Escherichia coli

Dev Growth Differ, 2001 Feb, 43(1), 13 - 23
Primary structure and developmental expression of Bufo arenarum cellular nucleic acid-binding protein: changes in subcellular localization during early embryogenesis; Armas P et al.; A Bufo arenarum cellular nucleic acid-binding protein (bCNBP) full-length cDNA was cloned . bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser-rich region . Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes . Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior . One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription . Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes . In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm . At mid-blastula stage, CNBP was mainly detected in the epiblast . At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining . Nuclei in this layer were stained even stronger than the cytoplasm . Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

Biochemistry, 2001 Jan 16, 40(2), 375 - 86
Solution structure and dynamic character of the histidine-containing phosphotransfer domain of anaerobic sensor kinase ArcB from Escherichia coli; Ikegami T et al.; An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions . Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPt(ArcB)), which has a phosphorylation site, His717 . The backbone dynamics were also investigated by analyses of the (15)N relaxation data and amide hydrogen exchange rates . Furthermore, the protonation states of the histidine imidazole rings were characterized by means of (1)H and (15)N chemical shifts at various pHs . The determined solution structure of HPt(ArcB) contains five helices and forms a four-helix bundle motif like other HPt domains . The obtained order parameters, S (2), {(1)H}-(15)N heteronuclear NOE values, and chemical exchange parameters, R(ex), showed that the alpha-helical regions of HPt(ArcB) are rigid on both picosecond to nanosecond and microsecond to millisecond time scales . On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D . These results suggest that HPt(ArcB) may undergo a small conformational change in helix D upon phosphorylation . It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other . This is consistent with the solution structure of HPt(ArcB), in which the imidazole ring of His717 is exposed to the solvent.

Biochemistry, 2001 Jan 16, 40(2), 353 - 60
Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase; Mizuguchi H et al.; Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues, Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK(a) value of the Schiff base formed between pyridoxal 5'-phosphate (PLP) and Lys258 . The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues . PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation . Mutation of Arg386 eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base . On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier . Because PLP-N-R lies between the two domains, the domain opening increases the strain of the Schiff base . Therefore, the true electrostatic effects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation . Through the analyses, we could dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pK(a) . The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively . Thus, the electrostatic effect of the arginine residues is not as strong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low pK(a) value of the Schiff base {Hayashi, H., Mizuguchi, H., and Kagamiyama, H . (1998) Biochemistry 37, 15076-15085}.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 441 - 4 Epub 2001 Jan 09.
A subunit of human nuclear RNase P has ATPase activity; Li Y et al.; Human nuclear RNase P purified from HeLa cells has ATPase activity . This activity is associated with one of the protein subunits of the enzyme, Rpp20 . Thus, human nuclear RNase P, which contains several proteins and one essential RNA, has at least one other enzymatic activity in addition to cleavage of phosphoester bonds in RNA . The amino acid sequence of Rpp20 has a signature motif found in an ATPase-containing subunit of a family of protein complexes (ABC transporters) that mediate a variety of trans-membrane traffic, as well as a segment, DIxxN, that resembles the DEAD box motif of many ATPases: together, these might represent an ATPase signature motif.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 525 - 30 Epub 2001 Jan 09.
Genetic architecture of thermal adaptation in Escherichia coli; Riehle MM et al.; Elucidating the genetic basis of adaptation on a genomewide scale has evaded biologists, but complete genome sequences and DNA high-density array technology make genomewide surveys more tractable . Six lines of Escherichia coli adapted for 2,000 generations to a stressful high temperature of 41.5 degrees C were examined on a genomewide scale for duplication/deletion events by using DNA high-density arrays . A total of five duplication and deletion events were detected . These five events occurred in three of the six lines, whereas the remaining three lines contained no detectable events . Three of the duplications were at 2.85 Mb of the E . coli chromosome, providing evidence for the replicability of the adaptation to high temperature . Four candidate genes previously shown to play roles in stress and starvation survival were identified in the region of common duplication . Expression of the two candidate genes examined is elevated over expression levels in the ancestral lines or the lines without the duplication . In the two cases where the duplication at 2.85 Mb has been further characterized, the timing of the genome reorganization is coincident with significant increases in relative fitness . In both of these cases, the model for the origin of the duplication is a complex recombination event involving insertion sequences and repeat sequences . These results provide additional evidence for the idea that gene duplication plays an integral role in adaptation, specifically as a means for gene amplification.

