (PLC-beta 1) pathway, PLC-beta 1 is both the effector regulated by Gq and acts as GTPase activating protein (GAP) for Gq . To rapidly evaluate in vitro PLC-beta 1 mutants constructed by oligonucleotide-directed mutagenesis, we established a quick expression and purification procedure . A pQE60/His6PLC-beta 1 construct was expressed in E . coli SG13009{pREP4} . Purification (approximately 160-fold) was obtained after high-salt extraction and chromatography over Ni(+)-agarose, Mono Q and Mono S columns . Several His6PLC-beta 1 mutants were equally responsive to alpha q . GTP gamma S, although the mutant His6PLC-beta 1-P57 (G758D) had only 2.5% the intrinsic PLC activity of the wild type . Also, His6PLC-beta 1 wild type and mutants acted as GAPs for Gq in a reconstitution assay . Thus, the present procedure provides a method to quickly assess phospholipase activity, alpha q-responsiveness, and GAP activity of PLC-beta 1.

Biochem Biophys Res Commun, 1996 Aug 14, 225(2), 608 - 16
Isolation of cDNA for a novel human protein KNP-I that is homologous to the E . coli SCRP-27A protein from the autoimmune polyglandular disease type I (APECED) region of chromosome 21q22.3; Nagamine K et al.; We have isolated cDNA clones for a novel human protein KNP-I from fetal brain and bone marrow cDNA libraries . Northern blot analysis indicated that the KNP-I gene is ubiquitously expressed in various human tissues . Significant homology of the KNP-I protein with Escherichia coli anti-sigma cross-reacting protein (SCRP-27A) (44% identity) and zebrafish (Brachydanio rerio) esl protein (49% identity) suggested that the KNP-I protein may be involved in a basic cellular function . Genomic sequencing revealed that the KNP-I gene consists of seven exons spanning 12 kb . Exon 5 was involved in alternative splicing . The KNP-I gene was mapped between D21S1460 and D21S25 on human chromosome 21q22.3, 26 kb distal to a Not 1 site of D21S1460 . Thus, this novel KNP-I gene could be a candidate gene for autoimmune polyglandular disease type I (APECED) and other disorders mapped to this region.

Vet Rec, 1996 Aug 10, 139(6), 139 - 41
Efficacy of a commercial competitive exclusion product against a chicken pathogenic Escherichia coli and E coli O157:H7; Hakkinen M et al.; The prevention of caecal colonisation of chicks by a poultry pathogenic Escherichia coli O20:K-:H8 and the human pathogenic E coli O157:H7 was studied in vivo in four one-week laboratory trials . Chicks were treated with a competitive exclusion product on the day of hatch and challenged one day later . The poultry pathogenic serotype showed higher levels of caecal colonisation than the human pathogenic serotype, but the protection against both pathogens was highly significant.

Cell, 1996 Aug 9, 86(3), 503 - 12
Maturation pathways for E . coli tRNA precursors: a random multienzyme process in vivo; Li Z et al.; tRNA maturation consists of the specific removal of precursor sequences from both the 5' and 3' termini of an initial RNA transcript . How this is accomplished has heretofore not been ascertained in any system . Using Northern analysis of RNA isolated from a variety of RNase-deficient E . coli strains, we have identified the processing intermediates that accumulate in the absence of specific processing nucleases . From this information we have established the maturation pathways for 12 different E . coli tRNAs including the specific role of each of the relevant RNases in the process . The surprising conclusion from this work is that tRNA maturation is a stochastic process that lacks a defined order and that can proceed with a variety of alternative 3' processing nucleases.

Cell, 1996 Aug 9, 86(3), 495 - 501
Base-specific recognition of the nontemplate strand of promoter DNA by E . coli RNA polymerase; Roberts CW et al.; RNA polymerase recognizes its promoters through base-specific interaction between defined segments of DNA and the sigma subunit of the enzyme . This interaction leads to separation of base pairs and exposure of the template strand for RNA synthesis . We show that base-specific recognition by the sigma 70 holoenzyme in this process involves primarily nontemplate strand bases in the -10 promoter region . We suggest that melting involves the persistence of these contacts as the bound duplex (closed) form is converted to the single-stranded (open) form of the enzyme-promoter complex.

Cell, 1996 Aug 9, 86(3), 485 - 93
Function of E . coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing; Ring BZ et al.; The sigma factor sigma 70 of E . coli RNA polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage lambda gene Q transcription antiterminator to act . We identify the signal in DNA that induces early pausing to be a version of the sigma 70 -10 promoter consensus, and we show that sigma 70 is both necessary for pausing and present in the paused transcription complex . Regions 2 and 3 of sigma 70 suffice to induce pausing . Since pausing is induced by the nontemplate DNA strand of the open transcription bubble, we conclude that RNA polymerase containing sigma 70 carries out base-specific recognition of the nontemplate strand as single stranded DNA . We suggest that sigma 70 remains bound to core RNA polymerase when the -10 promoter contacts are broken, and then moves to the pause-inducing sequence.

Biokhimiia, 1996 Aug, 61(8), 1483 - 8
{Regulatory properties of RNA polymerase from rifampicin-resistant E . coli strain rpoB403}; Kamzolova SG; Activities of RNA polymerases from wild type B/r strain of E . coli and its pleiotropic rpoB403 rif-r mutant were studied using five various DNA matrices and various groups of early region promotors of T4 and T7 phage DNA . The mutation differentially affects RNA polymerase-catalyzed RNA synthesis depending on the promotor used . Relative efficiency of A3 promotor of T7 phage DNA is enhanced in case of the mutant enzyme . rpoB403 mutation can be used for the classification analysis of promotors differing in their interaction with the enzyme.

Int J Biol Macromol, 1996 Aug, 19(2), 91 - 7
Effects of Mg2+ and denaturants on the unfolding pattern of DNA-T--a replication protein of E . coli; Antony T et al.; Escherichia coli-DNA-T protein is a key component of a multiprotein complex called the primosome which is involved in the initiation of DNA replication . The thermal and urea induced unfolding transition of this protein in the presence and absence of Mg2+ was studied using circular dichroism (CD) and fluorescence spectroscopy as probes . Quenching of the intrinsic fluorescence of DNA-T was observed in the thermal unfolding while formation of a hyperfluorescent form of the protein was found in the urea induced unfolding process . The CD studies showed a monophasic transition curve for thermal unfolding in the presence and absence of Mg2+ . Biphasic curves indicative of the formation of intermediates was observed in the urea induced unfolding . The results suggest that the pathways of unfolding of thermal- and urea-induced transitions are different . MgCl2, which affects the conformation of the protein and stabilises the secondary structure, also affects the unfolding pattern.

Nat Med, 1996 Aug, 2(8), 883 - 7
Microencapsulated genetically engineered live E . coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats; Prakash S et al.; Safety concerns about introducing genetically engineered cells into the body have prevented their use in medical treatments . To solve this problem, we prepared polymeric membrane artificial cells (semipermeable microcapsules) containing genetically engineered live cells from the bacteria Escherichia coli DH5 . When given orally, the cells remain at all times in the microcapsules and are finally excreted in the stool . During their passage through the intestine, small molecules like urea diffuse rapidly into the microcapsules and are acted on by the genetically engineered cells . This lowers the high plasma urea level to normal in uremic rats with induced kidney failure, and has exciting implications for the use of this and many other types of genetically engineered cells in a number of medical applications.

J Biotechnol, 1996 Jul 31, 48(3), 191 - 200
The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E . coli; Corchero JL et al.; The VP1 protein (23 kDa) of the foot-and-mouth disease virus has been produced in MC1061 and BL21 E . coli strains as beta-galactosidase fusion proteins, joined to either the amino and/or the carboxy termini of the bacterial enzyme . In BL21, devoid of La protease, all the recombinant fusion proteins are produced at higher yields than in MC1061, and occur mainly as inclusion bodies . The fusion of VP1 at the carboxy terminus yields a protease-sensitive protein whose degradation releases a stable, enzymatically active polypeptide indistinguishable from the native beta-galactosidase . On the contrary, when the same viral domain is fused to the amino terminus, the resulting chimeric protein is resistant to proteolysis even in the soluble form . These data demonstrate that the position of the heterologous domain in beta-galactosidase fusion proteins would not be irrelevant since it can dramatically influence properties of biotechnological interest such as solubility and proteolytic resistance.

Structure, 1996 Jul 15, 4(7), 861 - 72
The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E . coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr; Liao DI et al.; BACKGROUND: The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) transports exogenous hexose sugars through the membrane and tightly couples transport with phosphoryl transfer from PEP to the sugar via several phosphoprotein intermediates . The phosphate group is first transferred to enzyme I, second to the histidine-containing phosphocarrier protein HPr, and then to one of a number of sugar-specific enzymes II . The structures of several HPrs and enzymes IIA are known . Here we report the structure of the N-terminal half of enzyme I from Escherichia coli (EIN) . RESULTS: The crystal structure of EIN (MW approximately 30 kDa) has been determined and refined at 2.5 A resolution . It has two distinct structural subdomains; one contains four alpha helices arranged as two hairpins in a claw-like conformation . The other consists of a beta sandwich containing a three-stranded antiparallel beta sheet and a four-stranded parallel beta sheet, together with three short alpha helices . Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein . CONCLUSIONS: The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr . It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I . The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.

EMBO J, 1996 Jul 15, 15(14), 3529 - 37
In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA; Koebnik R; The two-domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well-established model for the study of membrane assembly . The N-terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight antiparallel transmembrane beta-strands . A set of 16 gene variants carrying deletions in the membrane-embedded domain of OmpA was constructed . When pairs of these mutant genes were co-expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments . Assembly was found when the polypeptide chain was split at the second or third periplasmic turn . All four protein termini were located in the periplasmic space . Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N-terminus of one fragment and the C-terminus of the other fragment were exposed at the cell surface . This is the first demonstration of correct membrane assembly of split beta-structured membrane proteins . These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.

J Mol Biol, 1996 Jul 12, 260(2), 113 - 9
CytR/cAMP-CRP nucleoprotein formation in E . coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP; Kristensen HH et al.; The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription . Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed . Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP . Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations . Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR . Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.

Bioorg Khim, 1996 Jul, 22(7), 483 - 8
{Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E . coli cells and comparison of their functional activity}; Zargarov AA et al.; A recombinant plasmid was constructed for expressing a gene for bovine recoverin under the control of the lac promoter . Coexpression of the recoverin and N-myristoyl transferase genes was performed to prepare recombinant myristoylated recoverin . The obtained systems provide high levels of biosynthesis of the recombinant recoverins in the E . coli cells . Using a reconstructed system, containing urea-washed rod outer segment membranes, purified rhodopsin kinase (RK), and a recoverin, it was shown that the three recoverin forms (natural, recombinant nonmyristoylated, and recombinant myristoylated ones) perform the calcium-dependent regulation of the activity of RK with half a maximum effect at a free calcium concentration of 2 microM . Interestingly, the N-terminal myristoylation of recoverin increased substantially its functional activity.

Plasmid, 1996 Jul, 36(1), 19 - 25
High expression of plasmid-encoded tetracycline resistance gene in E . coli causes a decrease in membrane-bound ATPase activity; Valenzuela MS et al.; Expression of the plasmid pBR322-encoded tetracycline-resistant gene (tet) is known to cause other pleiotropic effects in addition to mediating the efflux of tetracycline from bacterial host cells . We have recently reported that expression of the tet gene in plasmid pKH47, a high-copy-number derivative of pBR322, causes growth inhibition of Escherichia coli cells harboring this plasmid . In this paper we report that reduced membrane-bound ATPase activity is found in E . coli cells containing plasmid pKH47 . This effect is dependent on the presence of an intact tet gene and reduces the ability of the cells to grow in a minimum medium containing succinate as the sole carbon source . The same effect is more dramatically observed in cells containing an unrelated plasmid in which tet gene expression is under the control of the tac promoter.

J Biochem (Tokyo), 1996 Jul, 120(1), 26 - 8
A preliminary description of the crystal structure of gamma-glutamyltranspeptidase from E . coli K-12; Sakai H et al.; The structure of GGT {EC 2.3.2.2} from E . coli K-12 was studied at 3 A resolution by X-ray crystallography . Initial protein phases were calculated using two kinds of Pb2+ derivatives . The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching . The GGT molecule has overall dimensions of 60 x 50 x 40 A . There are two antiparallel beta-pleated sheets consisting of 6 and 7 beta-strands . The two beta-sheets form a wall-like structure . Twelve short alpha-helices were detected, of which the maximum length appears to be four helix turns.

Bioorg Med Chem, 1996 Jul, 4(7), 1015 - 20
Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E . coli P-protein; Zhang S et al.; Analysis of the active-site residues of a fully functional chorismate mutase representing the N-terminal 113 amino acids of the Escherichia coli P-protein suggests that Lys39 and Gln88 play critical roles in catalyzing the rearrangement of chorismate to prephenate . Five site-directed mutants at these positions have been constructed in which Lys39 was replaced with Arg, Asn, and Gln, and Gln88 was replaced with Arg and Glu . Although the Gln88Arg plasmid failed to produce detectable cross-reacting proteins in E . coli, the other four plasmids were expressed, and the mutant proteins purified to homogeneity . Their structures were similar to wild type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing a small deviation . In accordance with predictions, all mutations result in major loss of catalytic activity at pH 7.8 . However, activity of the Gln88Glu mutant at pH 4.5 exceeded wild-type EcCM . Implications for the mechanism of mutase catalysis are discussed.

Virus Res, 1996 Jul, 43(1), 91 - 6
A 27 amino acid coding region of JE virus E protein expressed in E . coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice; Seif SA et al.; We have recently shown that neutralizing epitope(s) exist near the C-terminal of JE virus E-protein by expressing the coding gene cDNA fragments as fusion proteins with protein A . Among four cDNA fragments, the fragment (B3) carrying the coding sequence of amino acid number 373-399 of E protein elicited the highest neutralizing (N) antibody titer (1:75) . To exclude the possible influence of protein A contained in the expressed gene products on the mouse immune response, we expressed (B3) using pGEX-3X expression vector as fusion with glutathione-S transferase (GST) . The mice immunized with recombinant GST-B3 fusion protein induced an immune response (mean average ELISA: 3364; N: 1:75) almost similar to that by recombinant protein A-B3 fusion protein (mean average ELISA: 3476; N: 1:75) . While hemagglutination-inhibition (HI) antibodies were not induced by this fusion protein . These results indicate that 27 amino acid sequence on the E protein (373-399) was sufficient to induce neutralizing antibodies without association with protein A moiety.

EMBO J, 1996 Jul 1, 15(13), 3477 - 85
Membrane regulation of the chromosomal replication activity of E . coli DnaA requires a discrete site on the protein; Garner J et al.; The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP . Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form . Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes . A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide . The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids . The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids . In contrast, the smaller tryptic fragment was inert to both forms of phospholipids . Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments . The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide . Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

Mol Cell Biochem, 1996 Jun 21, 159(2), 115 - 21
Hepatic response to the oxidative stress induced by E . coli endotoxin: glutathione as an index of the acute phase during the endotoxic shock; Portoles MT et al.; Reactive oxygen species are important mediators of cellular damage during endotoxic shock . In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat . A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase) . The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase . Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process . These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process.

FEBS Lett, 1996 Jun 17, 388(2-3), 217 - 8
The respiration-driven active sodium transport system in E . coli does not function with lithium; Verkhovskaya ML et al.; Comparison of respiration-driven active transport of alkali cations from E . coli cells loaded with Na+ or Li+ showed that Li+ could not be expelled from the cells like Na+ . K+ accumulation, which was fast in Na+-loaded cells, was strongly inhibited in Li+-loaded cells, despite high membrane potential and respiratory rate . When Li+-loaded cells were placed into medium containing Na+ instead of Li+, Li+/Na+ exchange took place initially, while K+ accumulation was delayed . Only after almost all inside Li+ was replaced by Na+ did active Na+ and K+ transport commence . These data confirm that it is a distinct active sodium transport system (AST) with Na+,K+/H+ antiporter activity, and not the Na+/H+ antiporters, that is responsible for active Na+ transport in E . coli {Verkhovskaya, M.L., Verkhovsky, M.I . and Wikstrom, M . (1996) Biochim . Biophys . Acta 1273, 207-216} . In contrast to the Na+/H+ antiporters, the AST system is inhibited by Li+.

J Mol Biol, 1996 Jun 14, 259(3), 422 - 33
Mutational analysis of the joining regions flanking helix P18 in E . coli RNase P RNA; Hardt WD et al.; We have studied variants of Escherichia coli RNase P RNA with base exchanges in the joining regions flanking helix P18, which form part of the ribozyme core structure . Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance in single turnover reactions; (3) structural perturbations utilizing Pb2+ -induced hydrolysis; and (4) in vivo function by complementation analysis in E . coli RNase P mutant strains . Our in vitro experiments revealed that the base moieties of nucleotides (nt) 303 and 331 to 333 neither significantly contribute to tRNA binding or structural stabilization of RNase P RNA nor to active site chemistry . Single base exchanges at nt 300, 301, and 330 reduced tRNA binding, while having little effect on the catalytic rate, which demonstrates that these nucleotides are involved in forming the high affinity (pre-)tRNA binding site . In contrast, point mutations at the strictly conserved positions nt 328, 329, 334 and 335 reduced tRNA binding affinity as well as the catalytic rate, suggesting that these mutations additionally disrupted important interactions in the catalytic center . Probing by Pb2+ revealed that particularly the mutations that affected catalytic function most strongly perturbed a more extended region (nt 248 to 335) known to be involved in tRNA binding . Under high salt conditions (> or = 0.8 M NH4+), catalytic defects of the mutant RNase P RNAs were much less pronounced, suggesting that structural perturbations leading to increased electrostatic repulsion between phosphate groups were the main cause for observed functional defects . Only mutant C334 retained a largely increased pre-steady-state K(m(pss)) under high salt conditions . We conclude that the base at position 334 is directly involved in a contact crucial to pre-tRNA binding . A complementation analysis demonstrated the important role in vivo of the joining regions flanking helix P18 . None of the bases could be mutated without affecting bacterial viability.

Mutat Res, 1996 Jun 12, 363(2), 115 - 23
Induction of E . coli oh8Gua endonuclease by oxidative stress: its significance in aerobic life; Kim HS et al.; The induction of 8-hydroxyguanine (oh8Gua) endonuclease, a DNA repair enzyme for an oxidatively modified guanine, oh8Gua was studied in various growth conditions in Escherichia coli (AB1157) . Anaerobically grown E . coli were found to have a very low activity of this enzyme while aerobically grown cells showed activity about 20 times that of the anaerobic level . Under the same condition, superoxide dismutase (SOD) showed about 6-fold increase in activity . A shift in growth conditions from anaerobic to aerobic resulted in rapid induction of this enzyme, and this induction was blocked (but not completely) by chloramphenicol . It is indicated that molecular oxygen is an effective stimulator to the induction of this enzyme and its induction depends partly on protein synthesis . Superoxide-producing compounds such as paraquat and menadione also increased the activity of endonuclease as well as SOD, but H2O2 showed no effect . Thus, superoxides are also implied as a stimulator . In contrast, hyperoxia induced only SOD not the endonuclease . This induction of the endonuclease by hyperoxia was only observed in a SOD-deficient strain (QC774) . The aerobic activity of the endonuclease in QC774 was the same as that of wild types (AB1157, GC4468) . It is implied that the responsiveness of oh8Gua endonuclease to superoxides is less sensitive than that of SOD . The endonuclease was also induced by a temperature shift from 30 to 43 degrees C and treatment with nalidixic acid . Among the stimuli used, molecular oxygen seems to be most effective for its induction . The inducible nature of this enzyme will serve as an important mechanism for the protection of oxidative DNA damage in the aerobic environment.

Biochem Biophys Res Commun, 1996 Jun 5, 223(1), 118 - 22
Replacement of the sole histidinyl residue in OmpF porin from E . coli by threonine (H21T) does not affect channel structure and function; Saint N et al.; The sole histidine residue in OmpF porin was replaced by threonine using site-directed mutagenesis . This exchange affected neither channel properties nor channel structure, as determined by X-ray analysis to 3.2 A . Conductance and critical voltage (Vc) were observed in the pH range 4.3-9.4, with results indistinguishable from those observed in the wild-type protein . The validity of these observations is supported by the independence of the methods used, and by the fact that mutants in residues located in the channel constriction yielded significantly different values from wild-type protein . The binding of a glycolipid molecule might be affected.