Cell Stress Chaperones, 2000 Apr, 5(2), 148 - 59
Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity; Fernando P et al.; Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones . Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C . An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli . These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells . Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays . Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity . In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity . Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution . Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates . Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins . These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.

Arch Biochem Biophys, 2000 Dec 1, 384(1), 174 - 80
Properties of the beta subunit of the proteasome activator PA28 (11S REG); Wilk S et al.; The proteasome activator PA28 (11S REG) is composed of two homologous subunits termed alpha and beta . The properties of the recombinant beta-subunit were explored and compared to the properties of the recombinant alpha-subunit . PA28beta produced in an Escherichia coli expression system migrates on a calibrated gel filtration column as an apparent heptamer (Mr = 250,000) . Low concentrations of SDS (0.005%), dissociate the protein to a monomer (Mr = 33,000) . PA28beta, has a complex effect on proteasome activity . At concentrations which favor oligomerization (> 2 microM), PA28beta is a strong proteasome activator although its affinity for the proteasome is about 10-fold less than recombinant PA28alpha . The catalytic properties of the PA28alpha and PA28beta-activated proteasome are similar . At low concentrations, PA28beta is a monomer and a potent allosteric proteasome inhibitor . These studies show that oligomerization of PA28beta is required for proteasome activation and that PA28beta monomers are potent proteasome inhibitors.

Arch Biochem Biophys, 2000 Dec 1, 384(1), 1 - 8
Purification and characterization of the recombinant form of Acyl CoA oxidase 3 from the yeast Yarrowia lipolytica; Luo YS et al.; The Acyl CoA dependent oxidase 3 (Aox3p) from the yeast Yarrowia lipolytica, expressed in Escherichia coli, as an active protein with a 6 His tag at its N-terminal region has been purified to electrophoretic homogeneity . The purified enzyme exhibits a specific activity of 1.95 microM/min/mg using hexanoyl-CoA as substrate, and it remains active for at least 1 month upon storage at -30 degrees C in the presence of 35% (V/V) glycerol . The pH and temperature optima of the enzyme are 7.4 and 28-38 degrees C, respectively . Aox3p catalyzes the oxidation of both aliphatic acyl-CoA substrates of different chain lengths (e.g., hexanoyl-CoA, decanoyl-CoA, myristyl-CoA) as well as of the aromatic/heterocyclic ring-substituted chromogenic substrates, such as furylpropionyl-CoA . Of the above substrates, the efficiency of the enzyme, as judged by its kcat to Km ratio, exhibits the following order: decanoyl CoA > myristyl CoA > hexanoyl CoA > furyl-propionyl-CoA (FPCoA) . Phenol, which is normally used in the coupled assay system for monitoring the H2O2 formation, functions as both an activator (at low concentrations) and a competitive inhibitor (at high concentrations) with respect to acyl-CoA substrates . The magnitude of activation and inhibition of the enzyme is dependent on the nature of the acyl-CoA substrates.

Hematol Oncol Clin North Am, 2000 Dec, 14(6), 1367 - 79, x
Recent clinical trials in acute lymphocytic leukemia by the Cancer and Leukemia Group B; Larson RA; The Cancer and Leukemia Group B (CALGB) has performed a series of studies evaluating different aspects of remission induction and postremission treatment in adults with acute lymphocytic leukemia (ALL) . In recent years, these clinical trials have been supplemented by systematic morphologic, immunophenotyping, cytogenetic, and molecular genetic studies leading to the identification of different risk groups of patients who may warrant individualized treatments . These protocols have enrolled all adult patients older than 15 years with ALL, without an upper age restriction, and did not exclude Philadelphia (Ph) chromosome-positive patients.