Biochem Genet, 1996 Jun, 34(5-6), 165 - 78
Both an altered DNA structure and cellular proteins are involved in protecting a triplex forming an oligopurine-rich sequence from Dam methylation in E . coli; Klysik J; When the 4-bp Dam recognition sequence was placed between two d(GA)7 tracts, it became severely undermethylated in JM101 Escherichia coli cells compared to other Dam sequences in the same plasmid DNA . This site specific undermethylation was also detected on supercoiled molecules in vitro . Mutational analysis indicated that undermethylation is related to the capacity of the oligopurine tract to adopt the H-DNA conformation . In addition, chemical probing of the cells was consistent with a cellular protein bound to the DNA . Therefore it is likely that the combination of altered DNA conformation and a cellular protein leads to Dam-site protection . We also found that the site-specific undermethylation is detectable in certain E . coli strains only.

Mol Immunol, 1996 Jun, 33(9), 819 - 29
Binding properties and solubility of single-chain T cell receptors expressed in E . coli; Schodin BA et al.; The diversity and domain structure of alpha beta T cell receptors (TCR) are similar to immunoglobulins based on sequence homologies, but the three-dimensional structure of the alpha beta-heterodimer has not been solved . To begin structure/function studies, we have compared the properties of a soluble single-chain V alpha V beta TCR (scTCR) expressed in three E . coli systems . The V alpha and V beta regions were expressed with pelB or ompA signal sequences or as a thioredoxin fusion protein . The scTCRs were detected only in the insoluble fraction of the cells and could be solubilized in guanidine and renatured to obtain properly folded scTCR from each system . Only a small fraction (1-5%) of the ompA and pelB scTCRs folded properly . In contrast, the thioredoxin fusion protein exhibited high total yields and a solubility that was ten times higher than the other scTCRs . The thioredoxin fusion protein also bound specifically to the peptide/MHC ligand with a KD of approximately 0.7 microM, as shown by a competitive inhibition assay with Fab fragments that recognize the MHC complex . Furthermore, estimates from saturation binding with antibodies that react with the native TCR indicated that up to 80% of the thioredoxin fusion protein was in the properly folded form . The improved yield, solubility, and binding activity of the thioredoxin-scTCR should make it useful for various structure/ function studies.

J Appl Physiol, 1996 Jun, 80(6), 2066 - 76
Controlled trials of rG-CSF and CD11b-directed MAb during hyperoxia and E . coli pneumonia in rats; Freeman BD et al.; We studied the effects of inhibiting and augmenting neutrophil function by using an immunocompetent rat model of infectious and hyperoxic lung injury . After intrabronchial Escherichia coli challenge at all fractional inspired O2 (FIO2) values studied (FIO2 = 0.21, 0.60, and 0.95) and after lethal O2 exposure alone (FIO2 = 0.90), lung injury, as measured by histological and physiological changes, was reduced by a CD11b/CD18-directed monoclonal antibody (MAb 1B6, P < 0.05 vs . controls) but was increased by recombinant granulocyte colony-stimulating factor (rG-CSF; P < 0.05 vs . control; MAb 1B6 vs . rG-CSF, P < 0.004) . Pulmonary neutrophil counts were reduced by MAb 1B6 (P < 0.04) and increased by rG-CSF (P < 0.0004) compared with control animals . However, despite antibiotics, MAb 1B6 and rG-CSF both significantly increased the relative risk of death, independent of O2 concentration, during E . coli pneumonia (1.74 {symbol: see text} 1.20 and 2.39 {symbol: see text} 1.19, respectively, each P < 0.01) . During lethal hyperoxia, MAb 1B6 increased the relative risk of death (1.76 {symbol: see text} 1.28, P < 0.16), whereas rG-CSF had no effect on survival (0.97 {symbol: see text} 1.28, P = 0.89) . Thus inhibition of neutrophil function attenuated and enhancement worsened lung injury in response to infectious and hyperoxic challenges, supporting a pathophysiological role of the neutrophil in these processes . However, it is problematic that MAb 1B6 therapy, despite preventing lung damage, ultimately worsened host defenses and survival . Furthermore, rG-CSF also adversely affected survival during infectious lung injury, demonstrating the inherent risks of inhibiting or augmenting neutrophil function in an immunocompetent host during infection.

J Biomol NMR, 1996 Jun, 7(4), 261 - 72
19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E . coli glucose/galactose receptor; Luck LA et al.; 19F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E . coli periplasmic glucose/galactose receptor (GGR) . The protein was derived from E . coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and {2,4,6,7-2H4}-5-fluorotryptophan . As a result of the large gamma-isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored . Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism . In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on {2,4,6,7-2H4}-5-fluorotryptophan . Analysis of the observed 19F powder pattern lineshape resulted in anisotropy and asymmetry parameters of delta sigma = -93.5 ppm and eta = 0.24 . Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S2 of approximately 1, and by correlation times for internal motion approximately 10(-11)s . Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with tau i set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S2 = 0.89 for fluorotryptophan residues 183, 127, 133, and 195 . By contrast, the calculated order parameter for FTrp284, located on the surface of the protein, was 0.77 . Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R1F183 > R1F127 approximately R1F133 approximately R1F195 > R1F284 . Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data . In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule . These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp284, located in the hinge region of the receptor.

Infect Immun, 1996 Jun, 64(6), 2167 - 71
In vivo cytokine response to Escherichia coli alpha-hemolysin determined with genetically engineered hemolytic and nonhemolytic E . coli variants; May AK et al.; Alpha-hemolysin is an Escherichia coli exotoxin that enhances bacterial virulence, has profound effects on leukocytes in vitro, and induces the release of interleukin-1 (IL-1) but not tumor necrosis factor (TNF) from human monocytes in vitro . The purpose of this study was to examine alpha-hemolysin's influence on virulence and TNF and IL-1 production in vivo . Two genetically engineered, isogeneic strains of E . coli were used; one variant produces alpha-hemolysin, and the other does not . Male BALB/c mice were injected with either of the two variants and serum TNF and IL-1 were assayed . These results were compared with those obtained from the injection of either of two serotypes of lipopolysaccharide (LPS) . The nonhemolytic E . coli strain produced no mortality and no significant elevation of serum TNF or IL-1 levels . In contrast, equal inocula of the hemolytic E . coli strain produced significant mortality and elevation of serum IL-1 levels . No significant elevation of TNF levels was detected in this group despite high-level mortality . A pattern of induction of mortality and elevation of serum IL-1 levels without elevation of serum TNF levels is distinct from the pattern typical of LPS . In these experiments, both serotypes of LPS caused elevations of TNF and IL-1 levels whether or not mortality was induced . Thus, alpha-hemolysin produces a cytokine response in vivo that is similar to that previously demonstrated in vitro by Bhakdi et al . (S . Bhakdi, M . Muhly, S . Korom, and G . Schmidt, J . Clin . Invest . 85:1746-1753, 1990) and appears to induce mortality independently of serum TNF.

J Mol Biol, 1996 May 31, 259(1), 7 - 14
The hexameric E . coli DnaB helicase can exist in different Quaternary states; Yu X et al.; The DnaB protein is the primary replicative helicase in Escherichia coli, and the active form of the protein is a hexamer . It has been reported that the protein forms a ring with strong 3-fold symmetry, which was suggested to be a trimer of dimers . We show that under different conditions, using either ATP, ATP gamma S, AMP-PNP or ADP as nucleotide cofactors, we always find two different forms of the DnaB ring; one with a 3-fold symmetry and one with 6-fold symmetry . We have used scanning transmission electron microscopy for mass analysis, and have found that both forms are hexamers, excluding the possibility that the 3-fold form is in fact a trimer of the 52 kDa monomer . We have also found rings that are in an intermediate state between these two . The existence of hexamers in discrete states shows that the transitions between these states must be cooperative . These observations suggest that there may be an equilibrium between two different conformations of the hexameric ring . The role of these two states in the mechanism of helicase action remains to be determined.

Boll Chim Farm, 1996 May, 135(5), 287 - 96
"Hybridization analysis for the quantitative determination of residual DNA in some recombinant proteins expressed in E . Coli cells"; Facchetti I et al.; The recent advances in the area of pharmaceutical recombinant DNA products have led to an impressive increase in the number of clinically used therapeutic proteins, for which stringent purity requirements are established by authorities . Nucleic acids are host cell derived contaminants and their allowed level in the final product is in the low pg range, because of the health risk to the patient . Even if fast non-radioactive methods have been recently developed as an alternative to the traditional hybridization assay, the latter is still widely used being a specific and sensitive method . However hybridization quantitative aspects are only partially reviewed in the literature as well as the procedures utilised to cope with the possible interference of the sample protein on the assay results . These topics are described in the present paper by detailing and comparing the methods set up for three recombinant proteins: human pro-Urokinase (r-h-proUK), human basic fibroblast growth factor (r-h-bFGF) and human granulocyte macrophage colony stimulating factor (r-h-GMCSF).

Biofizika, 1996 May-Jun, 41(3), 642 - 9
{A model of phase modulation of high frequency nucleoid oscillations in reactions of E . coli cells to weak static and low-frequency magnetic fields}; Matronchik AIu et al.; The method of anomalous viscosity time dependencies was used to investigated the influence of extremely low frequency alternating and static magnetic field on the genome conformational state of E . coli K12 AB1157 cells . The physical model was developed on the base of obtained results to describe effects of weak static and alternating magnetic fields on the living cells . In this model the high frequency of oscillations of a charged nucleoid are considered with a classical approach . The wave-like dependence of maximum effect on magnetic induction within the range from 0 to 110 microT was established.

Biochem Mol Biol Int, 1996 May, 39(2), 235 - 42
High-level expression of active human plasminogen activator inhibitor type 1 (PAI-1) in E . coli; Zhou A et al.; Plasminogen activator inhibitor 1 (PAI-1) is an important regulator of plasminogen activation, which inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) . The DNA sequence encoding mature PAI-1 protein was inserted into an inducible expression vector . This gene was highly expressed to produce a soluble active protein in E . coli cells . The amount of the recombinant protein was up to 20% of total cellular protein . By efficient purification with a yield of about 15-20%, the recombinant protein could be purified to homogeneity with its specific activity up to 6.1 x 10(4) (uPA) IU/mg . Its inhibitory activity declined during incubation at 37 degrees C with a half life of about 2 hr.

Biochem Mol Biol Int, 1996 May, 39(1), 171 - 80
Expression, purification and properties of recombinant E . coli ribonuclease III; Srivastava N et al.; Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over-expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography . The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification . The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3{H}poly{A}.poly{U}, and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild-type E . coli cells . The recombinant RNase III showed maximal activity at 37 degrees C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild-type cells . Recombinant RNase III efficiently hydrolyzed 3{H}.poly{A}.poly{U} in the presence of Mg2+ . However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+ . A concentration of Mn2+ ranging from 150 to 300 microM was found to be optimal; concentrations higher than 0.5 mM were inhibitory . Other divalent cations did not support RNase III activity . Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity . The thermal stability of the recombinant RNase III was examined at two temperatures, 37 degrees and 50 degrees C . Incubation of RNase III at 37 degrees C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 degrees C for 30 min . Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild-type RNase III.

Genet Anal, 1996 May, 13(1), 1 - 7
Detection of hammerhead ribozyme-mediated cleavage and reduced expression of LacZ' mRNA in E . coli; Junn E et al.; Hammerhead ribozymes have been shown to specifically suppress the expression of target genes in various cells, but their in vivo cleavage products have seldom been directly detected . A hammerhead ribozyme sequence was designed to cleave the phosphodiester bond just 3' to the GUC of SalI site of M13mp 18 . The ribozyme was inserted some base pairs upstream of the target region without disrupting the reading frame of the lacZ' gene and without introducing any translational stop codons . More than 90% RNAs synthesized in vitro were cleaved at the expected site after 1-h incubation in the presence of 10 mM magnesium ion at 37 and 50 degrees C . Inclusion of the designed ribozyme sequence was also shown to suppress the expression of the fused lacZ' gene in E . coli cells . When the cells were infected by the ribozyme-containing phage, they remained colourless in the presence of X-gal, and the cellular beta-galactosidase activity was reduced by more than 90% . Insertion of the same ribozyme sequence in reverse orientation showed little effect on beta-galactosidase activity . Furthermore, a primer extension by reverse transcriptase revealed a cleavage product that resulted from cleavage of LacZ' mRNA at the targeted site as designed . Thus, our data demonstrated that the designed hammerhead ribozyme cleaves and reduces the expression of a fused LacZ' mRNA in E . coli cells.

EMBO J, 1996 May 1, 15(9), 2313 - 21
Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein; Garner J et al.; The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP . Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form . Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes . A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide . The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids . The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids . In contrast, the smaller tryptic fragment was inert to both forms of phospholipids . Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments . The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide . Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

FEBS Lett, 1996 Apr 29, 385(1-2), 15 - 20
NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E . coli tRNA(Ala); Limmer S et al.; A ribooligonucleotide duplex representing the acceptor stem of E . coli RNA(Ala) with a G3-U70 wobble base pair, which is the main identity element for the recognition by the alanine-tRNA synthetase, has been characterized by 2D-NMR, as having two sequence variants with a regular Watson-Crick G3-C70 and an I3-U70 wobble pair, respectively . As compared to a regular A-RNA, the G-U base pair gives rise to variations of the local helix geometry which are reflected in distinct local chemical shift changes . Structural differences between the duplex possessing an I3-U70 base pair and the wild-type G3-U70 sequence have also been found . The nucleotides in the ubiquitous single-stranded NCCA terminus display a surprisingly high degree of stacking order, especially between A73, C74, and C75.

FEBS Lett, 1996 Apr 29, 385(1-2), 114 - 8
Initial analysis of 750 MHz NMR spectra of selective 15N-G,U labelled E . coli 5S rRNA; Grune M et al.; The overall folding of an RNA molecule is reflected in its base pairing pattern . The identification of that pattern provides a first step towards the determination of the structure of an RNA molecule . We show that the application of heteronuclear NMR methods at 750 MHz to E . coli 5S rRNA (120 nucleotides) selectively labelled with 15N in guanine and uridine allows observation of base pairing patterns for a larger RNA molecule . We also present evidence that the fold of the E-domain of the 5S rRNA (nt 79-97) as a contiguous part of the 5S rRNA and as an isolated molecule is virtually the same.

Biochem Biophys Res Commun, 1996 Apr 25, 221(3), 641 - 6
Identification of promoter in the 5'-flanking region of the E . coli thioredoxin-linked thiol peroxidase gene: evidence for the existence of oxygen-related transcriptional regulatory protein; Kim HK et al.; E . coli thiol peroxidase (Tpx) linked to the thioredoxin as an in vivo thiol regenerating system acts as an antioxidant enzyme removing peroxides and H2O2 . In order to elucidate the mechanism regulating tpx gene expression in E . coli in response to oxygen stress, we made 5' progressive deletions of upstream region from tpx gene, and fused to lacZ gene . LacZ activity was increased 6-fold by oxygen stress and inverted repeat sequence located between -47 and -33 nt was proven to be essential for the oxygen response of tpx promoter . Primer extension experiment and analysis of upstream sequence revealed transcription start point, -10, and -35 regions, which are in good agreements with the consensus sequences recognized by E sigma 70 . Northern hybridization showed that expression of tpx gene is regulated at the transcriptional level . DNA binding assays using inverted repeat sequence including -35 region provides preliminary evidence that expression of tpx requires additional transcriptional factor in response to oxygen stress.

FEBS Lett, 1996 Apr 15, 384(2), 155 - 61
Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E . coli; Huang W et al.; Methods are described for producing an active amino-terminal domain of tissue inhibitor of metalloproteinases-1 (N-TIMP-1) from inactive protein expressed as inclusion bodies in E . coli . Yields exceed 20 mg per litre of bacterial culture . Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis . The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2 and 3 is 6-8-fold less than that of the recombinant full-length protein, indicating that deletion of the C-terminal domain reduces the free energy of interaction by < 10%.

FEBS Lett, 1996 Apr 15, 384(2), 117 - 22
An 11.8 kDa proteolytic fragment of the E . coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity; Stoller G et al.; The 48 kDa trigger factor (TF) of E . coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase) . Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding . The trigger factor was investigated with regard to a domain carrying the catalytic activity . An enzymatically active fragment could be isolated after proteolysis by subtilisin . The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251 . The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32 . After expression in E . coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column . Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln-148 to Thr-249 . This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate . Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506.

Biokhimiia, 1996 Apr, 61(4), 721 - 6
{Participation of DnaK chaperone and ATP in in vivo folding of firefly luciferase, synthesized by E . coli cells}; Leont'eva OV et al.; The time-courses of protein accumulation and luciferase activity of Luciola mingrelica firefly luciferase synthesized on plasmid pJG lambda in E . coli strains omega 238 and B178groE7 (DnaK- and GroEL-deficient, correspondingly) and omega 237 and W3110 that are control to them, have been studied . It was shown that the amounts of the luciferase protein synthesized by all strains was approximately equal to 3.0-3.9% of the total cell protein . Luciferase was synthesized in a catalytically inactive form and transformed into an active enzyme during incubation at 21 degrees C . In the DnaK-deficient E . coli strain omega 238, the enzyme transformation into a catalytically active form did not occur which provides evidence for participation of the DnaK chaperone in transformation of newly synthesized luciferase into catalytically active form . Luciferase folding was accelerated with an increase in ATP concentration inside the cell . It is possible to increase the ATP concentration inside the cell by treatment with polymyxin and addition of ATP to the culture medium, which significantly accelerates the luciferase folding.

Infect Immun, 1996 Apr, 64(4), 1441 - 5
Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene sequences in enterotoxigenic E . coli strains pathogenic for humans; Yamamoto T et al.; The sequence of the enteroaggregative Escherichia coli enterotoxin 1 ( EAST1) gene was present in most (or all) strains of human-colonizing enterotoxigenic E . coli (ETEC) with colonization factor antigen II (CFA/II) or CFA/IV (CS6) . The EAST1 gene was also strongly associated with PCF09+ ETEC or CFA/I+ ETEC elaborating heat-liable enterotoxin (and heat-stable enterotoxin I) . In contrast, CFA/I+ ETEC elaborating heat-stable enterotoxin I, CFA/III+ ETEC, or CS17+ ETEC exhibited very weak or no associated . E . coli from healthy volunteers had no EAST1 gene sequence . A CFA/I+ ETEC strain (H10407) possessed multiple copies of the EAST1 gene on the CFA/I-encoding plasmid and chromosome . In one CFA/II+ ETEC strain, the EAST1 gene was present on the CFA/II-encoding plasmid . The EAST1 gene sequences of the CFA/I+ and CFA/II+ ETEC strains were identical to each other and 99.1% homologous to the reported gene sequence of enteroaggregative E . coli . The data indicate that the EAST1 gene is distributed among ETEC strains with a case of the presence of multiple copies in a single cell and that this distribution is associated with the adherence factor type.

FEBS Lett, 1996 Mar 11, 382(1-2), 171 - 4
Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of alpha subunit of E . coli-F(0)F(1); Hase B et al.; Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E . coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36-->E, alpha-R40-->K) or unrelated amino acids (alpha D-36-->K, alpha R40-->G), respectively, by employing oligonucleotide-directed mutagenesis . ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36-->K), but only slightly affected in the other mutants . The inhibition is not due to a lower content of F0F1 in HS2 . In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed . The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants . After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2 . This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36-->K mutation . In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine beta-R52 . The effect of alpha-D36-->K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the alpha and beta interaction.

FEBS Lett, 1996 Mar 11, 382(1-2), 167 - 70
Succinyl phosphonate inhibits alpha-ketoglutarate oxidative decarboxylation, catalyzed by alpha-ketoglutarate dehydrogenase complexes from E . coli and pigeon breast muscle; Biryukov AI et al.; Effects of a set of alpha-ketoglutarate phosphoanalogues on the activity of alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) complexes from E . coli and pigeon breast muscle, as well as on alpha-ketoglutarate dehydrogenase isolated from the pigeon breast muscle, have been studied . alpha-Ketoglutarate phosphoanalogues (succinyl phosphonate and its monomethyl ester) were found to be effective inhibitors of alpha-ketoglutarate oxidative decarboxylation, catalyzed by both muscle and bacterial alpha-ketoglutarate dehydrogenase complexes, as well as muscle alpha-ketoglutarate dehydrogenase . The ability of glutamate phosphoanalogues to inhibit alpha-ketoglutarate oxidative decarboxylation has been shown in E . coli extract and a model system.

Hua Xi Yi Ke Da Xue Xue Bao, 1996 Mar, 27(1), 10 - 6
{The PCR amplification, cloning, sequencing, expression in E . coli of gene encoding endoflagella subunit protein (fla B) from Leptospira interrogans serovar lai}; Chen Z et al.; A pair of oligonucleotide primers were designed by ourselves to amplify the endoflagella gene of L . interrogans serovar lai . A fragment about 840 bp was generated with PCR and inserted into plasmid pUC8 after the fragment and pUC8 were digested respectively with Bam HI and Pst I . A recombinant plasmid (designated as pLF1) was obtained . SDS-PAGE analysis indicated that a 33 kd was expressed in E . coli JM103 harboring pLF1 and the expression level of the protein was 11% of total bacterial soluble proteins . Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflagella (Axiall filament) of Leptospira interrogans serovar lai . Nucleotide seguence data showed an open reading frame encoding 282 aminoacids residues, corresponding to a protein of molecular weight 33.6 kd . The G + C content of endoflagella subunit protein gene was 48 mol% . Therefore, the G + C content of the leptospiral fla B Gene is significantly higher than the reported 39 mol% G + C content of leptospiral genome of L.interrogans serovar lai but similar to the G + C of the Treponema pallidum genome . Comparison of the deduced endoflagellar subunit protein (fla B) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum fla B proteins . Immunization/protection experiment was performed on the model of BALB/c mice and showed that the survival rate in the group JM103-pLF1 was higher than that in the group JM103-pUC8, but statistically the difference between them was significant (P < 0.05) and pLF1 did not induce significant levels of agglutinating antibodies against L.interrogans serovar lai.

J Clin Microbiol, 1996 Mar, 34(3), 689 - 94
Characteristics of Escherichia coli strains belonging to enteropathogenic E . coli serogroups isolated in Italy from children with diarrhea; Giammanco A et al.; Fifty-five Escherichia coli strains belonging to enteropathogenic E . coli (EPEC) serogroups were examined for phenotypic and genetic factors associated with virulence . The strains were isolated in Italy from children with diarrhea and identified as EPEC by clinical laboratories using commercially available antisera . O:H serotyping showed that 35 strains (27 of O26, O111, and O128 serogroups) belonged to 11 serotypes considered to be classical EPEC O:H serotypes . The other 20 isolates were classified as 15 nonclassical EPEC O:H serotypes . All the potential EPEC virulence factors associated with bacterial adhesion (localized adherence, fluorescentactin staining test positivity, presence of the attaching and effacing {eaeA} gene), the production of verotoxin, and the positivity with the enterohemorrhagic E . coli probe were significantly more frequent among isolates belonging to classical than nonclassical serotypes . Strains displaying an aggregative adhesion and hybridizing with the enteroaggregative DNA probe were found in serogroups O86, O111, and O126 . Verotoxin-producing isolates belonged to serogroups O26, O111, and O128 . Only one of the isolates hybridized with the EPEC adherence factor (EAF) probe, but 33 strains gave positive results with the eae probe, confirming that the former is more suitable in epidemiological studies in European countries . These results indicate that up to 75% of strains identified as EPEC by commercial antisera may possess potential virulence properties and/or belong to classical EPEC O:H serotypes and suggest that O grouping is still a useful diagnostic tool for presumptive identification of diarrheagenic E . coli in clinical laboratories.

Exp Physiol, 1996 Mar, 81(2), 313 - 5
Luminal capsaicin inhibits fluid secretion induced by enterotoxin E . coli STa, but not by carbachol, in vivo in rat small and large intestine; Nzegwu HC et al.; The enterotoxin E . coli STa induced fluid secretion in the rat jejunum, ileum and proximal colon in vivo that was greatly inhibited by the co-presence of luminal capsaicin, which is a specific neural toxin of afferent C fibres . The same dose of capsaicin had no effect on the fluid secretion activated by carbachol in the jejunum, ileum and proximal colon . Afferent C fibres appear to be involved in the activation by STa of fluid secretion in the rat intestine in vivo.

Berl Munch Tierarztl Wochenschr, 1996 Mar, 109(3), 108 - 11
{Management of E . coli dependent factorial diseases in weaning piglets}; Bolcskei A et al.; The most important postweaning factorial diseases are at least partly caused by E . coli . The term postweaning coli complex can be subcategorized into the following manifestations: postweaning diarrhoea, edema disease, postweaning wasting and hemorrhagic gastroenteritis . In the presented study the effect of prophylactic zootechnique alone and zoo- and biotechnique in combination was evaluated during the first weeks postweaning . The results showed that combined zoo- and biotechnique is superior to simple zootechnique regarding food conversion (1.41 kg versus 1.73 kg), average daily weight gain (390 g versus 325 g) and postweaning piglet mortality (3.1% versus 4.9%) . It is the opinion of the authors that combined postweaning zoo- and biotechnique should be performed in such pig production units where ETEC and/or SLTEC are present.

Arterioscler Thromb Vasc Biol, 1996 Mar, 16(3), 392 - 8
Lys and fibrinogen binding of wild-type (Trp72) and mutant (Arg72) human apo(a) kringle IV-10 expressed in E coli and CHO cells; Klezovitch O et al.; In a previous study, we identified a lysine (Lys)-binding-defective form of human lipoprotein(a) and attributed this defect to the presence of a Trp72-->Arg mutation in apolipoprotein(a) {apo(a)} kringle IV-10 . To document this relationship, we expressed both wild-type (wt) and mutant (mut) forms of kringle IV-10 in Escherichia coli (nonglycosylated form) and Chinese hamster ovary (CHO) cells (glycosylated form) . The Arg72 mut was prepared by introducing the T-->A mutation in apo(a) kringle IV-10 amplified from human liver mRNA by the reverse-transcriptase polymerase chain reaction technique . All expressed kringles were tested for their ability to bind Lys and plasmin-modified fibrinogen (PM-fibrinogen) . wt kringle IV-10 expressed in both E coli and CHO cells bound to Lys-Sepharose with comparable affinity . In contrast, the Arg72 mut expressed in both systems exhibited no Lys-binding capacity . Moreover, the wt kringle IV-10 expressed in both systems bound to PM-fibrinogen and exhibited two binding components, one Lys mediated (inhibitable by epsilon-amino-n-caproic acid) and one Lys insensitive, occurring in about the same proportions . Only the latter type of binding was present in the Arg72 mut expressed in E coli . We conclude that kringle IV-10 of human apo(a) has Lys- and PM-fibrinogen-binding capacities that are independent of glycosylation and require the presence of Trp72, one of the seven amino acids that constitute the Lys-binding site of kringle IV-10 . Our results also show that the binding of kringle IV-10 to PM- fibrinogen is more complex than that to Lys, in that the former requires an additional binding site or sites outside the Lys-binding site.

Kansenshogaku Zasshi, 1996 Mar, 70(3), 215 - 23
{Characteristics of enterotoxigenic Escherichia coli and E . coli harboring enteroaggregative E . coli heat-stable enterotoxin-1 (EAST-1) gene isolated from a water-borne outbreak}; Yatsuyanagi J et al.; A water-borne outbreak occurred in A Town in Akita prefecture on March 1995 . Enterotoxigenic Escherichia coli (ETEC) strains were isolated from 6 of 13 feces of patients with food poisoning disease and from 1 of 4 drinking water samples . In addition, E . coli strains harboring Enteroaggregative E . coli (EAggEC) heat-stable enterotoxin-1 (EAST-1) gene were isolated from 5 of 13 patient's feces and 1 feces sample obtained from the septic tank . Both of the E . coli strains were isolated from the 3 patient's feces, suggesting that this outbreak was a mixed infectious case . All of the ETEC strains possessed both heat-stable enterotoxin (ST) and EAST-1 genes and their serotype was O148:H28 . The EAST-1 gene was detected on a ca . 80 kb plasmid by a southern blot analysis using EAST-1 DNA probe in the 5 of 7 ETEC strains . The southern blot analysis suggested that the location of the EAST-1 gene was genome in the rest of the 2 ETEC strains . A southern blot analysis using ST DNA probe also suggested that the location of the ST gene was genome in all of the ETEC strains . On the other hand, all of the 6 E . coli strains harboring EAST-1 gene could not be serotyped with commercially available OH sera . The location of the EAST-1 gene in all of the isolates was suggested to be genome by the southern blot analysis . All of the isolates lacked aggA gene which has been demonstrated to be involved in expression of aggregative adherence phenotype in EAggEC, suggesting that the EAST-1 gene-harboring strains isolated in this case were distinct from EAggEC . These results indicated that the EAST-1 gene was also harbored by E . coli strains distinct from EAggEC . In addition, a possibility was also suggested that the EAST-1 gene might be a transposon, as well as ST gene . Further study should be conducted in order to elucidate the significance of EAST-1 as a vilurence factor of diarrheagenic E . coli.

Int J Radiat Biol, 1996 Mar, 69(3), 345 - 50
Methylene blue sensitizes E . coli cells to X-rays; Teixeira P et al.; Methylene blue (MB) is shown to sensitize E . coli cells to the effects of X-rays . This sensitization is dependent on factors such as dye concentration, incubation temperature, membrane permeability, and repair capacities, suggesting that the binding and/or penetration of the dye into the cells determines the potentiation of the lethal effects of X-rays by MB . It is also demonstrated that the presence of the polymerase 1 enzyme is essential for this sensitization to take place . Since MB is known to penetrate, accumulate, and be retained preferentially in some malignant tissues, the possibility of using this dye as a specific sensitizer to X-rays in the radiotherapy of some cancers is discussed.

Biochem Biophys Res Commun, 1996 Feb 27, 219(3), 876 - 83
E . coli growth inhibition by a high copy number derivative of plasmid pBR322; Valenzuela MS et al.; We have observed that plasmid pKH47, a pBR322-derivative containing a 100bp poly(dA)-poly(dT) insertion, causes growth inhibition of host E . coli cells harboring it . In this paper we show that this inhibitory effect is due to an increased copy number property of this plasmid, which is turn leads to an over expression of the plasmid-encoded tet gene . Our work also indicates that contrary to other pleiotropic effects caused by the tet gene product, which solely depend on the expression of the 5' end of the gene, growth inhibition requires an intact tet gene . In addition we present the isolation of an E . coli mutant that is refractive to the inhibitory effect of pKH47 and shares some properties with the parental bacteria containing plasmid pKH4.

FEBS Lett, 1996 Feb 26, 381(1-2), 161 - 4
High resolution surface structure of E . coli GroES oligomer by atomic force microscopy; Mou J et al.; Using atomic force microscopy (AFM) in aqueous solution, we show that the surface structure of the oligomeric GroES can be obtained up to 10 angstroms resolution . The seven subunits of the heptamer were well resolved without image averaging . The overall dimension of the GroES heptamer was 8.4 +/- 0.4 nm in diameter and 3.0 +/- 0.3 nm high . However, the AFM images further suggest that there is a central protrusion of 0.8 +/- 0.2 nm high and 4.5 +/- 0.4 nm in diameter on one side of GroES which displays a profound seven-fold symmetry . It was found that GroEL could not bind to the adsorbed GroES in the presence of AMP-PNP and Mg2+, suggesting that the side of GroES with the central protrusion faces away from the GroEL lumen, because only one side of GroES was observed under these conditions . Based on the results from both electron and atomic force microscopy, a surface model for the GroES is proposed.

Biochem Biophys Res Commun, 1996 Feb 15, 219(2), 359 - 65
Characterization of bovine endothelial nitric oxide synthase expressed in E . coli; Martasek P et al.; Bovine endothelial constitutive nitric oxide synthase (eNOS) was expressed in E . coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroELS, encoding the chaperonins groEL and groES . The E . coli BL21 cultures reproducibly synthesized 6-10 mg of recombinant enzyme per liter of culture . The eNOS protein was purified using 2'5'-ADP Sepharose 4B and appeared as a single band of apparent molecular mass 135 kDa on SDS/PAGE . The recombinant resting enzyme is predominantly high spin with an absorbance maximum at 406 nm . The dithionite-reduced, CO-bound form shows an absorbance maximum at 444 nm . The spectral properties of recombinant eNOS from E . coli are identical to those observed with eNOS from stably transfected HEK 293 cells or from baculovirus expression systems . Enzymatic activity of eNOS from E . coli ranged between 68-135 nmol product formed/min/mg at 25 degrees C, using hemoglobin-NO capture or L-citrulline formation assays . The enzyme is replete with heme and flavins and both activity and {3H}-nitroarginine binding were largely dependent on tetrahydrobiopterin . The heterologous expression of eNOS offers a number of advantages over tissue sources of the protein.

FEBS Lett, 1996 Feb 5, 379(3), 291 - 4
Topology of the secondary structure elements of ribosomal protein L7/L12 from E . coli in solution; Bocharov EV et al.; Topology of the secondary structure elements of ribosomal protein L7/L12 has been studied . The sequential assignment was obtained for main and side chain resonances . This allows the overall secondary structure to be described . The results of high resolution NMR studies show that dimer of the ribosomal protein L7/L12 from Escherichia coli has a parallel (head-to-head) orientation of subunits, and N-terminal domain (NTD, residues Ser1-Ser33) has no contacts with the C-terminal domain (CTD, residues Lys51-Lys120) . The NMR data for CTD are in line with crystallographic structure . The flexible interdomain (hinge) region (residues Ala34-Glu50) has an unordered structure, the Pro44 forming both cis and trans peptide bonds . Due to the conformational exchange the intensities of the peaks from the NTD are low . The conformation of the NTD, which is responsible for the formation of the L7/L12 dimer, is alpha-helical hairpin . the NTD dimer forms an antiparallel four-alpha-helix bundle.

Biochem Mol Biol Int, 1996 Feb, 38(2), 267 - 73
The X-ray induction of SOS repair activity in E . coli: euoxic vs . anoxic DNA damage; Ewing D et al.; The induction of DNA repair (the "SOS Response") by X rays has been studied in E . coli GE94, irradiated under euoxic versus anoxic conditions . GE94 contains a recA-lacZ gene fusion that allows the radiation-induced transcription of recA to be followed by measuring the changes in beta-galactosidase levels . At doses up to about 450 Gray, more recA transcription (indicating more DNA damage capable of inducing SOS repair activity) occurs in cells irradiated in air instead of 100% nitrogen . Although the presence of dissolved oxygen cannot change the initial number of DNA radicals (initial "energy-deposition events"), apparently more of these radicals become stable lesions when oxygen is present.

Glycoconj J, 1996 Feb, 13(1), 45 - 52
Susceptibility of infant mice to F5 (K99) E . coli infection: differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains; Grange PA et al.; Enterotoxigenic Escherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface . We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant . In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series . We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors . The two murine strains differed in the relative activities of galactosyltransferases (GbOse3Cer and GM1 synthases), N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialytransferases (GM3 and GD3 synthases) . An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility . Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.

Eur J Neurosci, 1996 Feb, 8(2), 424 - 8
Macrophage inflammatory protein-1beta (MIP-1beta) produced endogenously in brain during E . coli fever in rats; Minano FJ et al.; Macrophage inflammatory protein-1 (MIP-1) evokes an intense fever, independent of a prostaglandin mechanism, and is now thought to play an important role in the defence response to bacterial pyrogens . The purpose of this study was 2-fold: (i) to determine whether the potent doublet of this cytokine, MIP-1beta, is actually produced in the brain in response to a pyrogenic dose of a lipopolysaccharide of Escherichia coli and (ii) to determine the anatomical site of synthesis of this cytokine in the brain . Following the intense fever produced by intraperitoneal administration of lipopolysaccharide in the unrestrained rat, MIP-1beta immunoreactivity was identified post mortem in two regions of the brain implicated in fever: the organum vasculosum laminae terminalis (OVLT) and the anterior hypothalamic, preoptic area (AH/POA) . Microinjection of goat anti-mouse MIP-1beta antibody (anti-MIP-1beta) directly int the AH/POA markedly suppressed fever in rats in response to lipopolysaccharide . Further anti-MIP-1beta administered 180 min after the injection of lipopolysaccharide acted as an antipyretic and reversed the fever induced by the endotoxin . anti-MIP-1beta or control immunoglobulin G antibody microinjected into the hypothalamus immediately before the intraperitoneal injection of the control saline did not alter the temperature of the rats . Taken together, the present results demonstrate that MIP-1beta is produced in the brain in response to a bacterial endotoxin . These observations, in the light of earlier data on fever induced by MIP-1beta, further support the hypothesis that endogenously synthesized MIP-1beta acts as an intermediary factor in the evocation of fever by acting on the thermosensitive cells of the brain.

J Mol Biol, 1996 Jan 12, 255(1), 44 - 54
The mechanism of CAP-lac repressor binding cooperativity at the E . coli lactose promoter; Vossen KM et al.; The cyclic AMP receptor protein (CAP) and lactose repressor bind their regulatory sites in the lactose promoter with moderate cooperativity (omega C101 = 11.8(+/- 3.7)) . This cooperativity is significantly reduced by the removal of DNA located upstream of the CAP binding site or by substitution of the dimeric lacI-18 mutant repressor for the wild-type tetrameric protein . These results are consistent with a mechanism of interaction in which CAP bends the DNA and the lac repressor binds simultaneously to its operator site and to promoter-distal sequences . Similar values of omega C101 were obtained with a promoter truncation containing the O3 pseudooperator site and one in which the site is destroyed, suggesting that DNA contacts distal to the O3 site are necessary for cooperative binding.

Biochim Biophys Acta, 1996 Jan 4, 1292(1), 61 - 8
Pressure-induced inactivation of E . coli beta-galactosidase: influence of pH and temperature; Degraeve P et al.; In order to assess the feasibility of a high-pressure immunodesorption process using a beta-galactosidase-anti-beta-galactosidase complex as a model, the influence of high hydrostatic pressure on the activation of E . coli beta-galactosidase has been investigated . The irreversible activity loss of beta-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar) . This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme . The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of phosphate buffer employed for pressurisation . Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of beta-galactosidase was investigated by using a high-pressure stopped-flow device . It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa . An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed . For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using beta-galactosidase as a tag, are discussed.

FEBS Lett, 1996 Jan 2, 378(1), 69 - 73
High-level expression of fully active human glutaredoxin (thioltransferase) in E . coli and characterization of Cys7 to Ser mutant protein; Padilla CA et al.; Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation . To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E . coli BL21 (DE3) by IPTG induction . High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis . The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein . Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage . A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity . The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein . Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins . Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.

Mutat Res, 1996 Jan 2, 362(1), 1 - 8
A novel DNA N-glycosylase activity of E . coli T4 endonuclease V that excises 4,6-diamino-5-formamidopyrimidine from DNA, a UV-radiation- and hydroxyl radical-induced product of adenine; Dizdaroglu M et al.; We report on a novel activity of T4 endonuclease V . This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA . In this work, we show that T4 endonuclease V excises 4,6-diamino-5-formamidopyrimidine from DNA . 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation . DNA substrates were prepared by UV-or gamma-irradiation of DNA in aqueous solution . DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme . After incubation, DNA was precipitated and supernatant fractions were separated . Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry . The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradiated DNA . Kinetics of excision were also determined . Fifteen other pyrimidine- and purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme . It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation- and hydroxyl radical-induced monomeric product in DNA.