Brain Res Mol Brain Res, 2000 Dec 28, 85(1-2), 251 - 9
Loss of base excision repair in aging rat neurons and its restoration by DNA polymerase beta; Rao KS et al.; Synthetic staggered oligodeoxynucleotide duplexes are formed by annealing a 5'-32P-labeled 14-mer with four different 21-mers . These duplexes have either a correct or mismatched base pair at 3'-end of the primer . With these model template primers the ability of neuronal extracts, obtained from rats of different ages, to extend the primer to the predicted length was tested . While the neuronal extracts of all ages were able to degrade the 14-mer to shorter lengths, extension of the primers in general and in particular, the mismatched, is achieved only feebly by the young and adult neuronal extracts and undetectable with old neuronal extracts . The possibility of restoring the lost activity by supplementing the neuronal extracts with pure DNA polymerases was examined . Of the three polymerases tested (calf thymus alpha polymerase, E . coli DNA polymerase I and rat liver DNA polymerase beta) only polymerase beta gave consistent and encouraging results although the extension was slow and distributive in nature and mismatched primers were extended much less efficiently than the correctly paired primer . However, significantly improved extension, including those of mismatched primers, was achieved by prior removal of mismatched bases in a preincubation with just the neuronal extracts (3'-5'exonuclease activity) followed by extension by the added polymerase beta and dNTPs in the presence of Mn(2+) instead of the usual Mg(2+) . These results are taken to indicate that the activity of polymerase beta in brain cells is compromised with age and that this deficit can be corrected in vitro by the addition of pure recombinant rat liver polymerase beta under appropriate conditions.

Virology, 2001 Jan 5, 279(1), 210 - 20
Identification of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids; Mitra-Kaushik S et al.; The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells . We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E . coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells . Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c) . A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response . The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted . Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins . We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins . Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope . This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides .

J Immunol, 2001 Jan 15, 166(2), 1106 - 13
Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses; Simmons CP et al.; In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV . M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant . CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss . CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance . Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance . Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge . Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV . These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.

J Immunol, 2001 Jan 15, 166(2), 1009 - 15
New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling; Blunck R et al.; LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators . These regulate the immune response, but may also cause severe harm leading to septic shock . The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14 . As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior . Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages . The channel is activated by endotoxically active LPS in a dose-dependent manner . Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs . Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K(+) channel blockers.

Microbiol Immunol, 2000, 44(11), 905 - 14
Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli; Yoda T et al.; The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans . The virus capsid is composed of a single 60 kDa protein . The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it . The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E . coli . All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions . Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548) . Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples . It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.

Redox Rep, 2000, 5(5), 299 - 301
The effect of electromagnetic field exposure on the formation of DNA lesions; Lourencini da Silva R et al.; In an attempt to determine whether electromagnetic field (EMF) exposure might lead to DNA damage, we exposed SnCl2-treated pBR322 plasmids to EMF and analysed the resulting conformational changes using agarose gel electrophoresis . An EMF-dependent potentiation of DNA scission (i.e . the appearance of relaxed plasmids) was observed . In confirmation of this, plasmids pre-exposed to EMF also were less capable of transforming Escherichia coli . The results indicate that EMF, in the presence of a transition metal, is capable of causing DNA damage . These observations support the idea that EMF, probably through secondary generation of reactive oxygen species, can be clastogenic and provide a possible explanation for the observed correlation between EMF exposure and the frequency of certain types of cancers in humans.

Redox Rep, 2000, 5(5), 287 - 93
Sensing and protecting against superoxide stress in Escherichia coli--how many ways are there to trigger soxRS response?
Touati D.
Reactive oxygen species (ROS) are produced as an inescapable consequence of aerobic life . Their levels are kept low enough to be harmless by specific enzymes, such as superoxide dismutase, which eliminate them . Expression of these defence enzymes is modulated depending on the environmental oxidative threat . This basic protection, however, is not sufficient to protect against sudden large increases in ROS production . To cope with oxidative stress, rapid global responses are induced that enable bacteria to survive the stress period by multiple means: elimination of ROS, repair of oxidative damage, bypass of damaged functions and induction of adapted metabolism . The soxRS response, which protects against superoxide (O2.-)-generating agents and nitric oxide (.NO), is triggered by activation of a sensor molecule, SoxR, containing two essential {2Fe-2S} clusters . The soxRS regulon is induced in a two-stage process . Upon activation, SoxR induces soxS expression and SoxS, in turn, activates transcription of genes of the regulon . The mechanism of signalling has been under debate for years . Evidence for several pathways of SoxR activation, mediated by the modifications of {2Fe-2S} centres, has emerged from recent data . The direct oxidation of {2Fe-2S} centres, any event that may interfere with the pathway maintaining SoxR in a reduced inactive form, and direct nitrosylation by .NO can trigger SoxR activation . The multiple possibilities for SoxR activation, along with signal amplification via the two-stage process, constitute a unique, and particularly sensitive, system enabling cells to induce rapidly a protective response to a broad range of environmental changes indicative of possible oxidative stress.

Cell Transplant, 2000 Sep-Oct, 9(5), 657 - 67
The X-gal caution in neural transplantation studies; Sanchez-Ramos J et al.; Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue . The lacZ gene encodes the bacterial (E . coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction . LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells . If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g. . pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells . Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction . Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker . We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E . coli beta-gal activity.

Planta, 2000 Nov, 211(6), 883 - 6
Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase; Milkowski C et al.; A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.) . The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36 . The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate . Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated . The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (beta-acetal esters).

J Biotechnol, 2000 Sep, 74(3), 137 - 58
Functional tethered lipid bilayers; Knoll W et al.; Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support . Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support . An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite . And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates . All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques . For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer . Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step . Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e . the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively . The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes . The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies . The results obtained for reconstituted H(+)-ATPase from chloroplasts and E . coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 95 - 7
Enterotoxigenic Escherichia coli--an overview; Guth BE; Enterotoxigenic Escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world . In this presentation we will focus on the main virulence attributes of this pathogenic category of E . coli, and discuss the evolution of studies conducted in our laboratory.

Dev Growth Differ, 2000 Dec, 42(6), 603 - 11
Conversion from mitosis to meiosis: morphology and expression of proliferating cell nuclear antigen (PCNA) and Dmc1 during newt spermatogenesis; Yazawa T et al.; The conversion from mitosis to meiosis is a phenomenon specific to the cellular progenitors of gametes; however, the mechanism or mechanisms responsible for this conversion are poorly understood . To this end, some morphological and molecular changes that occur during the initiation of meiosis in newt spermatogenesis are reported in the present paper . In situ morphologic studies revealed that spermatogonial stages comprise two phases: early mitotic generations (G1-G4) and late mitotic generations (G5-G8) . Morphologic conversion from secondary spermatogonia to primary spermatocytes occurred during the intermediate stage of premeiotic DNA replication . The expression of proliferating cell nuclear antigen (PCNA), a DNA polymerase-delta auxiliary protein, in spermatogonia was weak in G1, highest during DNA synthesis (S), decreased in G2 and was not detectable in dividing cells . Complementary DNA for newt homologs of DMC1 (disrupted meiotic cDNA), which is an Escherichia coli RecA-like protein specifically active during meiosis, were isolated . The newt Dmc1 mRNA was first expressed significantly during the preleptotene stage and this continued into the spermatid stage . These observations present a basis for investigating the mechanism(s) controlling the conversion of newt spermatogonial cells from mitosis to meiosis.

J Biomol NMR, 2000 Nov, 18(3), 239 - 52
Assessment of molecular structure using frame-independent orientational restraints derived from residual dipolar couplings; Skrynnikov NR et al.; Residual dipolar couplings measured in weakly aligning liquid-crystalline solvent contain valuable information on the structure of biomolecules in solution . Here we demonstrate that dipolar couplings (DCs) can be used to derive a comprehensive set of pairwise angular restraints that do not depend on the orientation of the alignment tensor principal axes . These restraints can be used to assess the agreement between a trial protein structure and a set of experimental dipolar couplings by means of a graphic representation termed a 'DC consistency map' . Importantly, these maps can be used to recognize structural elements consistent with the experimental DC data and to identify structural parameters that require further refinement, which could prove important for the success of DC-based structure calculations . This approach is illustrated for the 42 kDa maltodextrin-binding protein.

Can J Microbiol, 2000 Dec, 46(12), 1101 - 7
Phase variation of F165(1) (Prs-like) fimbriae from Escherichia coli causing septicaemia in animals; Harel J et al.; Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and synthesize fimbriae involved in virulence, designated as F165(1) . F165(1) fimbriae belong to the P fimbrial family and are encoded by the foo gene cluster . The foo regulatory region of strain 5131 possesses characteristics similar to that of members of the P regulatory family, including papI and papB homologues, and two GATC sites separated by 102 bp, targets of differential Dam methylation . In wild-type strains, the synthesis of F165(1) is repressed by leucine and the fimbriae undergo phase variation . Immunofluorescence staining showed that phase variation of F165(1) results in a majority of cells (98%) in the ON phase, in contrast with phase variation of other members of this regulatory family, for which the majority of the cells are in the OFF state . Using a translational fusion in strain 5131 between phoA and fooA, encoding for the major structural subunit of F165(1), it was shown that leucine inhibits the OFF to ON switch and modulates the basal transcription of the foo operon.