J Tongji Med Univ, 1996, 16(2), 70 - 4, 86
Construction of expressing plasmids of recombinant FN polypeptides with bifunctional-domain and the characterization of the products expressed in E . coli; Feng Z et al.; Two expressing plasmids have been constructed and used to express two bifunctional-domain recombinant polypeptides of human fibronectin (FN) in E . coli . One was CH50 (Pro1239-Ser1515 of FN linked with Ala1690-Thr1960 of FN through Met) and the other was CH56 (Pro1239-Thr1960 of FN) . Both of two polypeptides were capable of binding heparin and were purified by heparin-agarose affinity chromatography . The purified products were capable of binding cells . The production of CH50 and CH56 polypeptides provided a fundamental basis for further study of the anti-metastatic function of recombinant fibronectin polypeptides.

Biochem Cell Biol, 1996, 74(2), 295 - 8
The activation of beta-galactosidase (E . coli) by Mg(2+) at lower pH values; Martinez-Bilbao M et al.; The activation of beta-galactosidase (E . coli) by Mg(2+) at pH values below 7.6 was studied . If the Mg(2+) concentration was high enough, the k(cat) values at pH values down to 5.0 remained at the same high level as at pH 7 and 7.6 (600-620 s-(1) with o-nitrophenyl-beta-D-galactopyranoside as the substrate) . Very high concentrations of Mg(2+) (greater than 100 mM at pH 5) were, however, needed to saturate the Mg(2+) site at lower pH values . The Km values at low levels of Mg(2+) were high at every pH but they decreased and approached the same low value at every pH (about 0.13 mM) as the {Mg(2+)} was increased . These data indicate that it is difficult to bind Mg(2+) at lower pH values, but the k(cat) and K(m) values of the enzyme, and therefore the rates of galactosylation (k(2)), degalactosylation (k(3)), and binding (K(s)), do not change substantially as a function of pH provided that a Mg(2+) is bound to the enzyme . The data also showed that Mg(2+) and protons compete for the same site . Analysis by plotting log {Mg(2+)}(mid) vs . pH showed that the binding of Mg(2+) to the free enzyme involves two groups with pK(a) values in the vicinity of 7 and one group with a pK(a) value near 5.5 . (The values referred to as {Mg(2+)}(mid) are the Mg(2+) concentrations that resulted in k(cat) values midway between basal and maximum.) The "apparent" pK(a) values of the groups when a Mg(2+) was bound (at saturating {Mg(2+)}) all appeared to be below 5.0.

Basic Life Sci, 1996, 64, 149 - 74
Structural model of the 50S subunit of E . coli ribosomes from solution scattering; Svergun DI et al.; The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described . The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins . From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety . Based on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety . The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40 A . The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles . The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Chin J Biotechnol, 1996, 12(2), 73 - 9
The cloning, sequence analysis, and expression in E . coli of coat protein gene of grapevine fanleaf virus Gh; Guan H et al.; Fragments of 5' end and 3' end of the coat protein gene of a Chinese strain grapevine fanleaf virus (GFLV-Gh) were separately amplified through RT-PCR . The two products were cloned and ligated into a complete full-length coat protein gene via an inherent BgIII restriction site . The entire gene contains 1512 nucleotides which encode a 504 amino acids coat protein, molecular weight approximately 56 kDa . Comparison with GFLV F-13 shows 88.4% homology in nucleotides and 95.8% similarity in deduced amino acid residues . Also this coat protein gene was expressed in E . coli DH 5 alpha.

Virus Genes, 1996, 13(2), 135 - 42
Expression in E . coli and purification of the active autoprotease P20 of classical swine fever virus; Muyldermans G et al.; The genes for Flaviviridae structural proteins are located at the 5' terminus of the genome, while the 3' terminus contains the genes for the non-structural proteins . The first protein product of the large ORF of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity . Here we report the cloning of the p20/p14 genes behind the strong Trc promotor and expression of the p20 at high levels in E . coli . The autoprotease p20 was responsible for its own release from the nascent polyprotein in E . coli and was further purified by chromatography techniques.

Bioelectromagnetics, 1996, 17(5), 384 - 7
Difference in frequency spectrum of extremely-low-frequency effects on the genome conformational state of AB 1157 and EMG2 E . coli cells; Alipov YD et al.; The effect of weak extremely-low-frequency (ELF) magnetic fields (sinusoidal, 30 microT amplitude) on the genome conformational state (GCS) of E . coli mutant and wild type cells was studied by using the method of anomalous viscosity time dependency (AVTD) in the 6-37 Hz frequency range . We confirmed the existence of three resonance frequencies of 8.9, 15.5, and 29.4 Hz when mutant cells of K12 AB1157 strain were exposed . In the same frequency range, the wild type K12 EMG2 cells displayed only two effective windows, with resonance frequencies of 8.3 and 27 Hz . The resonance frequencies differed significantly (P < .001-.000001) in the strains studied, whereas other resonance parameters did not . It was concluded that mutations in the AB1157 strain resulted in a significant rearrangement in the ELF action spectrum, including the appearance of a new resonance.

Biochimie, 1996, 78(6), 408 - 15
The lacZ mRNA can be stabilised by the T7 late mRNA leader in E coli; Lopez PJ et al.; Previous work from this laboratory has shown that T7 RNA polymerase outpaces ribosomes in vivo, generating naked mRNA stretches which may be nuclease-sensitive . In particular, lacZ transcripts synthesised this way are highly unstable and yield little beta-galactosidase . We have argued that most of these transcripts are prematurely inactivated via an RNase E cleavage that occurs ahead of the leading ribosome, whereas a few escape this initial cleavage and are translated normally . Presumably, these rescued transcripts are later inactivated non-nucleolytically and subsequently scavenged by a process partially controlled by RNase E, as for the natural lacZ mRNA . In contrast, despite being synthesised by T7 RNA polymerase, T7 late transcripts are stable . The 5' regions of several of these transcripts, exemplified by the gene 10 mRNA, harbour hairpin structures which may act as barriers against RNase E action . To test whether these structures are indeed 5' stabilisers, we replaced the lacZ leader sequence by the corresponding region from T7 gene10 . This replacement yielded a ca 2.9-fold increase in beta-galactosidase yield per transcript . This increase vanished in the presence of the rne-50 mutation which inactivates RNase E, and therefore it reflects a protection of the transcript against RNase E-dependent inactivation . Yet, the leader replacement did not stabilise the transcript chemically . We propose that this replacement inhibits the initial cleavage step but somehow facilitates the subsequent scavenging process.

Int J Legal Med, 1996, 109(1), 37 - 41
E . coli endotoxin enhances cardiomyopathy in rats with chronic alcohol consumption; Kita T et al.; The purpose of the study was to show whether it was possible to produce alcoholic cardiomyopathy by short-term alcohol ingestion combined with an infinitesimally low endotoxin injection . Wistar rats were fed an alcoholic liquid diet according to the formula of Lieber and Decarli, and challenged with an injection of E . coli lipopolysaccharide (LPS) endotoxin (1.0 microgram/g body weight per day for ten weeks) . After ten weeks alcohol diet combined with LPS challenge, light microscopical examination showed changes commonly seen in alcoholic cardiomyopathy such as hypertrophy, oedema and disarray of myofibers . By electron microscopy, degeneration of mitochondria and degeneration of myocardial fibers were observed, the latter showing disturbance of the myofibrilla arrangement and interstitial fibrosis . Rats on an alcoholic liquid diet and rats challenged with a single identical doses of LPS did not show characteristic histological findings of alcoholic cardiomyopathy . These results suggest that short-term alcohol ingestion combined with an infinitesimally low endotoxin injection experimentally produces alcoholic cardiomyopathy, and may support the idea that endotoxin plays an important role in the aetiology of alcoholic cardiomyopathy.

Biochimie, 1996, 78(4), 227 - 35
RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli; Cruz AA et al.; In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay . We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough . The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis . The half-life of total cyc mRNA intermediates was increased in the RNase E mutant . A number of degradation intermediates were stabilised, and new species arose . However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation . The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity . This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.

Eur Biophys J, 1996, 24(3), 179 - 84
Resonance assignment and structural analysis of acid denatured E . coli {U-15N}-glutaredoxin 3: use of 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC; Nordstrand K et al.; Virtually complete sequence specific 1H and 15N resonance assignments are presented for acid denatured reduced E . coli glutaredoxin 3 . The sequential resonance assignments of the backbone rely on the combined use of 3D F1-decoupled ROESY-15N-HSQC and 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC using a single uniformly 15N labelled protein sample . The sidechain resonances were assigned from a 3D TOCSY-15N-HSQC and a homonuclear TOCSY spectrum . The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without 13C/15N double labelling . Chemical shifts, 3J(alpha H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation . The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.

Biochimie, 1996, 78(3), 201 - 3
Recognition of the primers containing different modified nucleotide units by the Klenow fragment of DNA polymerase I from E coli; Kolocheva TI et al.; A comparison of Km values and maximal rates of extension (Vmax) for primers containing different modified bases or mismatches, and fully complementary primers of the same length catalyzed by the Klenow fragment of E coli DNA polymerase I was carried out . Base modifications include T-T dimers and apurinic sites . In the case of mismatch, the number of complementary bases from the 3'-terminus to the non-complementary nucleotide determines the efficiency of substrate incorporation, which is a measure of degree of interaction of the enzyme with its primer template . Differently, removal of one base in any position from the 3'-terminus of the primer is equivalent to shortening of the primer by one nucleotide unit, and decreases the affinity to the enzyme by 1.8-fold . Since apurinic sites fail to interfere with the efficiency of DNA synthesis, we suppose that the Klenow fragment of E coli DNA polymerase I does not participate in the correction of DNAs containing apurinic nucleotides units . Finally, the efficiency of elongation of the d(p primer was shown to decrease with an increase in T-T dimers in the primer . When the d(pT)10m primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

Free Radic Biol Med, 1996, 21(2), 133 - 8
Effects of pteridines on chloramine-T-induced growth inhibition in E . coli strains: correlations with molecular structure; Horejsi R et al.; Little is known about the biological significance of most pteridines, despite their ubiquitous occurrence in living cells . Seventeen different pteridines were tested for their ability to modulate the growth inhibitory effect of the disinfectant chloramine-T on three different strains of Escherichia coli bacteria . We found striking differences between the pteridine derivatives: whereas aromatic pterins with a hydroxy function at side chain atom C2' increased the growth inhibition, those with a 7,8-dihydro structure exerted a suppressive effect . These results are in excellent agreement with previously observed effects of pteridine derivatives on chloramine-T-induced luminol-dependent chemiluminescence, and together, are highly suggestive of a general interaction of these compounds with oxygen or chlorine free radicals . This interaction is likely to have biological significance and might offer an explanation for the widespread occurrence of pteridines.

Biosens Bioelectron, 1996, 11(10), 991 - 1000
The development of a new biosensor based on recombinant E . coli for the direct detection of organophosphorus neurotoxins; Rainina EI et al.; A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E . coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents . The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed . This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass . In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor . A pH glass electrode was used as the physical transducer . The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM) . The response times were 10 min for the batch reactors and 20 min for the flow-through systems . It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions . The cryoimmobilized E . coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Trends Genet, 1996 Jan, 12(1), 20 - 6
Exchanging partners: recombination in E . coli; Eggleston AK et al.; Examination of the many proteins involved in recombination in Escherichia coli has provided detailed information concerning how homologous DNA is paired and exchanged between different molecules . Recent studies have begun to resolve long-standing issues, such as how a DNA helicase with rampant nuclease activity is able to promote the initiation of recombination, how the four-stranded intermediate arising from DNA strand exchange is migrated and resolved and how ancillary proteins assist RecA protein-mediated activities . In addition, the identification of eukaryotic homologues of RecA protein, similar both in structure and in function, shows that at least some of the fundamental steps of recombination have been conserved in all organisms . This finding holds promise that the development of in vitro systems for recombination by eukaryotic proteins lies in the not-too-distant future.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1996 Jan, 16(1), 37 - 8
{Experimental study of R plasmid eliminating action of Coptis chinensis on E . coli}; Chen Q et al.; The R plasmid curing experiment was performed in vitro with E . coli strain E . 102 bearing R plasmid as target bacteria and Coptis chinensis as elimination agent . The influence of different time on R plasmid elimination was also observed . Results showed that when the acting time was 24 hours, the cure rate of R plasmid was 2.42% and when the acting time increased to 48 hours, the cure rate elevated to 22.57% . The Missing patterns of R plasmid might be occurred in disappearence of either multiple or single resistance.

Thromb Haemost, 1996 Jan, 75(1), 196 - 202
Primary binding domain of bovine von Willebrand factor fragment expressed in E . coli; Bakhshi MR et al.; Bovine vWF cDNA has been cloned from a bovine endothelial cell library . A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E . coli . The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb . Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF . In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay . The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.

J Steroid Biochem Mol Biol, 1996 Jan, 57(1-2), 43 - 50
Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E . coli in the presence of heat shock proteins shows typical native receptor characteristics; Jaglaguier S et al.; Domain E, considered as the putative hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR) was expressed in Escherichia coli as a fusion protein with either maltose binding protein (MBP) or glutathione S-transferase (GST) . These bacterially-produced MR constructs had no steroid binding activity per se . In fact, heat shock protein association (hsp) is required for high affinity ligand-binding of the MR . After incubation of purified MBP- or GST-HBD with rabbit reticulocyte lysate, known to be rich in heat shock proteins, we obtained saturable binding of {3H}aldosterone . The Kd value for aldosterone was 0.3 nM and the Bmax = 32 pmol/mg . Hormone binding specificity was assessed by competition studies with various steroid ligands . Sucrose gradient assays performed with {3H}aldosterone-MBP-HBD revealed complex sedimenting at 8.3S and 4.9S with {3H}progesterone-MBP-HBD . Western-blot analysis of the sedimentation peak showed the concomitant presence of MBP-HBD by a monoclonal anti-MBP antibody, and hsp90 by a monoclonal anti-hsp antibody . Moreover, following incubation with the anti-rabbit hsp90 monoclonal antibody the sedimenting gradient showed a 10.4S sedimenting complex . These analyses demonstrated that the {3H}aldosterone-MBP-HBD complex is at least associated with hsp90 in reticulocyte lysate and that the HBD of hMR is sufficient to bind hsp90 . Deletions of a relatively short amino- (729-766) or carboxy-terminal (940-984) region of the HBD fragment eliminated all steroid-binding properties . Overall, these results indicate that the integrity of domain E is necessary and sufficient to bind steroid ligands, agonists or antagonists, with characteristics similar to the entire native MR.

Arch Virol, 1996, 141(2), 315 - 29
Nucleotide sequence and expression in E . coli of the complete P4 type VP4 from a G2 serotype human rotavirus; Mahajan NP et al.; The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined . Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic . Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types . To date, expression of complete VP4 in E . colic has not been achieved . In this study we present successful expression in E . coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography . The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it . Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.

Eur J Haematol, 1996 Jan-Feb, 56(1-2), 35 - 8
Inhibition of hypercoagulation by antithrombin substitution in E . coli L-asparaginase-treated children; Nowak-Gottl U et al.; Acquired deficiency of antithrombin (AT), which in some patients could lead to thrombosis, has been a serious side effect of protocols which incorporate E . coli L-asparaginase (ASP) for the treatment of acute lymphoblastic leukaemia (ALL) . In a longitudinal, prospective, non-randomized study children with ALL (n=27) were treated according to the protocol ALL-BFM-90 . During the induction phase using prednisone, vincristine, daunorubicin and ASP, AT substitution was performed in 15/27 patients, when their plasma concentration decreased below 60% of normal with a concomitant increase of D-dimer formation . After the administration of the AT concentrate the patients, plasma concentration of AT increased and remained elevated after 18, 48, and 72 h . In addition, the plasma concentration of enhanced thrombin generation, D-dimer formation and plasminogen activator inhibitor 1 decreased towards normal levels . Although the observed laboratory findings may serve as evidence for a possible clinical benefit of AT substitution during ASP treatment, further randomized studies are requested to evaluate whether the use of prophylactic AT administration could reduce the incidence of thromboembolic events in childhood acute leukaemia.

Biophys Chem, 1996 Jan, 57(2-3), 269 - 78
Conformational changes of E . coli RNA polymerase during transcription initiation; Sen R et al.; Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription . We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process . A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37 degrees C showed that, starting with initial 'closed complex', there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation . The equilibrium from closed complex (RPc) to open complex (RPo) consists of at least the following two intermediate complexes: {formula: see text} Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide . In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RPc, RPi1, RPi2 and RPo, respectively . From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results . Together these results suggest that formations of RPc and RPi1 do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RPil-->RPi2 and RPi2-->RPo . The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies.

DNA Res, 1995 Dec 31, 2(6), 277 - 84
Improved large-scale preparation of phage T4 endonuclease VII overexpressed in E . coli; Golz S et al.; Using PCR, we cloned T4 gene 49, which encodes the endonuclease VII, and the inactive mutant gene 49 amE727 into vector pET-11a . In combination with Escherichia coli host strain BL21 (DE3), this system provided excellent repression of the expression of the highly toxic protein before induction with IPTG . After induction, the proteins were made in high quantities while remaining soluble . Dilution of the crude lysate at 1:10,000 continued to show a highly specific activity in the case of the wild-type enzyme . The protein was purified to homogeneity with a recovery of 33% using two chromatography steps . The yield was 20 times higher and the specific activity 500 times higher than that obtained by using the previously published protocol.

J Mol Biol, 1995 Dec 8, 254(4), 771 - 92
Molecular dynamics simulation of E . coli ribonuclease H1 in solution: correlation with NMR and X-ray data and insights into biological function; Philippopoulos M et al.; A 500 ps molecular dynamics simulation of Escherichia coli RNase H1 in the presence of explicit water molecules has been carried out to aid in the interpretation of NMR N-H backbone model free parameters and X-ray B-factor values of the free enzyme . Both experimental techniques have revealed unusual structural and dynamic features of the protein . Atomic fluctuations (B-factors) and re-orientational motions of the backbone heteronuclear bonds (order parameters) computed from the simulation are compared with results obtained from experiments . Qualitative agreement is obtained between the computed and X-ray B-factors, whereas the agreement between the computed and NMR generalized order parameters is as good as quantitative for most residues . Reasons for significant discrepancies, the physical basis and the plausible biological consequences of the observed protein dynamics are discussed.

Acta Biotheor, 1995 Dec, 43(4), 285 - 97
Control of threonine pathway in E . coli . Application to biotechnologies; Rais B et al.; Threonine is an essential amino acid for mammals and birds and an adequate supply is necessary for growth and maintenance . Its production has become the aim of metabolic bioengineering and genetic manipulations . We propose in this paper a rational approach for increasing threonine production in an E . coli strain based on metabolic control theory . We have derived a way to measure the control coefficients of threonine pathway in vivo . The method consists in modelling the results of presteady-state experiments . The in vivo concentrations and activities of the enzymes can then be measured and introduced into the model, so that the in vivo steady-state of the pathway can be evaluated . With such a model it is possible to calculate the theoretical values of the control coefficients of the threonine synthesis flux in vivo.

EMBO J, 1995 Dec 1, 14(23), 6058 - 65
Cadaverine induces closing of E . coli porins; delaVega AL et al.; We have used the electrophysiological technique of patch-clamp to study the modulation of Escherichia coli porins by cadaverine . Porin channels typically have a very high probability to be open, and were not known to be inhibited by specific compounds until the present study . Experiments performed on patches of outer membrane reconstituted in liposomes reveal that cadaverine applied to the periplasmic side increases the frequency of channel closures in a concentration-dependent fashion, and thereby decreases the total amount of ion flux through a porin-containing membrane . The positive charge on cadaverine is important for inhibition, because the effect is relieved at higher pH where fewer polyamine molecules are charged . Modulation is observed only at negative pipet voltages, and therefore confers voltage dependence to porin activity . Cadaverine increases the number and duration of cooperative closures of more than one channel, suggesting that it does not merely block the pore but exerts its kinetic effect allosterically . As a biological assay of porin inhibition, E . coli behavior in chemotaxis swarm plates was tested and found to be impaired in the presence of cadaverine . Polyamines are naturally found associated with the outer membrane of E.coli, but are lost upon fractionation . We postulate that cadaverine might be a natural regulator of porin activity.