RNA, 2000 Dec, 6(12), 1870 - 81
Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells; Gutgsell N et al.; Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) established that TruB, a pseudouridine (psi) synthase from Escherichia coli, was able to make psi55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs . By deletion of the truB gene, we now show that TruB is the only protein in E . coli able to make psi55 in vivo . Lack of TruB and psi55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type . The negative selection did not appear to be acting at either the exponential or stationary phase . Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to approximately 0.2% of its original value . The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB . Direct analysis showed that this mutant did not make psi55 under the conditions of the competition experiment . Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of psi55.

RNA, 2000 Dec, 6(12), 1714 - 26
Magnesium ions mediate contacts between phosphoryl oxygens at positions 2122 and 2176 of the 23S rRNA and ribosomal protein L1; Drygin D et al.; The complex of ribosomal protein L1 with 23S rRNA from Escherichia coli is of great interest because of the unique structural and functional aspects of this ribonucleoprotein domain . We have minimized the binding site for protein L1 on the 23S rRNA to nt 2120-2129, 2159-2162, and 2167-2178 . This RNA fragment consists of two helices as well as an interconnecting loop of unknown structure . RNA molecules corresponding to the minimized L1 binding site, in which G, A, U, or C were individually replaced by their deoxyribo- (dN) or alpha-thio- (rNaS) analogs have been synthesized by T7 transcription in vitro and analyzed for their ability to bind protein L1 . It has been demonstrated that the substitution of rNaS at position 2122 or 2176 decreases the affinity of the RNA for the protein in the presence of magnesium five- to tenfold, whereas the same changes have little effect on binding in the presence of manganese . This suggests that Rp oxygens in the phosphates preceding positions 2122 and 2176 are coordinated with Mg2+ and may participate in L1-23S rRNA interaction via magnesium bridges . We have also shown that this interaction is impaired by the presence of dC at position 2122 coupled with the presence of deoxyribonucleotide(s) at other positions in the RNA . This study demonstrates that the ribose-phosphate backbone of the helix encompassing nt 2120-2124/2174-2178 is intimately involved in the interaction of protein L1 with the 23S rRNA . In particular, we suggest that this helix is positioned in the cleft between the two domains of protein L1.

RNA, 2000 Dec, 6(12), 1704 - 13
The ribosomal binding and peptidyl-tRNA hydrolysis functions of Escherichia coli release factor 2 are linked through residue 246; Wilson DN et al.; Replacing a cassette of 31 residues from Escherichia coli release factor 1 with the equivalent residues in release factor 2 gave a protein active in codon-specific binding to the ribosome but inactive in peptidyl-tRNA hydrolysis . Such a phenotype is also found unexpectedly with release factor 2 when expressed at high concentration in bacteria . Substituting threonine with the release factor 1 equivalent serine at position 246 within the cassette restored the impaired activity of the chimeric protein, and also that of inactive recombinant release factor 2, both in vitro and in vivo . The differences in activity are not due to posttranslational modifications or a lack of it at this residue . Random mutagenesis of codon 246 suggests that this position is pivotal for the function of the release factor, being able to affect differentially both its binding to the ribosome and its peptide release activities . We propose that amino acid 246 is close to a sharp turn (GGQ motif at position 250), and is essential for transmitting the signal from cognate codon recognition by correctly positioning the peptidyl-tRNA hydrolysis domain of the release factor into the peptidyltransferase center.

J Exp Bot, 2000 Dec, 51(353), 1979 - 90
Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I; Balestrazzi A et al.; Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus carota) cDNA library and two classes of nucleotide sequences were found . One component of the first class, pTop9, perfectly matches the open reading frame of pTop28, a truncated top1 cDNA previously described, and extended it by 594 nucleotides (top1alpha) . A member of the second class, pTop11, contains an open reading frame 2727 bp long (top1ss) with a coding capacity for a second putative DNA topoisomerase I of 101 kDa . Both pTop9 and pTop11 clones are full length cDNAs . The two deduced amino acid sequences share a relevant similarity (89%) only at the C-terminal domain, whereas the similarity is reduced to 32% in the N-terminal region . Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the presence of two distinct loci . Northern blot analysis revealed the presence of two distinct transcripts of 3.0 and 3.2 kb in both cycling and starved cell populations . Three fusion peptides corresponding to the N-terminal domain of the alpha and ss forms and from the common C-terminal domain of carrot topoisomerases I were overexpressed in E . coli cells and used to raise antibodies in rabbit . Immunolocalization seems to suggest the presence of two topoisomerases I in carrot nuclei.

J Med Chem, 2001 Jan 4, 44(1), 36 - 46
Toward a rational design of peptide inhibitors of ribonucleotide reductase: structure-function and modeling studies; Pender BA et al.; Mammalian ribonucleotide reductase, a chemotherapeutic target, has two subunits, mR1 and mR2, and is inhibited by AcF(1)TLDADF(7), denoted P7 . P7 corresponds to the C-terminus of mR2 and competes with mR2 for binding to mR1 . We report results of a structure-function analysis of P7, obtained using a new assay measuring peptide ligand binding to mR1, that demonstrate stringent specificity for Phe at F(7), high specificity for Phe at F(1), and little specificity for the N-acyl group . They support a structural model in which the dominant interactions of P7 occur at two mR1 sites, the F(1) and F(7) subsites . The model is constructed from the structure of Escherichia coli R1 (eR1) complexed with the C-terminal peptide from eR2, aligned sequences of mR1 and eR1, and the trNOE-derived structure of mR1-bound P7 . Comparison of this model with similar models constructed for mR1 complexed with other inhibitory ligands indicates that increased F(1) subsite interaction can offset lower F(7) subsite interaction and suggests strategies for the design of new, higher affinity inhibitors.

Biochemistry, 2001 Jan 9, 40(1), 153 - 9
Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity; Zhang K et al.; DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage . One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates . Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem . We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector . To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification . The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light . In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E . coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E . coli endonuclease IV . Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I . This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps . In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and multiply damaged sites that might otherwise be difficult to prepare by other methods due to their lability.

Biochemistry, 2001 Jan 9, 40(1), 15 - 27
Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase; Edwards RA et al.; Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase . Biochemical and spectroscopic characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme . X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures . The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel . Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects . The Q146E mutant is isolated as an apoprotein lacking dismutase activity . Q146L and Q146H mutants retain only 5-10% of the dismutase activity of the wild-type enzyme . The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms . Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH . Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.

Nature, 2000 Dec 21-28, 408(6815), 1008 - 12
Structural basis of IAP recognition by Smac/DIABLO; Wu G et al.; Apoptosis is an essential process in the development and homeostasis of all metazoans . The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins . The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions . Amino-terminal sequences in Smac/DIABLO are required for this function, as mutation of the very first amino acid leads to loss of interaction with IAPs and concomitant loss of Smac/DIABLO function . Here we report the high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP . Our results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighbouring residues on BIR3 . These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper . In conjunction with other observations, our results reveal how Smac may relieve IAP inhibition of caspase-9 activity . In addition to explaining a number of biological observations, our structural analysis identifies potential targets for drug screening.

Nature, 2000 Dec 21-28, 408(6815), 1004 - 8
Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain; Liu Z et al.; The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes . Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation . Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function . To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac . The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein . The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions . This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.

Nucleic Acids Res . 2001 Jan 15;29(2):E8.
Isolation of segments of homologous genes with only one conserved amino acid region via PCR; Laging M et al.; We present a method which allows the isolation of fragments from genes coding for homologous proteins via PCR when only one block of conserved amino acids is available . Sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences . The second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by DNA synthesis . The cassette is ligated to partially-digested chromosomal DNA . The second primer is biotinylated to allow elimination of PCR products carrying degenerated primers on both sides via streptavidin binding . Fragments obtained after amplification and enrichment are cloned and sequenced . The feasibility of this method was demonstrated in a model experiment, where degenerated primers were deduced from six conserved amino acids within the family of homologs to the Escherichia coli Vsr protein.

Nucleic Acids Res, 2001 Jan 15, 29(2), 553 - 64
Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain; Chmiel NH et al.; The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs . The N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain . Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition . In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions . In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments . Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release . However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme . In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type . The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G . Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms . Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo . This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

Nucleic Acids Res, 2001 Jan 15, 29(2), 536 - 44
Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4; Tocchetti A et al.; DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha . In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation) . Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death . Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs . Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization . Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control.