Biochem Mol Biol Int, 1995 Dec, 37(6), 1127 - 35
Use of UV spectroscopy for the study of nucleic acid cleavage by E . coli RNase H and restriction endonucleases; Petrauskene OV et al.; A one-step spectrophotometric method for monitoring of nucleic acid cleavage by ribonuclease H from E . coli and type II restriction endonucleases has been proposed . It is based on recording of the increase in the UV absorbance at 260 nm during the course of enzymatic reaction . Duplexes stable under the reaction conditions were chosen as substrates for the enzymes being studied . In order to obtain duplex dissociation following their cleavage by the enzyme appreciate temperature conditions were selected . The spectrophotometric method may be applied for rapid testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzymes . This method may be successfully employed in kinetic studies of nucleic acid-protein interactions.

Glycobiology, 1995 Dec, 5(8), 775 - 82
alpha-galactosyl (Galalpha1-3Galbeta1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate alpha1,3galactosyltransferase expressed in E.coli; Galili U et al.; Developing methods for in vitro synthesis of the carbohydrate structure Galalpha1-3Galbeta1-4GlcNAc-R (termed the alpha-galactosyl epitope) on human tumour cells may be of potential clinical significance in cancer immunotherapy . Tumour vaccines with this epitope would be opsonized in vivo by the natural anti-Gal antibody, which is present in large amounts in humans, and which interacts specifically with alpha-galactosyl epitopes . Binding of anti-Gal to alpha-galactosyl epitopes on tumour cell membranes is likely to increase uptake of the cell membranes by antigen-presenting cells, such as macrophages, via the adhesion of the Fc portion of anti-Gal to Fc receptors on these cells . This, in turn, may increase processing and presentation of tumour-associated antigens by antigen-presenting cells, and induce an effective immune response against tumour cells with these antigens . The present study describes a method for the synthesis of alpha-galactosyl epitopes on human cells (red cells used as a model) by recombinant alpha1,3galactosyltransferase (rec . alpha1,3GT) expressed in bacteria . Escherichia coli was transformed with cDNA of the luminal portion of New World monkey rec . alpha1,3GT linked to six histidines (His)6 at the N-terminus . The enzyme produced by the bacteria was isolated from bacterial lysates on a nickel-Sepharose column and eluted with imidazole . This recombinant enzyme displayed acceptor specificity similar to that of rec . alpha1,3GT produced in COS cells . Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine acceptors, then subjected to rec . alpha1,3GT activity . This enzyme synthesized at least 4 x 10(4) alpha-galactosyl epitopes/red cell . These epitopes were found to be accessible for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4 lectin . It is argued that the method presented can be used for the synthesis of alpha-galactosyl epitopes on membranes of autologous tumour vaccines in humans.

Indian J Biochem Biophys, 1995 Dec, 32(6), 343 - 50
Identification of the elongation factor Tu binding site on 70S E . coli ribosomes by chemical crosslinking; Nag B et al.; Elongation factor Tu (EF-Tu), in the presence of Phe-tRNA, GMPPCP, and Poly (U), binds to 70S ribosomes at the recognition (R) site . In order to identify the ribosomal proteins adjacent to the EF-Tu occupying the R site, EF-Tu:Phe-tRNA:GMPPCP:ribosome complexes were crosslinked by modification with 2-iminothiolane and mild oxidation to form disulfide bridges between neighbouring proteins whose endogenous or introduced SH groups were appropriately located . The binding of Phe-tRNA to the ribosome was shown to be largely dependent on the presence of Poly(U) . The total protein from the complexes was extracted and separated by two-dimensional gel electrophoresis by non-equilibrium pH gradient electrophoresis (NEpHGE) in the first dimension, followed by gradient SDS gel electrophoresis in the second dimension . Comparison of control samples crosslinked without Poly(U) to those crosslinked with Poly(U) present showed a single crosslinked complex in the region of the gel near EF-Tu . No cross-links in the vicinity of EF-Tu were visible in the absence of Poly(U) . The crosslinked proteins in this region were recovered by electroelution, radiolabeled and their identity was confirmed by 2D gel electrophoresis and immunoblot analyses . Two major 50S ribosomal proteins, L7/L12 and L10 were found to be covalently linked to EF-Tu . The isolated crosslinked complex did not contain any protein from the 30S subunit . These results demonstrate that L7/L12 and L10 are the major, if not only, ribosomal protein cross-links to EF-Tu in the R site . In contrast to previous crosslinking results obtained by others, our results define a unique location for the EF-Tu binding site, one compatible with functional data and near that of the EF-G binding site on the ribosome.

Mol Biotechnol, 1995 Dec, 4(3), 239 - 45
Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments in E . coli; Molloy PE et al.; This work describes protocols for the production of single-chain antibody and T-cell receptor fragments in E . coli . A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography . The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.

FEBS Lett, 1995 Nov 27, 376(1-2), 67 - 73
Expression of an archaeal chaperonin in E . coli: formation of homo- (alpha, beta) and hetero-oligomeric (alpha+beta) thermosome complexes; Waldmann T et al.; Co-expression of the two genes encoding the alpha- and beta-subunit of the Thermoplasma acidophilum thermosome in Escherichia coli yielded fully assembled hetero-oligomeric complexes (alpha+beta) . Surprisingly, also separate expression of both genes resulted in formation of hexadecameric complexes (alpha, beta) in the bacterial cytoplasm . On electron micrographs these complexes were indistinguishable from each other and from the native thermosome . The recombinant alpha-complex as well as the native thermosome could be reconstituted in vitro from their dissociated subunits in the presence of Mg-ATP.

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1013 - 7
Expression of fungal Mn peroxidase in E . coli and refolding to yield active enzyme; Whitwam RE et al.; The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was expressed in Escherichia coli . The portion of the cDNA encoding the enzyme's signal peptide, not found in the processed holoenzyme, was deleted from the cDNA . The polypeptide was produced as inactive inclusion bodies that could be solubilized in 8 M urea and the reducing agent dithiothreitol . Reconstitution of activity was accomplished by diluting the urea concentration to 2M in the presence of hemin, calcium, and oxidized glutathione . All of the additives were required for recovery of activity . The activity of the recombinant enzyme was dependent on both Mn2+ and H2O2.

EMBO J, 1995 Nov 15, 14(22), 5494 - 505
Early events in preprotein recognition in E . coli: interaction of SRP and trigger factor with nascent polypeptides; Valent QA et al.; In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins . In this study we use cross-linking to demonstrate that E . coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins . Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo . A second E . coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins . We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 648 - 54
Mutation of the carboxy terminal zinc finger of E . coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity; Zhou L et al.; Escherichia coli isoleucyl-tRNA synthetase has been shown to contain two enzyme-bound zinc atoms per polypeptide chain . To investigate the structural and functional significance of the C-terminal enzyme-bound zinc, mutagenesis was used to alter Cys 922 to Ser {IleRS(C922S)} and to replace Cys 922 through Ala 939 with a 33 amino acid peptide unable to bind zinc (AIleRS) . Both IleRS(C922S) and AIleRS were found to contain only a single enzyme-bound zinc per polypeptide chain . Substitution of Co2+ for Zn2+ in IleRS(C922S) gave a visible spectrum characteristic of that expected for a single tetrahedrally coordinated enzyme-bound Co2+ atom . Little or no effect on the Km values for ATP or Ile and only a 5 fold reduction of the (kcat/Km)Ile was observed for IleRS(C922S) and AIleRS in the isoleucine-dependent ATP-pyrophosphate exchange reaction . In the tRNA-dependent aminoacylation reaction, Km values for tRNA(Ile) were only slightly affected in the mutant proteins . However, kcat/Km values were decreased approximately 200 and 2500 fold for IleRS(C922S) and AIleRS, respectively . These results suggest that both the C-terminal enzyme-bound zinc and the C-terminal peptide play important roles in aminoacylation of tRNA(Ile).

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 549 - 53
The directed evolution of radiation resistance in E . coli; Ewing D; E . coli AB1157 (a wild-type, K-12 strain having no known defects in DNA repair capability) was irradiated daily with a very large X-ray dose to develop a series of strains unusually resistant to both X rays and ultraviolet (UV) photons . An understanding of how wild-type strains mutate and become more resistant should lead to a better understanding of DNA repair processes and their effects on radiation sensitivity.

J Theor Biol, 1995 Nov 7, 177(1), 29 - 43
A mechanism for induction of the SOS response in E . coli: insights into the regulation of reversible protein polymerization in vivo; Kuzminov A; During normal DNA replication, RecA, the principal recombinational repair enzyme of E . coli, cannot assemble its filament on SSB-bound single-stranded DNA at the replication forks . This behavior is paralleled in vitro, where at low Mg2+ concentrations RecA can not polymerize on SSB-bound single-stranded DNA . Inhibition of DNA replication in vivo renders RecA able to polymerize on SSB-bound single-stranded DNA and to activate the SOS response . Although the mechanism of SOS induction is still obscure, abundant in vitro observations indicate that RecA filament formation on SSB-bound single-stranded DNA is facilitated at elevated concentrations of ATP, Mg2+ and spermidine . It is proposed here that inhibition of DNA synthesis in vivo leads to a similar accumulation of ATP and its counter-ions, Mg2+ and spermidine, resulting ultimately in SOS induction . When DNA synthesis is restored, the concentration of ATP, Mg2+ and spermidine returns to normal levels, favoring RecA depolymerization . On the basis of the known structure of RecA, a mechanism for reversible RecA polymerization is presented . In a RecA polymer, the monomers are known to interact with each other primarily through hydrophobic, oppositely charged surfaces . In conditions suboptimal for polymerization, these hydrophobic surfaces of the monomers are possibly masked by electrostatic interactions with other, oppositely charged domains of the monomers . There are known recombinational repair proteins whose specific functions are likely to assist in RecA polymerization or depolymerization . Features of reversible polymerization of eukaryotic proteins tubulin and actin are consistent with the possibility that RecA exploits a general principle for the regulation of reversible protein polymerization.

Cell, 1995 Nov 3, 83(3), 365 - 73
Three-dimensional structure of E . coli core RNA polymerase: promoter binding and elongation conformations of the enzyme; Polyakov A et al.; The structure of E . coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals . The structure reveals extensive conformational changes when compared with the previously determined E . coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure . While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E . coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E . coli core and yeast RNAPII form part of a ring that surrounds the channel . This may define promoter-binding and elongation conformations of RNAP, as E . coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E . coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

Biochem Biophys Res Commun, 1995 Nov 2, 216(1), 406 - 13
Recombinant human pancreatic ribonuclease produced in E . coli: importance of the amino-terminal sequence; Futami J et al.; Human pancreatic ribonuclease 1 (hRNase 1) in the mature form has been produced in E . coli using T7 expression system . The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns . The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity . The activity was perfectly inhibited by human placental RNase inhibitor . Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra . The present study has revealed the important contribution of the amino-terminal sequence of hRNase 1 to the characteristics of the protein.

Sci China B, 1995 Nov, 38(11), 1321 - 31
Cloning, expression and purification of the ligand-binding region of human IL-6R in E . coli and its preliminary functional identification; Duan J et al.; The ligand-binding region of human IL-6R is taken as the target gene fragment to be cloned and expressed . With pET-3b as expressing vector, two recombinants pET-6R(B) and pET-6R(B)4 have been constructed encoding the ligand-binding region (28 kD) of hIL-6R and its dimmer (53 kD), respectively . After induction with IPTG, they produced two proteins rIL6R-28 of 28 kD and rIL6R-53 of 53 kD amounting to 50% and 30% of total bacteria proteins, respectively . The expressed products were mainly recovered as inclusion bodies . After purification and renaturation, both of them were capable of augmenting the growth-stimulating effect of IL-6 on 7TD1 cells, an IL-6 dependent cell line . The result of ELISA also revealed that both rIL6R-28 and rIL6R-53 had the obvious ligand-binding activity.

Biochem Mol Biol Int, 1995 Nov, 37(5), 991 - 1000
E . coli D-glyceraldehyde-3-phosphate dehydrogenase modified by 2,3-butanedione: manifestation of a pairwise of non-equivalence of active centers; Levashov PA et al.; Chemical modification of E . coli d-glyceraldehyde-3-phosphate dehydrogenase by an arginine-specific reagent, 2,3-butanedione, stabilized the tetrametric enzyme in an asymmetric state, with only two of the four active centers able to catalyze oxidative phosphorylation of D-glyceraldehyde-3-phosphate . The catalytically incompetent active centers retain the capacity of binding NAD+, forming charge transfer complex, and be alkylated by iodoacetamide . Analogous results have been previously obtained with the rabbit muscle D-glyceraldehyde dehydrogenase modified at a single arginine residue per subunit (Kuzminskaya, E.V., Asryants, R.A., and Nagradova, N.K . (1991) Biochim . Biophys . Acta 1075, 123-130), the only differences being inaccessibility of the catalytically incompetent pair of active centers to the alkylating reagent, on one hand, and lower residual activity exhibited by the functioning active centers (3-4%), on the other . In the case of E . coli enzyme, activity loss upon arginine modification never exceeded 80-82% . These results are consistent with the idea that the two enzymes share common principles of the protein design, but differ in the peculiarities of their active centers conformations . An improved method for D-glyceraldehyde-3-phosphate dehydrogenase purification from a wild type E . coli strain is described.

Microbiol Res, 1995 Nov, 150(4), 429 - 36
Colonization factors of enterotoxigenic E . coli (ETEC) from residents of northern Egypt; Oyofo BA et al.; Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem to children in developing countries . Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA) . The importance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs induced protective immunity against diarrhea in animal models with preformed antibodies . In humans, ETEC containing CFA/I, II, III and IV have been identified . The aim of this study was to define CFAs of ETEC isolated in Alexandria, Egypt . One hundred and seven ETEC isolates from 132 human residents in Alexandria, Egypt were isolated during a birth cohort study . ETEC isolates were screened for heat labile (LT) and heat stable (ST) toxins using a 32P oligonucleotide hybridization probe and a GM1 ELISA . These isolates were examined using monoclonal antibodies against CFA/I, II, III, IV, and against the putative colonization antigens PCF0159 and PCF0166, CS 7 and CS 17 . CFAs were found in 48% of ETEC strains . CFA/I was found in 18% of the strains, CFA/II in 10% and CFA/IV in 14% . CFA III was not found . All fifteen strains expressing CFA/IV expressed CS6 and produced ST . CFA/IV was not found in non-ST producing strains, while CFA/I was absent in ST-only producing strains.

Appl Environ Microbiol, 1995 Nov, 61(11), 4124 - 7
Responses to toxicants of an Escherichia coli strain carrying a uspA'::lux genetic fusion and an E . coli strain carrying a grpE'::lux fusion are similar; Van Dyk TK et al.; A transcriptional fusion of the Escherichia coli uspA promoter to luxCDABE was characterized and compared with a heat shock-responsive grpE'::lux fusion . Similarities in range and rank order of inducing conditions were observed; however, the magnitude of induction was typically greater for the grpE'::lux fusion strain.

FEBS Lett, 1995 Oct 23, 374(1), 95 - 9
Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E . coli by p-alkoxyphenols; Potsch S et al.; The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E . coli, mouse, and herpes simplex virus (HSV-2) by a series of p-alkoxyphenols with different alkyl chains, have been studied by stopped-flow UV-vis and stopped-flow EPR spectroscopy . The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe2+ . p-Alkoxyphenols reduce the mouse R2 tyrosyl radical 1-2 orders of magnitude faster than the HSV-2 and E . coli radical . In contrast to E . coli, the iron center in R2 from mouse and HSV-2 is reduced by the inhibitors . For mouse R2, the rate of reduction of the tyrosyl radical increases in parallel with increasing alkyl chain length of the inhibitor, an observation which may be important for the design of new antiproliferative drugs.

FEBS Lett, 1995 Oct 23, 374(1), 25 - 8
Cloning, expression and characterization of human thioltransferase (glutaredoxin) in E . coli; Chrestensen CA et al.; PCR primers were designed from the known amino acid (aa) sequence for human red blood cell thioltransferase (hRBC TTase) and the known cDNA sequence for pig liver TTase (82% homologous) and used to amplify thioltransferase from a pool of human brain cDNAs . The PCR product was inserted into the pKK233-2 expression vector . The DNA sequence of the insert agreed with the aa sequence . High level expression of the enzyme was accomplished in E . coli, and Western blot analysis confirmed its identity . Recombinant TTase displayed catalytic properties indistinguishable from natural hRBC TTase.

J Mol Biol, 1995 Oct 20, 253(2), 277 - 90
Stabilised secondary structure at a ribosomal binding site enhances translational repression in E . coli; Brunel C et al.; The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase is negatively autoregulated at the translational level . The negative feedback is due to the binding of the synthetase to an operator site on its own mRNA located upstream of the initiation codon . The present work describes the characterisation of operator mutants that have the rare property of enhancing repression . These mutations cause (1) a low basal level of expression, (2) a temperature-dependent expression, and (3) an increased capacity of the synthetase to repress its own expression at low temperature . Surprisingly, this enhancement of repression is not explained by an increase of affinity of the mutant operators for the enzyme but by the formation, at low temperature, of a few supplementary base-pairs between the ribosomal binding site and a normally single-stranded domain of the operator . Although this additional base-pairing only slightly inhibits ribosome binding in the absence of repressor, simple thermodynamic considerations indicate that this is sufficient to increase repression . This increase is explained by the competition between the ribosome and repressor for overlapping regions of the mRNA . When the ribosomal binding site is base-paired, the ribosome cannot bind while the repressor can, giving the repressor the advantage in the competition . Thus, the existence of an open versus base-paired equilibrium in a ribosomal binding site of a translational operator amplifies the magnitude of control . This molecular amplification device might be an essential component of translational control considering the low free repressor/ribosome ratio of the low affinity of translational repressors for their target operators.

J Mol Biol, 1995 Oct 20, 253(2), 259 - 65
Heterogeneity of E . coli RNA polymerase revealed by high pressure; Erijman L et al.; The activity and subunit association of Escherichia coli RNA polymerase has been investigated by high pressure techniques (up to 2000 atm) . The extent of subunit dissociation in the presence and absence of DNA was monitored by carrying out electrophoresis directly at elevated pressure . The degree of inactivation brought about by high pressure was determined by measuring the enzyme activity following decompression . The loss of activity if the enzyme molecules are not actively involved in transcription is correlated with the extent of association of the polymerase subunits . At any particular pressure only a fraction of polymerase molecules becomes inactivated; the remaining fraction retains its original activity characteristics . If the enzyme molecules are actively involved in transcription when the high pressure is applied, the RNA synthesis can be completely halted, but the elongation activity is fully recovered on decompression . The experimental results are consistent with the existence of a broad distribution of protein conformers that are differentially sensitive to the level of pressure . RNA polymerase molecules that display similar catalytic properties have large differences in their free energy of subunit association in the absence of substrates.

Mol Membr Biol, 1995 Oct-Dec, 12(4), 349 - 53
A quantitative assay to determine the amount of signal peptidase I in E . coli and the orientation of membrane vesicles; van Klompenburg W et al.; The number of Signal Peptidase I (SPasel) molecules per E . coli cell was determined using western blot techniques . Different strains were found to contain approximately 1000 SPasel molecules per cell during exponential growth . Based on the activity of SPasel in vitro it could be estimated that this amount is sufficient to process all translocated precursors . SPasel did not appear to be under growth phase dependent control, but was constitutively expressed . The quantitative western blot technique was also used to establish the orientation and intactness of isolated inner membrane vesicles.

Wei Sheng Wu Xue Bao, 1995 Oct, 35(5), 390 - 3
{Cloning of a cluster of genes encoding coli-surface antigen 6(CS6) of human enterotoxigenic E . coli}; Yang X et al.; A gene library was constructed from large plasmid DNA of wild strain E519/66A of human enterotoxigenic Escherichia coli (ETEC) . Positive colonies containing CS6 antigen were obtained . The CS6 gene fragment was identified by restriction endonuclease mapping . It was approximately 4.6 kb in length and responsible for encoding CS6 genes and regulating the CS6 antigen . Two forms of fimbriae protein with different molecular weight were produced by the selected clones . Both of them could react with the same CS6 antiserum . So we expect to use the recombinant strain as candidate for human ETEC vaccine development, and it is also useful for further research on the expression and regulation of genes encoding CS6 fimbriae protein.

Biol Pharm Bull, 1995 Oct, 18(10), 1328 - 34
The binding specificity and affinity of E . coli integration host factor (IHF) are influenced by the flexibility of flanking regions of its recognition sites; Shindo H et al.; To understand the roles of the 5'-flanking region of the recognition sites in binding specificity and the affinity of integration host factor (IHF), a variety of DNA fragments with a 13-bp consensus sequence, 5'-WATCAAN4TTR-3'{Friedman, Cell, 55, 545 (1988)}, but with different 5'-flanking sequences were investigated by gel retardation and methylation interference assays . It has been well-established that the putative A/T rich element distal from the 5'-end of the consensus made a significant contributions to the binding of IHF . However, many of the DNA fragments used here revealed specific binding to IHF without such an A/T element . Several bases neighboring to the 5'-end of the consensus sequence had significant effects on the binding specificity as well as its affinity, and these results indicate that the sequence-directed bendability of the flanking region plays an important role in the specific recognition by IHF.

Sci China B, 1995 Oct, 38(10), 1202 - 9
Expression of human interleukin-11 cDNA in E . coli; Miao J et al.; A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signal polypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vector pEx31B of E . coli . The authors identified the recombinant plasmid, designated pEx31-IL11, by restriction endonucleases digestion and DNA sequencing . The resulting recombinant plasmids were then used to transform E . coli strain HB101, and expression in the PL promoter system, which is temperature-regulated, was achieved . The expressed fusion protein amounts to 50% of total bacterial proteins . The hIL-11 protein expressed in E . coli was fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body . These recombinant proteins can be purified to about 80% by extracting inclusion body with urea . One IL-6-dependent cell line 7 TD1 was used for bioassay . The recombinant hIL-11 protein was preliminarily purified and renatured to a specific activity of 10(5)U/mg, even in the presence of an excess of a neutralizing anti-IL-6 antibody.

FEBS Lett, 1995 Sep 25, 372(2-3), 253 - 8
The functioning of the SRP receptor FtsY in protein-targeting in E . coli is correlated with its ability to bind and hydrolyse GTP; Kusters R et al.; In this study, we have established that FtsY, the E . coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity . GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change . FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP . Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY . These observations suggest that in E . coli GTP is important for proper functioning of FtsY in protein-targeting.

FEBS Lett, 1995 Sep 25, 372(2-3), 199 - 202
Molecular cloning and functional expression in E . coli of a novel plant enzyme mediating zeta-carotene desaturation; Albrecht M et al.; We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of zeta-carotene and neurosporene to lycopene when expressed in E . coli cells accumulating zeta-carotene or neurosporene . This enzyme is unable to dehydrogenate either phytoene or lycopene . The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca . 59 kDa and which is synthesized as a precursor with a NH2-terminal extension resembling transit peptides for plastid targeting . Sequence comparison reveals 33-35% similarity with previously cloned plant or cyanobacterial phytoene desaturases . In contrast, only limited sequence similarity is found with a zeta-carotene desaturase from the cyanobacterium Anabaena.

Cell, 1995 Sep 22, 82(6), 927 - 36
E . coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration; Slater S et al.; The seqA gene negatively modulates replication initiation at the E . coli origin, oriC . seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle . Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication . SeqA protein purification and DNA binding are described . SeqA interacts with fully methylated oriC strongly and specifically . This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation . SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly . Also, binding of hemimethylated oriC by membrane fractions is due to SeqA . Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

Sci China B, 1995 Sep, 38(9), 1084 - 93
High expression of synthetic human interferon-gamma cDNA in E . coli; Wang Z et al.; Human interferon-gamma (IFN-gamma) cDNA was synthesized, and it makes the usage of favorable codons in E . coli . The authors got 9 different expression plasmids which contain the synthetic IFN-gamma-cDNA and have different spaces between SD sequence and ATG . The free energies G0f298 in the formation of stable secondary structure in the translation initiation region (TIR) are different in various expression plasmids . One of them, pLY4-gamma 5, can highly yield INF-gamma which will be about 60%-80% of the total bacterial proteins, such a high expression was hardly noted in literature . The reasons of high expression in this work are optimal spaces between SD and ATC, favorable delta G0f298, favourable condons usage for E . coli.

Public Health, 1995 Sep, 109(5), 381 - 8
A widespread community outbreak of E coli O157 infection in Scotland; Davis BS et al.; Between the end of March and the end of May 1994, 22 cases of laboratory-confirmed infection by E coli O157, phage type 4, verotoxin type 1 & 2, were reported to the Scottish Centre for Infection and Environmental Health . Although cases were distributed throughout six health board areas, pulsed field gel electrophoresis indicated that the causative organisms were clonal or very closely related . The eight earliest cases had a connection with a chain of butchers' shops although no organisms were isolated from food or surfaces . A case-control study of 9 cases and 27 controls showed a statistically significant association between consumption of burgers and illness, leading to a statement from the Chief Medical Officer emphasising the importance of thorough cooking of burgers and other meat . This outbreak highlights the importance of a unified approach to epidemiological investigation when several administrative areas are involved.

Gene, 1995 Aug 30, 162(1), 161 - 2
The sigma-70 subunit from Escherichia coli C differs from that of E . coli K-12; Christie GE et al.; The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (sigma 70) subunit of RNA polymerase) from Escherichia coli C was determined . This gene differs from that of E . coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a sigma 70 subunit with three amino acid (aa) changes and a deletion of ten acidic aa.

Biochem Pharmacol, 1995 Aug 25, 50(5), 609 - 18
Bioactivation of dinitrobenzamide mustards by an E . coli B nitroreductase; Anlezark GM et al.; A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g . 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives . In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine . This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA . In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases . Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E . coli enzyme and cofactor (NADH) . Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme . None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells . For those compounds which were substrates for the E . coli nitroreductase, there was a positive correlation between kcat and IC50 ratio . Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively) . An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1 . This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

Biochim Biophys Acta, 1995 Aug 17, 1245(1), 29 - 36
Internalization of E . coli ST mediated by guanylyl cyclase C in T84 human colon carcinoma cells; Urbanski R et al.; Internalization of Escherichia coli heat-stable enterotoxin (ST) mediated by guanylyl cyclase C was examined in T84 human colon carcinoma cells . Surface-associated, receptor-bound ST was quantitatively separated from intracellular ligand employing acidic guanidine-HCl . ST was internalized in a time-, temperature-, and ligand concentration-dependent fashion only by cells specifically expressing guanylyl cyclase C . Only receptors which bound reversibly to ST appeared to mediate endocytosis . The rate of internalization of ST empirically determined in these studies was 0.23 min-1 . The density of surface receptors for ST was similar at 4 degrees C and 37 degrees C, suggesting that these receptors recycle back to the cell surface following internalization of ligand . Similarly, internalized ST was rapidly cleared from the intracellular compartment following endocytosis . These studies demonstrate that ST undergoes ligand-dependent receptor-mediated endocytosis in human colon carcinoma cells.

J Mol Biol, 1995 Aug 4, 251(1), 30 - 40
Analysis of E . coli rho factor: mutations affecting secondary-site interactions; Pereira S et al.; To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1 . They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively . Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235) . Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein . We found both KE352 and GV99 to be defective in secondary-site RNA activation, with Km values for r(C)10 of 100 microM and approximately 650 microM, respectively, compared to the wild-type value of 4 microM . These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro . By contrast, PH235 was very efficient at interacting with r(C)10 at the secondary site, with a measured Km of 0.5 microM, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities . Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination.

Nature, 1995 Aug 3, 376(6539), 441 - 4
A model of protein synthesis based on cryo-electron microscopy of the E . coli ribosome; Frank J et al.; The ribosome is formed by assembly of proteins and nucleic acids, and synthesizes proteins according to genetic instructions in all organisms . Many of the biochemical steps of this fundamental process are known, but a detailed understanding requires a well-defined structural model of the ribosome . Electron microscopy combined with image reconstruction of two-dimensional crystals or single ribosomes has been the most promising technique, but the resolution of the resulting models has been insufficient . Here we report a 25-A reconstruction of the ribosome from Escherichia coli, obtained by combining 4,300 projections of ice-embedded single particles . Our new reconstruction reveals a channel in the small ribosomal subunit and a bifurcating tunnel in the large subunit which may constitute pathways for the incoming message and the nascent polypeptide chain, respectively . Based on these new findings, a three-dimensional model of the basic framework of protein synthesis is presented.

Immunobiology, 1995 Aug, 193(5), 400 - 19
Expression of monovalent fragments derived from a human IgM autoantibody in E . coli . The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity; Jahn S et al.; A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia . The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes . To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E . coli . In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1 . fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody . The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody . Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM . We discuss the expression of recombinant scFv fragments in E . coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.

Bioessays, 1995 Aug, 17(8), 733 - 41
Instability of inhibited replication forks in E . coli; Kuzminov A; Inhibiting the progress of replication forks in E . coli makes them susceptible to breakage . Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway . These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E . coli chromosome in rnh mutants . Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication . A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E . coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.

J Mol Biol, 1995 Jul 28, 250(5), 617 - 26
Membrane assembly of circularly permuted variants of the E . coli outer membrane protein OmpA; Koebnik R et al.; The two-domain, 325 residue outer membrane protein OmpA is one of the most abundant proteins of Escherichia coli, playing a role in the maintenance of the integrity of the cell surface . The N-terminal domain, consisting of about 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight amphipathic transmembrane strands . Pairs of these proposed transmembrane strands were permuted at the DNA level, in order to dissect the process of membrane assembly . All three possible circular permutations led to variants, which were, in comparison with the wild-type protein, less efficiently assembled . In contrast, no membrane assembly could be detected in any of 18 non-circularly permuted variants . We take this as an indication that the "right" (wild-type) order of beta-strands is a necessary and sufficient prerequisite for at least partially successful membrane assembly . This may be the consequence of packing constraints and/or a failure to adopt the wild-type arrangement of beta-strands, which require crossing of the periplasmic turns.

EMBO J, 1995 Jul 17, 14(14), 3572 - 84
Structure of the dsRNA binding domain of E . coli RNase III; Kharrat A et al.; The double-stranded RNA binding domain (dsRBD) is a approximately 70 residue motif found in a variety of modular proteins exhibiting diverse functions, yet always in association with dsRNA . We report here the structure of the dsRBD from RNase III, an enzyme present in most, perhaps all, living cells . It is involved in processing transcripts, such as rRNA precursors, by cleavage at short hairpin sequences . The RNase III protein consists of two modules, a approximately 150 residue N-terminal catalytic domain and a approximately 70 residue C-terminal recognition module, homologous with other dsRBDs . The structure of the dsRBD expressed in Escherichia coli has been investigated by homonuclear NMR techniques and solved with the aid of a novel calculation strategy . It was found to have an alpha-beta-beta-beta-alpha topology in which a three-stranded anti-parallel beta-sheet packs on one side against the two helices . Examination of 44 aligned dsRBD sequences reveals several conserved, positively charged residues . These residues map to the N-terminus of the second helix and a nearby loop, leading to a model for the possible contacts between the domain and dsRNA.

J Antibiot (Tokyo), 1995 Jul, 48(7), 635 - 7
Direct transfer of plasmid DNA between Streptomyces spp . and E . coli by electroduction; Vujaklija D et al.; We describe a simple and reliable method which allows the direct transfer of shuttle plasmids between Streptomyces spp . and E . coli . The method is based on the fact that plasmid DNA molecules can be released or taken up from cells under conditions of electroporation . When a suspension of a plasmid-containing Streptomyces spp . is mixed with electroporation-competent E . coli and submitted to an electric pulse, plasmid DNA transfer to the E . coli recipient takes place . Two different Streptomyces spp . (S . lividans TK23, or TK24 and S . rimosus 554w) were effective donors, and the method was successfully employed to transfer four different bifunctional vectors (pPM801, pFD666, pRL270X and pZG5) varying in size from 5.2-14.4 kb, to E . coli . This provides a convenient method for the analysis of Streptomycete transformants.

Plant Cell Physiol, 1995 Jul, 36(5), 849 - 56
Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors (ARFs) and characterization of the binding of nucleotides by its product after expression in E . coli; Kiyosue T et al.; A cDNA clone, designated DcArf1, was isolated from a cDNA library prepared from embryogenic cell clusters of Daucus carota and characterized . The cDNA (883 bp) contained an open reading frame that encoded a protein of 181 amino acid residues with significant homology to the ADP-ribosylation factors (ARFs) of animals and yeast . The DcArf1-related transcripts were detected at a nearly constant level during somatic embryogenesis . The DcArf1-encoded protein was expressed as a fusion protein in E . coli and was shown to have dithiothreitol-independent {alpha-32P}GTP-binding activity . This binding was completely prevented by a 100-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding . The binding of {35S}GTP gamma S to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl2 and dimyristoylphosphatidylcholine (DMPC) . The amino acid sequence and the GTP-binding characteristics of the DCARF1 protein suggest that the DcArf1 cDNA encodes an ARF of carrot rather than a protein that only resembles such a factor, ADP-ribosylation factor-like protein (ARL).

FEBS Lett, 1995 Jun 26, 367(2), 183 - 7
Atomic structure at 2.5 A resolution of uridine phosphorylase from E . coli as refined in the monoclinic crystal lattice; Morgunova EYu et al.; Uridine phosphorylase from E . coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form . The structure was determined by the molecular replacement method at 2.5 A resolution . The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R-factor of 18.6% . The amino acid fold of the protein was found to be the same as that in the trigonal crystals . The positions of flexible regions were refined . The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.

Biochem Pharmacol, 1995 Jun 16, 49(12), 1759 - 67
Purification of recombinant human N-acetyltransferase type 1 (NAT1) expressed in E . coli and characterization of its potential role in folate metabolism; Ward A et al.; Human arylamine N-acetyltransferase type 1 (NAT1) has been cloned from human genomic DNA, into the vector pET(5a) and expressed in Escherichia coli . The recombinant protein has been purified to apparent homogeneity using anion exchange chromatography . The arylamine acceptor specificity, and the effect of potential NAT1 inhibitors has been investigated using purified recombinant protein . The Km of the recombinant NAT1 protein for the substrates para-aminobenzoate (p-aba) and 4-aminosalicylate are 14.3 and 11.8 microM, respectively . Folate and amethopterin were found to be potent competitive inhibitors of p-aba acetylation, with Ki values of 13.3 and 9.5 microM, respectively . The pteroate moiety of folate, in contrast is a poor inhibitor, with 100 microM pteroate inhibiting only 40% of NAT1 activity . A catabolite of folate para-aminobenzoly-L-glutamate has also been shown to be a NAT1 substrate with a Km value of 263 microM.

Acta Biotheor, 1995 Jun, 43(1-2), 143 - 53
{Control of the metabolic pathway of threonine in E coli . Application of biotechnology}; Rais B et al.; This paper deals with the application of the metabolic control theory, especially the measurement of control coefficients, to the threonine pathway in E . coli . The control coefficient of a step on a metabolic flux quantitatively assesses the flux response to the step variations . This concept is particularly relevant both in pathological situations (decrease in the activity of an enzymatic step in the metabolism) and in biotechnologies, where, on the contrary steps are amplified . Measurement of the control coefficients of the steps of a metabolic network makes it possible to know those whose amplification should lead to a simultaneous increase in the fluxes . We have applied these concepts to threonine biosynthesis from aspartate in E . coli . The threonine pathway starting from aspartate involves five steps catalyzed by five enzyme activities: aspartokinase (AK), aspartate-semialdehyde-dehydrogenase (ASA-DH), homoserine dehydrogenase (HDH), homoserine kinase (HK) and hreonine synthetase activity (TS) . Measurement of the control coefficient of the first step (AK, insensitive to threonine inhibition in the studied strain) has shown that it controls threonine production weakly . Our study has revealed a hitherto unknown inhibition of homoserine kinase activity by lysine . Mathematical modeling of this metabolic pathway has been undertaken to better understand our experimental results.

Nat Genet, 1995 Jun, 10(2), 213 - 8
Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E . coli; Kang S et al.; Several human hereditary neurological and neurodegenerative disease genes are associated with the expansion of CTG repeats . Here we show that the frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication . Large expansions occur predominantly when the CTGs are in the leading strand template rather than the lagging strand . However, deletions are more prominent when the CTGs are in the opposite orientation . Most deletions generated products of defined size classes . Strand slippage coupled with non-classical DNA structures may account for these observations and relate to expansion-deletion mechanisms in eukaryotic chromosomes for disease genes.

J Steroid Biochem Mol Biol, 1995 Jun, 53(1-6), 583 - 94
Receptor mediated genomic action of the 1,25(OH)2D3 hormone: expression of the human vitamin D receptor in E . coli; Hsieh JC et al.; The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitamin D-responsive element (VDRE) . VDREs in such positively controlled genes as osteocalcin, osteopontin, beta 3 integrin and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides . The present studies of VDR/VDRE interaction utilized full-length human vitamin D receptor (hVDR) that was overexpressed in E . coli, purified to near homogeneity (> 95%), and its authenticity confirmed by demonstrating high affinity hormone binding and reactivity to monoclonal antibody 9A7 gamma . The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-responsive element (VDRE) in the rat osteocalcin gene . Similar overexpression in E . coli of the DNA binding domain (delta 134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity . Both full-length and delta 134 hVDRs retain similar DNA binding specificities when tested with several natural hormone responsive elements, indicating that the N-terminal zinc finger region determines hVDR-DNA sequence selectivity . The C-terminal region of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via heterodimerization between hVDR and RXR . A natural ligand for the RXR co-receptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodimers.

Mutat Res, 1995 Jun, 347(1), 13 - 5
Starvation-associated mutation in E . coli strains with and without reverse transcriptase; Bridges BA; Starvation-associated mutation to Tyr+ in E . coli WU3610 is identical to that in an isogenic derivative lacking reverse transcriptase encoded by a retron phage.

FEBS Lett, 1995 May 15, 364(3), 255 - 8
A novel activity of E . coli uracil DNA N-glycosylase excision of isodialuric acid (5,6-dihydroxyuracil), a major product of oxidative DNA damage, from DNA; Zastawny TH et al.; We describe a novel activity of E . coli uracil DNA N-glycosylase (UNG) that excises isodialuric acid from DNA . Isodialuric acid is formed in DNA as a major oxidative product of cytosine . DNA substrates, which were prepared by gamma-irradiation, were incubated with UNG . Following precipitation of DNA, analyses of pellets and supernatant fractions by gas chromatography/mass spectrometry showed an efficient excision of isodialuric acid from DNA by UNG . None of the other 15 identified DNA base lesions was excised . The excision of isodialuric acid indicates that the non-aromaticity of a substrate may not be a limiting factor for UNG.

Cell, 1995 May 5, 81(3), 331 - 40
Comparison of recombination in vitro and in E . coli cells: measure of the effective concentration of DNA in vivo; Hildebrandt ER et al.; Despite the extremely high concentration of DNA in nucleoid/nuclear regions, chromosomal dimerization and entanglement are avoided . To help understand this, we measured the effective concentration of DNA in E . coli, a value that reflects the functional impact of the cellular milieu on DNA site reactivity . We used as probes plasmid fusion reactions by two site-specific recombinases . The normalized extents and rates of fusion in these systems were much lower in vivo than in analogous in vitro reactions . We calculate that the effective concentration of plasmid DNA is about one order of magnitude lower than the chemical concentration . We suggest that in bacterial cells DNA accessibility is highly restricted and that this dominates the forces that increase DNA activity, such as macromolecular crowding.