Nucleic Acids Res, 2001 Jan 15, 29(2), 506 - 14
DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli; Plumbridge J; The NagC and Mlc proteins are homologous transcriptional regulators that control the expression of several phosphotransferase system (PTS) genes in Escherichia coli . NagC represses nagE, encoding the N:-acetylglucosamine-specific transporter, while Mlc represses three PTS operons, ptsG, manXYZ and ptsHIcrr, involved in the uptake of glucose . NagC and Mlc can bind to each others operator, at least in vitro . A binding site selection procedure was used to try to distinguish NagC and Mlc sites . The major difference was that all selected NagC binding sites had a G or a C at positions +11/-11 from the centre of symmetry . This is also the case for most native NagC sites, but not the nagE operator, which thus looks like a potential Mlc target . The nagE operator does exhibit a higher affinity for Mlc than NagC, but no regulation of nagE by physiological concentrations of Mlc was detected in vivo . Regulation of wild-type nagE by NagC is achieved because of the chelation effect due to a second high affinity NagC operator covering the nagB promoter . Replacing the A/T at +11/-11 with C/G allows repression by NagC in the absence of the nagB operator.

Nucleic Acids Res, 2001 Jan 15, 29(2), 430 - 8
Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair; Hill JW et al.; 8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species . Human OGG1 excised the damaged base from an 8-oxoG . C-containing duplex oligo with a very low apparent k(cat) of 0.1 min(-1) at 37 degrees C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product . Excision of 8-oxoG by OGG1 alone did not follow Michaelis-Menten kinetics . However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased approximately 5-fold and Michaelis-Menten kinetics were observed . Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity . The affinity of OGG1 for its product AP.C pair (K:(d) approximately 2.8 nM) was substantially higher than for its substrate 8-oxoG.C pair (K:(d) approximately 23 . 4 nM) and the affinity for its final ss-elimination product was much lower (K:(d) approximately 233 nM) . These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover . These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.

Nucleic Acids Res, 2001 Jan 15, 29(2), 407 - 14
Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII; Venkhataraman R et al.; Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine . DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes . It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair . However, yNtg2p, E . coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion . Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair . Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3'- or 5'-terminus of the cleaved fragment . On the other hand, yeast yNtg2p can remove DHU remaining on the 5'-terminus of the 3' cleaved fragment, but is unable to remove DHU remaining on the 3'-terminus of the cleaved 5' fragment . In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3'-terminus of a cleaved 5' fragment, but are unable to remove DHU remaining on the 5'-terminus of a cleaved 3' fragment . Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species . The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.

Mutagenesis, 2001 Jan, 16(1), 65 - 9
Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo{c}phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2; Ponten I et al.; The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo{c}phenan-threne (benzo{c}phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3' . The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates . The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion . The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques . Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed . Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.

Mutagenesis, 2001 Jan, 16(1), 1 - 6
Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichia coli; Tanaka M et al.; Using purified photolyases for pyrimidine (6-4) pyrimidone photoproducts {(6-4)PP} and cyclobutane pyrimidine dimers (CPD), the effects of photoreactivation on mutagenesis were examined in the supF gene on a plasmid transfected into repair-deficient SOS-induced Escherichia coli host cells . More than 95% of CPD and (6-4)PP were removed from plasmid DNA by treatment with CPD photolyase and (6-4)photolyase, respectively . In each photolyase treatment, base substitutions at dipyrimidine sequences were predominantly observed . Of the single base substitutions observed after CPD photoreactivation, 83% were A:T-->G:C transitions at 5'-TT-3' sites . After (6-4)photolyase treatment, 81% were G:C-->A:T transitions at 5'-CC-3' and 5'-TC-3' sequences . Thus, the major mutagenic photoproducts of single-base substitutions were CPD at 5'-CC-3' or 5'-TC-3' sites and (6-4)PP at 5'-TT-3' sites . Tandem double mutations occurred mainly at 5'-CC-3' sites and were CPD-photoreactivated, suggesting that CPD at 5'-CC-3' was responsible for tandem double mutations . After photoreactivation of both CPD and (6-4)PP, single-base substitutions were primarily G:C-->A:T transitions at 5'-CC-3' or 5'-TC-3' sites and A:T-->G:C transitions at 5'-TT-3' sites, and secondarily G:C-->T:A transversions at 5'-CC-3' sites, G:C-->C:G transversions at 5'-CC-3' sites and A:T-->T:A transversions at 5'-TT-3' sites, which were essentially the same as those observed after photoreactivation of CPD alone, (6-4)PP alone and without photoreactivation . Thus, these transversions were not derived from unknown UV adducts but from incompletely repaired CPD and (6-4)PP.