Indian Pediatr, 1995 May, 32(5), 539 - 42
Contamination of weaning foods and transmission of E . coli in causation of infantile diarrhea in low income group in Chandigarh; Ghuliani A et al.; Samples of weaning foods and other sources of contamination, such as water, mother's nails, utensils and swab samples of feeding bottle nipple, mother's teats and child's hands were collected from a total of 100 houses of Low Income Group (LIG) in Chandigarh . A high incidence of E . coli isolation (72.3%) was noticed amongst the collected samples . Seventy nine per cent of storage containers of water exhibited the presence of E . coli . Eighty per cent of the children had diarrhea even when exclusively breastfed . Sixty six per cent children were weaned within 3-6 months; the ratio increasing with increase in the educational qualification of the mother . Eighty out of the total 100 households which had a history of infantile diarrhea exhibited 80.9% E . coli isolationPIP: In Chandigarh, India, samples of weaning food, water, utensils, and mother's nails and swab samples from the bottle nipple, mother's teats, and infant's hands were collected from randomly selected houses where the household income was less than Rs . 2000/month . Researchers aimed to determine sources of contamination of child's weaning food and the various modes of transmission of Escherichia coli into the infant's body and these modes' roles in causing infantile diarrhea . 99% of the infants were breast fed at birth; 68% at age 1 . 66% of the infants were introduced to weaning foods at age 3-6 months, 25% at age 6-12 months, and 2% at age 12-18 months . E . coli was isolated in 72.4% of all samples collected . It was isolated in 79% of containers used to store drinking water . The most common method of feeding was finger feeding (67%) . E . coli was isolated from 80.9% of the households where the infants had a history of diarrhea (p .05) . It was also isolated in 38.5% of households where the infants had no history of diarrhea . Only 2 of the 100 households sampled had neither history of infantile diarrhea nor E . coli isolation . E . coli was isolated from 91.5% of the mother's teats . The E . coli isolation rate for weaning food, water, mother's nails, utensils, feeding bottle, and child's hands was 56%, 79%, 79%, 66%, 71.8%, and 88.6%, respectively . 80% of infants had diarrhea even during exclusive breast feeding . 44% of mothers did not reheat weaning foods before serving those food to their baby if the foods had already been cooked . Only 10% boiled it for 5-7 minutes before feeding it to the infant . These findings show several opportunities for cross-contamination from one source to the other, indicative of poor personal hygiene . They also suggest that knowledge about hygiene and sanitation are low among low income mothers .

Cytokine, 1995 May, 7(4), 331 - 7
Induction of metalloproteinase activity, cartilage matrix degradation and inhibition of endochondral mineralization in vitro by E . coli lipopolysaccharide is mediated by interleukin 1 alpha; Scheller M et al.; Chronic inflammation and degradation of connective tissue in the course of periodontitis are maintained by bacterial products such as lipopolysaccharides (LPS), which probably act via inflammation mediators, e.g . cytokines . We investigated the effects of lipopolysaccharide (LPS) from E . coli and mouse recombinant interleukin 1 alpha (mrIL-1) on chondrogenesis, endochondral mineralization, matrix metalloproteinase activation and matrix degradation in vitro using cartilage organoid cultures . Mesenchymal cells of limb buds from mouse embryos (day 12) were grown at high density on a membrane filter at the medium/air interphase for 14 days . Chondrogenesis occurred during the first 6 days of culture . Endochondral mineralization took place upon addition of 5 mM beta-glycerophosphate from day 7 to 14 . Treatment of the cultures with LPS and mrIL-1 on days 2 to 14 and during mineralization on days 7 to 14 resulted in a marked decrease of types I and II collagen, matrix mineralization and proteoglycan content . In the medium, proteoglycan content and metalloproteinase activity were enhanced . LPS induced IL-1 alpha production and release into the medium . LPS antagonist polymyxin B partly abolished the LPS effect, whereas IL-1 receptor antagonist (IL-1ra) partly abolished both LPS and mrIL-1 effects . Reversal of LPS-induced effects by IL-1ra was comparable to the reversal of mrIL-1 effects, only the decrease in type II collagen after LPS treatment was abolished to a lesser extent by IL-1ra.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Anaesthesiol Scand, 1995 May, 39(4), 472 - 8
Effects of arterial hypoxia and beta-adrenoceptor blockade on cerebral blood flow and oxygen uptake following E . coli endotoxin in dogs; Westerlind A et al.; Earlier studies in normoxia have shown that an endotoxin injection in dogs leads to an increase in cerebral metabolic rate of oxygen (CMRo2), a decrease in cerebral blood flow (CBF) and increased concentrations of monoamines in blood and cerebrospinal fluid (CSF) . In animals pretreated with propranolol (PPL) the CMRo2 increase was abolished and thus beta-adrenoceptor mediated . Arterial hypoxia normally increases CBF without any influence on CMRo2 . The aim of this study was to investigate the effects of moderate arterial hypoxia on CBF, CMRo2 and catecholamine concentrations in blood and CSF after endotoxin with and without pretreatment with PPL . Three groups of dogs were studied . Group 1: Six animals were subjected to arterial hypoxia without any other intervention . Group 2: Six animals were given an endotoxin injection (E . coli lipopolysaccharide O 111: B 4), before the induction of hypoxia . Group 3: Eight animals were pretreated with PPL per os, 12.5 mg.kg-1 twice a day for one week before the experiments, and the effects of arterial hypoxia were studied both before and after an intravenous injection of endotoxin . Two levels of hypoxia were studied; oxygen saturation in arterial blood aiming at 75 and 50% . Endotoxin was given intravenously in a dose of 1 mg.kg-1 bodyweight over a 5 minute period . After an endotoxin injection, the response to arterial hypoxia was an increase in CMRo2, in contrast to the unchanged CMRo2 without endotoxin . After pretreatment with PPL the increase in CMRo2 after endotoxin was prevented . The CBF reaction to hypoxia was uniformly an increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Anaesthesiol Scand, 1995 May, 39(4), 467 - 71
Effects of propranolol pretreatment on cerebral blood flow, oxygen uptake and catecholamines during metabolic acidosis following E . coli endotoxin in dogs; Westerlind A et al.; After an intravenous injection of E . coli endotoxin in dogs a decrease in cerebral blood flow (CBF) and an increase in cerebral metabolic rate of oxygen (CMRo2) have been shown to occur . In metabolic acidosis following endotoxin CMRo2 increased with decreasing pH . A possible explanation for the increased CMRo2 after endotoxin and metabolic acidosis seems to be a damage of the blood-brain barrier (BBB) by endotoxin . This gives possibilities for a leakage of hydrogen ions and circulating monoamines from the blood to the brain, thus affecting the cerebral blood flow and metabolism . The effects of an E . coli endotoxin injection on CBF and CMRo2 during metabolic acidosis and beta-adrenoceptor blockade were studied in eight anaesthetized dogs . All the dogs were pretreated with propranolol (PPL), per os 12.5 mg.kg-1 twice a day for one week . Metabolic acidosis (pH 7.01-7.30) was achieved by an intravenous infusion of hydrochloric acid . Endotoxin E . coli lipopolysaccharide O 111:B 4 was given as an intravenous injection of 1 mg.kg-1 bodyweight over a 5 min period . Another five animals, published earlier, with the same experimental protocol but without PPL, constituted a control group . After endotoxin no increase in CMRo2 or CBF was observed with increasing acidosis in the PPL-group . In the control group, after endotoxin, both CBF and CMRo2 increased with decreasing pH . This resulted in a significant difference in both CBF and CMRo2 between the groups in the pH range 7.01-7.15 . The present results indicate that the increase in CMRo2 and CBF with metabolic acidodis in endotoxinaemia is mediated via beta-adrenoceptors.

Biochem Mol Biol Int, 1995 May, 36(1), 21 - 30
Ceramide quantitation: evaluation of a mixed micellar assay using E . coli diacylglycerol kinase; Van Veldhoven PP et al.; The phosphorylation of ceramide solubilized in octylglucoside/phosphatidylglycerol mixed micelles by E . coli diacylglycerol kinase was evaluated . Ceramide containing non-hydroxy fatty acids appeared to be phosphorylated quantitatively over a broad range from 25 to 2000 pmoles . If a 2-hydroxy fatty acid was present in the ceramide molecule, phosphorylation was not quantitative . When applied to cellular lipid extracts, TLC of the phosphorylated products is needed to separate ceramide-phosphates from the other labelled compounds (i.e . phosphatidate and lysophosphatidate) and revealed the presence of ceramides containing long and very long chain fatty acids . The mass levels of these ceramides in different cultured cells varied between 0.2 to 0.6 mol% (normalized to phospholipids) . Changes in these levels were observed under different stress conditions such as heat treatment or addition of DMSO or detergents to the cell cultures.

Bioorg Khim, 1995 May, 21(5), 354 - 8
{Chemical synthesis of a proteinase inhibitor (eglin C) gene without use of T4-DNA-ligase and its expression in E . coli}; Veiko VP et al.; A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors . A high yield of the polypeptide (110-130 mg/l) was attained in E . coli strains.

Bioorg Med Chem, 1995 May, 3(5), 525 - 32
Mapping the structural domains of E . coli carbamoyl phosphate synthetase using limited proteolysis; Mareya SM et al.; The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis . Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern . Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit . The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions . Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein . However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage . MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis . Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine . The small subunit has been shown to be protected from proteolysis by the large subunit . Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity . These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Biochem Cell Biol, 1995 May, 27(5), 469 - 73
Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E . coli; Alexandrova R et al.; The AGG and AGA are the least used arginine codons in E . coli but they are the most preferable ones in eukaryotes . The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA(AGG) (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992) . The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes . The two tandems of AGG codons (Arg12 Arg13 and Arg163 Arg164) of hIF alpha 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site . The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.

Electrophoresis, 1995 May, 16(5), 813 - 6
Expression of the human urokinase-type plasminogen activator receptor in E . coli and Chinese hamster ovary cells: purification of the recombinant proteins and generation of polyclonal antibodies in chicken; Magdolen V et al.; The receptor for urokinase-type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase-type plasminogen activator (uPA) to the cell surface . uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases . Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor . A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail . For this purpose, uPAR (lacking the GPI anchor) was expressed in E . coli and Chinese hamster ovary (CHO) cells . Recombinant uPAR from E . coli (corresponding to amino acids 1-284 of human uPAR) was expressed with an N-terminal histidine-tag insertion and purified by nickel chelate affinity chromatography . Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography . Expression in E . coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . Prior to immunization the N-termini of the recombinant uPAR variants were determined by amino acid sequence analysis . Polyclonal antibodies were generated in chickens and purified from egg yolk . The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.

Infect Immun, 1995 May, 63(5), 2070 - 4
Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E . coli; McKee ML et al.; In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined . EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam . The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E . coli strains but not K-12 strains . The log jam phenotype may represent a basal means by which E . coli bacteria attach to the human intestine.

FEBS Lett, 1995 Apr 17, 363(1-2), 46 - 8
A novel antiporter activity catalyzing sodium and potassium transport from right-side-out vesicles of E . coli; Verkhovskaya ML et al.; Downhill sodium efflux from right-side-out E . coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier {Bassilana et al., Biochemistry 23 (1984) 1015-1022}, and in agreement with the concept of electrogenic Na+/nH+ antiporters with n > 1 . However, sodium efflux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system . delta pH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efflux against its concentration gradient, but only when the vesicles had been loaded with both Na+ and K+ . Efflux of K+ against the concentration gradient was also observed under these conditions . When the vesicles were loaded separately with sodium tricine or potassium tricine, no K+ efflux and insignificant Na+ efflux were observed . We propose that there are at least two different mechanisms responsible for Na+ efflux in E . coli vesicles . One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.

FEBS Lett, 1995 Apr 17, 363(1-2), 33 - 6
Role of lysine-195 in the KMSKS sequence of E . coli tryptophanyl-tRNA synthetase; Chan KW et al.; Lysine 195 in the K195 MSKS sequence of E . coli tryptophanyl-tRNA synthetase (TrpRS) was replaced with alanine . The resulting K195A mutant TrpRS had essentially unchanged Km values for ATP and Trp, but a 1500-fold decreased kcat in a pyrophosphate-ATP exchange reaction . This large decrease in kcat reduces the rate of aminoacyladenylate formation (step 1) to a rate comparable to the rate of aminoacylation of tRNA(Trp) (step 2) by the K195A mutant enzyme . Both the TIGN and KMSKS sequences are important for step 1 of class I aminoacyl-tRNA synthetase reactions.

Biochim Biophys Acta, 1995 Apr 4, 1229(1), 64 - 72
Characterization of the interaction of NADH with proton pumping E . coli transhydrogenase reconstituted in the absence and in the presence of bacteriorhodopsin; Hu X et al.; (1) Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was purified in a reconstitutively active form employing affinity chromatography on immobilized palmitoyl-Coenzyme A . Reconstituted transhydrogenase showed an active proton pumping and a stimulation of the rate of reduction of 3-acetylpyridine-NAD+ by NADPH by uncouplers . Reconstitution in the absence of a thiol-reducing agent, e.g . dithiothreitol, abolished proton pumping without affecting catalytic activity, giving a decoupled transhydrogenase . (2) Co-reconstitution of transhydrogenase with bacteriorhodopsin gave vesicles which catalyzed a 5-10-fold increased rate of reduction of thio-NADP+ by NADH in the light . The Km for NADH, but not that for thio-NADP+, decreased markedly in the light, indicating an effect of the electrochemical proton potential on the affinity of the enzyme for NADH . Inhibition by substrate derivatives in the absence or presence of light supported this conclusion . Replacement of NADH with 2'-deoxy-NADH gave a strongly sigmoidal concentration dependence, indicating an allosteric change induced by binding to the NAD(H)-site . (3) Reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, previously demonstrated to be catalyzed by both reconstituted bovine transhydrogenase and detergent-dispersed E . coli transhydrogenase, occurred at a pH below 6.5 . This reaction did not pump protons . Proton pumping by 3-acetylpyridine-NAD+ plus NADPH occurred at a pH above 5.5 . The two reactions were thus close to mutually exclusive, with a cross point at pH 5.8 . Assuming a helix bundle structure of the membrane domain of transhydrogenase, a model is proposed involving histidine 91 of the beta subunit which previously was shown to be essential by site-directed mutagenesis . According to the model the extent of protonation of this histidine determines whether proton pumping or the NADH-3-acetylpyridine-NAD+ reaction takes place.

J Rheumatol, 1995 Apr, 22(4), 684 - 8
Comparative evaluation of adsorption with E . coli on ELISA tests for Lyme borreliosis; Fawcett PT et al.; OBJECTIVE . To evaluate prospectively in a clinical setting the use of a soluble fraction of E . coli to adsorb nonspecific antibodies which can cause false positive ELISA tests for Lyme borreliosis . METHODS . The patient population tested was obtained from individuals referred to or initially presenting at a pediatric Lyme disease clinic in Wilmington, DE . Patients were followed for a minimum of 6 months subsequent to primary presentation at the clinic . RESULTS . A total of 209 met criteria for study inclusion, 93 of whom were diagnosed as having Lyme borreliosis and 116 of whom had other diagnoses . Results of ELISA tests were compared with different diagnoses and, when available, ELISA results from commercial laboratories . Findings indicate that some commercial laboratories have excessively high rates of false positive results (> 90% of positives were found to be false positives) . CONCLUSION . Adsorption with E . coli antigens effectively removed antibodies causing false positive results including those occurring at commercial laboratories and did not cause any significant reduction in assay sensitivity.

J Photochem Photobiol B, 1995 Apr, 28(1), 77 - 85
Effects of vacuum UV and UVC radiation on dry E . coli plasmid pUC19 . I . Inactivation, lacZ- mutation induction and strand breaks; Wehner J et al.; Using Escherichia coli plasmid pUC19 as a test system to study the effects of radiation on DNA at the molecular level, the wavelength (160-254 nm) dependence of inactivation (loss of the ability to transform E . coli), mutation induction in the target gene lacZ and induction of single-strand breaks and double-strand breaks was investigated . The same fluences were applied for all endpoints tested . In the UVC range, the cross-sections of inactivation and mutation induction match the DNA absorption curve, whereas the cross-section for single-strand break induction deviates from the DNA curve, especially at 220 nm . In the vacuum UV range, with increasing energy of the photons, the cross-sections of inactivation and single-strand breaks increase sharply (from 190 to 160 nm by more than one order of magnitude), which is not reflected by the DNA curve . In this UV range, the shape of the action spectrum is similar to that of the absorption curve of the sugar phosphate moiety of DNA . Only after irradiation with vacuum UV at 160 nm are double-strand breaks detected . Their induction rate is about one order of magnitude lower than that of single-strand breaks at the same wavelength; however, their induction rate is at least twice that of single-strand breaks at longer wavelengths . Concerning mutation induction, the increment in the vacuum UV range is less well expressed . The data suggest the contribution of different kinds of photochemical injury to inactivation and mutation induction.

Bioorg Khim, 1995 Apr, 21(4), 282 - 8
{Dependence of the level of gene expression in E . coli on the structure of the translation initiation segment (TIR)}; Gurevich AI et al.; The expression levels of genes that are transcribed to give mRNAs with identical leader sequences and even with identical extended coding regions may differ considerably . In order to determine the mechanism of this phenomenon, secondary structures of some mRNAs synthesized from a series of expression plasmids were studied . It was shown that the effect of the mRNA secondary structure in the translation initiation region on the initiation efficiency is due not only to the hairpin formation in this region but also to long-range interactions . When complementary structures tighter than those resulted from the interaction of regions SD, UB1, UB2, and DB with 16S rRNA are formed, the efficiency of the translation initiation and, consequently, the expression level decrease.

Int J Radiat Biol, 1995 Apr, 67(4), 393 - 401
Role of charge in the radioprotection of E . coli by thiols; Prise KM et al.; The role of net charge (zeta) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E . coli . This strain, 7, is deficient in the enzyme gamma-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied . Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells . The first-order chemical repair rate in strain 7 is 280 s-1 in comparison with 1100 s-1 in the wild-type strain 1157 . From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, kr, of 1.23 +/- 0.3 x 10(5) dm3mol-1s-1 . Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min . The ratios of the intracellular to extracellular concentrations (Cin/Cout) were 0.74 for 3-mercaptopropionic acid (zeta = -1), 0.56 for 2-mercaptoethanol (zeta = 0), 1.47 for cysteamine (zeta = +1) and 1.04 for WR1065 (zeta = +2) . The kr's for these thiols were 1.3 +/- 0.5 x 10(5) dm3mol-1s-1 for 30-mercaptopropionic acid, 3.3 +/- 1.6 x 10(5) dm3mol-1s-1 for 2-mercaptoethanol, 3.9 +/- 1.1 x 10(5) dm3mol-1s-1 for cysteamine and 2.7 +/- 1.1 x 10(6) dm3mol-1s-1 for WR1065 . These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E . coli . However, the approximate three-fold increase in kr per unit increase in zeta shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E . coli.

Mol Cell Endocrinol, 1995 Apr 1, 109(2), 237 - 41
Overexpression of a human prostate-specific glandular kallikrein, hK2, in E . coli and generation of antibodies; Saedi MS et al.; The genomic and the cDNA clones of human glandular kallikrein (hK2), a member of the kallikrein family, have been isolated; however, the hK2 protein has not yet been identified and characterized . The deduced sequence of hK2 is highly homologous to prostate specific antigen (PSA), a widely accepted prognostic indicator of prostate carcinoma . Also, hK2 mRNA, like PSA mRNA, is exclusively expressed in prostatic epithelia . These two properties make hK2 a potentially useful marker for studying prostate cancer . In this paper, we describe for the first time the overexpression of the entire hK2 protein (pre-pro hK2:pphK2) in the E . coli system . Our system yields high levels of authentic pphK2 (as determined by partial amino acid sequence analysis) comprising about 40% of total cellular protein . pphK2 was purified to near homogeneity by preparative SDS/PAGE and used to generate anti-pphK2 antibodies in rabbits . The antibodies recognize the recombinant hK2 protein and a major band of approximately 34 kDa in seminal fluid.

Biochem Mol Biol Int, 1995 Apr, 35(4), 899 - 912
Expression and purification of active form of HIV-1 protease from E.coli; Wan M et al.; We have subcloned an N-terminal extended protease gene of human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame into expression vector pGEX-KG . A relatively high level of expression of recombinant HIV-1 protease (PR) was achieved with isopropyl beta-D-thiogalactoside (IPTG) induction and glucose supplement . An isolation method consisting of denaturation of protein and followed by refolding was developed for releasing this recombinant HIV-1 PR into the soluble phase since most of the expressed protease was initially present in insoluble inclusion bodies . High purity of this recombinant HIV-1 PR was obtained by sequential purification using Sephadex G-50 gel filtration and CM-23 cellulose cation exchange chromatography, yielding the protease more than 1 mg per liter culture . N-terminal amino acid sequence analysis showed that the recombinant HIV-1 PR underwent autocleavage from the fusion protein during expression . SDS-PAGE indicated that the molecular weight of this recombinant HIV-1 PR is 11 kDa . This recombinant HIV-1 PR showed proteolytic activity for the synthetic peptide substrates corresponding to the sequence at the Gag MA/CA and Pol p6*/PR junctions . The purified enzyme whose specific activity for the heptapeptide SQNYPIV was 848.7 nmol*min-1*mg protease-1 also processed recombinant polyprotein Gag41 as its substrate.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 215 - 24
Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E . coli ribonucleotide reductase; Swarts JC et al.; Kinetic studies at 25 degrees C, I = 0.100 M (NaCl), on the reduction of the tyrosyl radical of the R2 protein of E . coli ribonucleotide reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported . There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine which itself protonates in this range . At pH 7.6 the rate constant (0.46 M-1 s-1) for the HU reaction is in agreement with earlier values . Slower reactions are observed with the bulkier acetohydroxamic (0.020 M-1 s-1) and benzohydroxamic acids (0.040 M-1 s-1) . In the case of N-methylhydroxylamine the rate constant (0.41 M-1 s-1 at pH 7.6) decreases with pH, and it is concluded that the protonated form CH3NH2+OH(pKa = 6.2) has little or no reactivity with Tyr . For this reaction under air-free conditions a second-stage (0.027 M-1 s-1) corresponding to reduction of Fe(III)2 is observed . Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered . The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexed products . The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox . The mechanism of reduction of R2-Tyr by all of the reagents studied is discussed.

Nucleic Acids Res, 1995 Mar 11, 23(5), 779 - 84
Genetic selection for active E.coli amber tRNA(Asn) exclusively led to glutamine inserting suppressors; Martin F et al.; Suppressor tRNAs are useful tools for determining identity elements which define recognition of tRNAs in vivo by their cognate aminoacyl-tRNA synthetases . This study was aimed at the isolation of active amber tRNA(Asn) . Nineteen mutated tRNA(Asn)CUA having amber suppressor activity were selected by an in vivo genetic screen, and all exclusively inserted glutamine . From analysis of the different mutations it is concluded that glutamine accepting activity was obtained upon reducing the interaction strength between the first base pair of the tRNA(Asn)CUA by direct or indirect effects . Failure to isolate tRNA(Asn)CUA suppressors charged with asparagine as well as other evolutionary related amino acids is discussed.

Scand J Immunol, 1995 Mar, 41(3), 281 - 7
Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E . coli-mycobacteria shuttle vector; Matsumoto S et al.; MPB70 is known to be an immunogenic mycobacterial protein secreted in large amounts from Mycobacterium bovis BCG (BCG) Tokyo . The analogous gene for MPT70 was cloned from Mycobacterium tuberculosis H37Rv which produces this protein in only a small amount . The gene encoding 193 amino acid residues including 30 amino acids for the signal peptide, the promoter-like sequence, and the ribosome-binding site, was completely identical to that of BCG Tokyo . Computer analysis revealed that the carboxy-terminal half of MPT70 was homologous to amino acid sequences of fasciclin I, osteoblast-specific factor 2 (OSF-2), and human transforming growth factor-beta induced gene product (beta IG-H3) . Escherichia coli (E . coli) -mycobacteria shuttle vectors containing mpt70 or mpb70 genes 0.7kbp upstream of the 5' end of them were able to be expressed in BCG Pasteur which is a MPB70 low-producer, but the extent of the expression was not that of a high-producer.

Shi Yan Sheng Wu Xue Bao, 1995 Mar, 28(1), 77 - 83
{Cloning and expression of an antifreeze peptide gene of winter flounder in E . coli}; Jing HL et al.; Antifreeze peptide (AFP) produced in some animals is able to lower freezing temperature to prevent serum from freezing . There is a great potential to apply AFP in various circumstances wherever tolerance to cold environment is required . cDNA copies of AFP and proAFP coding sequence were synthesized according to the published sequence from a winter flounder (Pseudopleuronectes americanus) . Expression of AFP or proAFP cDNA was initially tested in E . coli using a procaryotic expression vector . Level of the expression was essentially low indicated by SDS-PAGE and protein staining procedures . In order to detect the low level expression, the AFP (or proAFP) cDNA was fused, in frame, to the 5'-end of the CAT reporter gene resulting in a chimeric AFP/CAT (or proAFP/CAT) cDNA . The product translated from such a chimeric mRNA must contain a N'-terminal AFP (or proAFP) portion and C'-terminal CAT portion . Thus, low level of expression of AFP (or proAFP) in the fused form may be detected indirectly by sensitive CAT assay as long as the chimeric CAT still remains the activity . As expected, CAT activity has been clearly detected in protein extract from induced E . coli indicating that AFP (or proAFP) cDNA clones be expressed in E . coli.

Biochem Biophys Res Commun, 1995 Feb 27, 207(3), 957 - 62
Hydroxyl radicals and DNA double-strand breaks in X-irradiated E . coli; Ewing D et al.; We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E . coli strains . Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.

Mol Cell Endocrinol, 1995 Feb 27, 108(1-2), 75 - 85
Production of a biologically active recombinant teleostean growth hormone in E . coli cells; Cheng CM et al.; We have isolated and characterized several recombinant lambda phage clones carrying growth hormone (GH) cDNA of striped bass (Morone saxatilis) . Nucleotide sequence and the predicted amino acid sequence of sbGH was determined from a recombinant clone carrying the longest cDNA insert . The sbGH cDNA encodes a pre-hormone of 204 amino acid residues . Comparison of the predicted amino acid sequence of sbGH with those of other vertebrates revealed different degrees of sequence identity: approximately 98% with European sea bass; 90% with bluefin tuna; bonito and red seabream; 71% with winter flounder; 64% with salmonids; 55% with carp; and 38% with human . Expression of the mature sbGH cDNA (without the signal peptide sequence) in E . coli cells under regulation of the lambda phage PL promoter produced a polypeptide of 20 kDa . Following renaturation, this recombinant hormone was shown to be biologically active in a radioreceptor competition binding assay and in the induction of hepatic insulin-like growth factor I (IGF-I) mRNA synthesis in vivo.

FEBS Lett, 1995 Feb 20, 360(1), 52 - 6
Synthesis and refolding of human TIMP-2 from E . coli, with specific activity for MMP-2; Negro A et al.; Tissue inhibitors of metalloproteinase (TIMPs) are inhibitory counterparts of collagenases, containing 12 cysteine residues paired to six internal disulphide bridges . TIMP-2, an inhibitory protein of 72 kDa gelatinase/type IV collagenase (MMP-2), was expressed in Escherichia coli as a fusion protein with a 34 amino acid NH2-linked tail containing six consecutive histidine residues . The protein was purified in a single-step using an ion metal affinity column (IMAC) in denaturing conditions . The immobilized fusion TIMP-2 protein was refolded at a high concentration in the column, producing about 5 mg of protein per litre of bacterial cells . It shows specific binding and inhibitory activity against MMP-2, but has no effect against 92 and 45 kDa gelatinases.

Virology, 1995 Feb 20, 207(1), 185 - 90
The Gag-Pol encoded proteinase of an avian retrovirus expressed in E . coli can produce a novel proteinase (PR + IleGly) that is two amino acids larger at its carboxy-terminal region than the major Gag proteinase (PR); Brynda J et al.; Gag-Pol frameshift translational products of avian retroviruses (e.g., myeloblastosis associated virus, MAV) contain a putative proteinase species of 131 amino acids that maps between the NC/PR and the PR/RT processing sites . Expression in Escherichia coli of an in-frame PR precursor that contains the natural NC/PR processing site and is translationally terminated at the PR/RT site leads to formation of a Gag-Pol proteinase of the expected molecular size (131 amino acids) and a novel PR product of 126 amino acids . This product extends 2 amino acids downstream of the gag-encoded 124 amino acids, and its proteolytic cleavage is promoted by conditions favorable for enzyme catalysis, is blocked by a specific MAV proteinase inhibitor, and can be demonstrated also for corresponding peptide substrates . The new self-processing cleavage product is termed PR(+IleGly) and exhibits similar, but slower, catalytic parameters than those of the Gag PR.

Mol Immunol, 1995 Feb, 32(3), 185 - 98
Paratope characterization by structural modelling of two anti-cortisol single-chain variable fragments produced in E . coli; Le Calvez H et al.; Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortisol, have been obtained; 6D6 is used in an assay kit for cortisol . The antibodies also recognize other, structurally related steroids present in the sample assayed . To improve the specificity of the assay, we aimed to minimize the recognition of non-cortisol steroids by the two anti-cortisol mAbs . Our strategy consisted in constructing an efficient expression vector in E . coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space . We demonstrated that temperature and inducer concentration of the bacterial culture influenced dramatically the yield of active scFv . From the nucleotide sequence we constructed a three-dimensional model of the two variable fragments in order to understand why related steroids are, or are not recognized by the antibody . For both antibodies, we have identified chemical groups which are probably involved in the binding of the steroid haptens and the antibodies . The hydrophobic pocket formed by the antibody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking . The serine at position 35 of the heavy chain is buried in the back of the pocket and can form a hydrogen bond with the 20-keto group of the cortisol . The stacking interactions and the hydrogen bond orient the steroid in the pocket . This reactivity of the binding site is sustained by the analysis of the cross-reactions of related steroids with the mAbs.

Infect Immun, 1995 Feb, 63(2), 700 - 2
Use of heme and hemoglobin by Escherichia coli O157 and other Shiga-like-toxin-producing E . coli serogroups; Law D et al.; The virulence properties of Escherichia coli O157 isolates were compared with those of Shiga-like-toxin-producing E . coli of non-O157 serogroups . The growth of all E . coli O157 isolates was stimulated by both heme and hemoglobin, and all produced enterohemolysin . The incidence of these properties was significantly lower in the non-O157 isolates . This may contribute to the greater virulence and higher incidence of human infection caused by E . coli O157.

Tierarztl Prax, 1995 Feb, 23(1), 80 - 2
{Treatment of chronic diarrhea in dogs and cats under field conditions using oral E . coli vaccines}; Weber A et al.; An autogenous oral E . coli vaccine was tested for efficacy in the treatment of chronic diarrhoea in dogs (n = 82) and cats (n = 50) under field conditions . The data were collected through evaluation of questionnaires completed and returned by veterinarians . After oral application of the E . coli vaccine the symptoms of diarrhoea were stopped in 71% of the treated animals within two to five days . In further 15% of the cases the enteric symptoms continued but were not so severe as in the beginning of the treatment . There was no therapeutic success with the oral E . coli vaccine in further 14% of the animals in cause of food allergy, ascariasis, volvulus or pancreatic insufficiency.

Proteins, 1995 Feb, 21(2), 130 - 9
Affinities of phosphorylated substrates for the E . coli tryptophan synthase alpha-subunit: roles of Ser-235 and helix-8' dipole; Sarker KD et al.; The roles of Ser-235 and helix-8' (residues 235-242) in the functional binding and turnover of phosphorylated substrates by the alpha-subunit of the E . coli tryptophan synthase (TSase) alpha 2 beta 2-holoenzyme complex are examined . Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP) . The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8' . The activities and substrate Kms of mutant alpha-subunits altered in this region by site-specific mutagenesis are reported here . Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a non-peptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold Km increases for IGP and D-glyceraldehyde-3-phosphate; no Km defects for indole were observed . kcat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value . Steric considerations may explain the results with the SE235 and SK235 mutant alpha-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amido group during catalysis, A helix-8' dipole role was explored following proline substitutions separately at the first six (of eight) residues . Proline substitutions at positions-1 through -4 in helix-8' have normal indole Kms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins . By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations . Km increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L-serine at the beta-subunit active site . In the absence of L-serine, substitution only at the first position results in binding defects; in the presence of L-serine, substitutions at the first, second and third positions, show binding defects of decreasing magnitude, sequentially . Substitutions at the fourth and fifth position have no effect on substrate binding . It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via intersubunit-induced conformational changes due to ligand (L-serine) binding to the beta-subunit.

Bioorg Khim, 1995 Feb, 21(2), 117 - 23
{Dependence of the level of gene expression in E . coli on the structure of the translation initiation segment (TIR) . I . Primary structure of TIR}; Gurevich AI et al.; The comparison of expression levels of two genes-interleukin-3 (il3) and epidermal growth factor connected to leader peptide of OmpF (lompegf)-was carried out using specially constructed plasmids contained various structures of translational enhancers . It was shown that besides already known binding sites from mRNA translation initiation region (TIR) to 16S rRNA (SD, UB1 and DB), there is additional binding site of TIR disposed from -30 to -60 nt upstream of start codon AUG (UB2) having significant increasing effect on translation initiation and correspondingly on expression level.

J Biochem Biophys Methods, 1995 Feb, 30(1), 59 - 68
A method for production of 13C/15N double labelled RNA in E . coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates; Nyholm T et al.; In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs . High amounts of labelled RNA was obtained from E . coli cells grown in 13C/15N enriched medium and treated with chloramphenicol . Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer . To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA . CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol . The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

Nucleic Acids Res, 1995 Jan 25, 23(2), 211 - 6
Probing the molecular mechanism of action of co-repressor in the E . coli methionine repressor-operator complex using surface plasmon resonance (SPR); Parsons ID et al.; We have studied quantitatively the effect of the corepressor, S-adenosylmethionine (SAM), on the interaction between the E . coli methionine repressor, MetJ, and an idealised operator fragment, by recording measurements of surface plasmon resonance using a BIAcore instrument . We have recorded kinetic binding data in the presence of SAM, which carries a net positive charge, and two corepressor analogues, adenosylornithine (AO) and aza-SAM, which differ in the location of the atom carrying the positive charge . Our data support the hypothesis that the effect of the corepressor is electrostatic in origin . The difference in electrostatic interaction energy between the SAM- and AO-repressor-operator complexes of approximately 3.5 kJ/mol calculated from the known three-dimensional structure is within the range of our experimentally determined values of 2.8-4.3 kJ/mol . These results illustrate the potential of SPR measurements for studying protein-nucleic acid interactions.

FEBS Lett, 1995 Jan 23, 358(2), 137 - 41
Nuclear factors specifically favor thyroid hormone binding to c-ErbA alpha 1 protein (thyroid hormone receptor alpha) over-expressed in E . coli; Daadi M et al.; A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E . coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha) . It was found that nuclear extracts (NE) added to rec-TR alpha markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported . This T3 binding amplifying effect on rec-TR alpha occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g . ovalbumin or cytosol) only moderately enhanced T3 binding . The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TR alpha . A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha to acquire and/or stabilize a conformation that allows the high affinity T3 binding . The nature of this nuclear amplifying factor is still unknown: RXR alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.

Biochem Biophys Res Commun, 1995 Jan 17, 206(2), 781 - 5
Can an X-ray dose threshold be measured for the induction of SOS repair activity in E . coli; Ewing D; Survival ("clonogenic ability") and induced DNA repair (SOS repair) have been studied at low X-ray doses in two Escherichia coli strains (GE94 and KY943), both of which contain a recA-lacZ protein fusion . X-ray doses greater than about 0.5 Gray clearly induce SOS repair activity . However, the threshold dose that just activates SOS repair is probably much lower than 0.5 Gray, perhaps as low as 0.001 Gray . It seems unlikely that this dose threshold can be more accurately measured with the recA-lacZ fusion technique.

Jpn J Ophthalmol, 1995, 39(4), 353 - 9
Effects of a combined use of steroidal and nonsteroidal anti-inflammatory drugs on E . coli endotoxin-induced uveitis in pigmented rabbits; Ogawa T et al.; The effects of a combined use of steroidal and nonsteroidal anti-inflammatory topical drugs on uveitis were studied . Uveitis was induced by intravenous injection of E . coli endotoxin (10 micrograms/kg) in pigmented rabbits . A solution of 0.5% indomethacin (IM) and/or 0.1% betamethasone (BM) was instilled 4 times: at 1 hour before and at 0, 2 and 4 hours after the endotoxin injection . The aqueous protein was quantified by laser flare-cell measurements hourly after the injection . The prostaglandin E2 concentration was assayed and venous leukocytes and thrombocytes were counted 5 hours after the endotoxin injection . The aqueous protein reached a peak concentration (27 mg/mL) about 4 hours after the endotoxin injection . IM, BM and a combination of IM and BM (IM + BM) inhibited the aqueous protein rise by 17, 63 and 94%, respectively . IM, BM and IM + BM almost completely inhibited the increase of aqueous prostaglandin E2 . IM + BM inhibited both leukocytopenia and thrombocytopenia . IM alone had no effect on these counts, and BM alone inhibited leukocytopenia only . The data suggest that the combined use of topical IM and BM is more useful for the therapy of endotoxin-induced uveitis than each drug alone . The inhibitory effects may be synergistic, probably acting on unknown mediators which may be derived from the systemic circulation.

Mol Biol Rep, 1995-96, 22(2-3), 111 - 4
Genetic analysis of the structure and function of RNase P from E . coli; Shiraishi H et al.; A brief review of the genetic studies on ribonuclease P (RNase P) from Escherichia coli is presented . Temperature-sensitive mutants of E . coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature . Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity . Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzyme consists of two subunits . Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.

Nucleic Acids Symp Ser, 1995, (34), 87 - 8
The recognition of DNA containing an AP site by E.coli endonuclease VI (exonuclease III); Shida T et al.; The major apurinic/apyrimidinic (AP) DNA-repair endonuclease of Escherichia coli is the endonuclease VI (exonuclease III) protein . To elucidate the substrate specificity of the AP endonuclease, we used the double- and single-stranded oligo deoxyribonucleotides containing an AP residue such as 2-deoxyribosylformamide (1), 2-deoxyribose (2), 1,2-dideoxyribofuranose (3), and propanediol (4) as the substrate . The endonuclease VI cleaved the phosphodiester bond 5' at these AP sites of the duplexes . The endonucleolytic activity was not influenced by the kind of nucleotide residue on the opposite side of the AP site . Further, it was observed that the AP endonuclease cleaved single-stranded oligomers containing an AP site.

Receptors Channels, 1995, 3(4), 291 - 7
Ligand binding analysis of human neuropeptide Y1 receptor mutants expressed in E . coli; Munch G et al.; Site-directed mutants of the human neuropeptide Y1 (NPY Y1) receptor expressed as a maltose binding protein fusion protein in E . coli show identical ligand binding parameters compared with the same mutants expressed in mammalian cells using a vaccinia virus expression system . However, it was remarkable that two receptor mutants, which were initially classified as non-binding when expressed in an eukaryotic expression system, could actually be revealed to have wild-type binding activity when expressed in E . coli . Re-expression and retesting of these mutants in mammalian cells confirmed this result . This shows that bacterial expression can be used as a fast, versatile and valuable alternative to mammalian expression systems for the analysis of ligand binding sites in G-protein coupled receptors.

Autoimmunity, 1995, 22(3), 137 - 47
Collagen-induced arthritis in mice: synergistic effect of E . coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen; Terato K et al.; DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis . We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11 . MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E . coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice . LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice . A single i.p . injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones . These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11 . Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.

J Tongji Med Univ, 1995, 15(3), 138 - 42
Construction of shuttle expression plasmid and stable expression of foreign gene in mycobacteria and E . coli; Huangfu YM et al.; By employing the pUC19 as a backbone, the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19 . The recombinant E . Coli-mycobacteria shuttle expression plasmid pBCG-8000 was constructed . The pBCG-8000 was able to replicate in both E . Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants . The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.

Mol Biol Rep, 1995, 21(2), 105 - 12
Expression of cDNA fragment encoding sperm membrane peptide in E . coli; Li Y et al.; A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector . Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence . The cloned foreign gene is under the control of the PR-PL promoter while the expression of the gene is regulated by the cI-gene product . The products are secreted into the periplasmic space of bacteria or into the medium . A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20 . The E . coli cells transformed with pRSD-220 were propagated at 30 degrees C, then incubated at 42 degrees C for several hrs . The cloned gene product was secreted into the culture medium at a high rate . The yield was about 60 mg of gene product per liter of cultured medium.

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