Biochem J, 2001 Jan 15, 353(Pt 2), 345 - 55
Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers; Hughes RK et al.; A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase) . Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state . Typical of other potato tuber lipoxygenases, it produced 5-HPETE {5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid} from arachidonate . In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate . We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity . We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase . This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases . We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access . Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Biochem J, 2001 Jan 15, 353(Pt 2), 207 - 13
Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide; Mills CE et al.; A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents . H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents . H(2)O(2) probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product . In the absence of agents that scavenge superoxide and peroxide, the mean K(m) for oxygen was 80 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O(2) . Purified Hmp consumed NO in the absence of added FAD (approx . 1 O(2) per NO), which is consistent with NO oxygenation . However, half-maximal rates of NO-stimulated O(2) consumption required approx . 47 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx . 30 microM O(2)) . On exhaustion of O(2), NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx . 1% of that for oxygenase activity . These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.

Biochem J, 2001 Jan 15, 353(Pt 2), 181 - 91
Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221-->Met mutant; Howes BD et al.; A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli . In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom . The Phe-221-->Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid . Significant differences between the mutant and wild-type enzymes can be detected spectroscopically . These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pK(a) of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms . The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by (1)H-NMR data, and the loss of the pi-pi interaction between His-170 and Phe-221.

Clin Diagn Lab Immunol, 2001 Jan, 8(1), 178 - 80
Assessment of neutrophil function in patients with septic shock: comparison of methods; Wenisch C et al.; Patients with septic shock are shown to have decreased neutrophil phagocytic function by multiple assays, and their assessment by whole-blood assays (fluorescence-activated cell sorter analysis) correlates with assays requiring isolated neutrophils (microscopic and spectrophotometric assays) . For patients with similar underlying conditions but without septic shock, this correlation does not occur.

Clin Diagn Lab Immunol, 2001 Jan, 8(1), 143 - 9
The AIDA autotransporter system is associated with F18 and stx2e in Escherichia coli isolates from pigs diagnosed with edema disease and postweaning diarrhea; Niewerth U et al.; Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets . Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, E . coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD . In light of these observations we investigated whether another E . coli adhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causing E . coli isolates . For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed . When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcine E . coli isolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects in orfB were associated with the absence of disease . Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.

Ecotoxicol Environ Saf, 2000 Nov, 47(3), 246 - 52
Impact of lead stress and adaptation in Escherichia coli; Kumar M et al.; The growth rate of Escherichia coli was stimulated when cells were in media containing lead up to a concentration of 300 ppm . Higher concentrations inhibited growth . Metal analysis revealed that in the presence of lead E . coli concentrates 22.8 mg of lead per gram (dry weight) of cells . Analysis of cellular subfractions indicated that membrane fraction concentrated over 95% of the lead taken up by cells, of which a major portion was found to be associated with membrane lipids . Alterations in alkaline phosphatase, Ca2+-Mg2+ -ATPase activities and the carbohydrate and phospholipid contents in membrane fractions were also observed when cells were grown in the presence of lead . A time- and concentration-dependent increase in the release of carbohydrates by the cells was also evident . The results suggest that besides thriving in higher lead surroundings, E . coli possess a marked ability to concentrate substantial amount of inorganic lead.

Extremophiles, 2000 Dec, 4(6), 333 - 41
Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications; Bhuiya MW et al.; NADP-dependent glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization . The enzyme retained its full activity on heating at 95 degrees C for 30 min, and the maximum activity in L-glutamate deamination was obtained around 100 degrees C . The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination . The Km values for NADP, L-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively . On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A . pernix K1 genome, cloned, and expressed in Escherichia coli . Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46170 . Phylogenetic analysis revealed that the glutamate dehydrogenase from A . pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota . The branching pattern of the enzymes from A . pernix K1, S . solfataricus, S . shibatae, and Pb . islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms.

Pancreas, 2001 Jan, 22(1), 32 - 9
Altered cytokine response in rat acute pancreatitis complicated with endotoxemia; Okabe A et al.; We demonstrated that the dynamic aspects of cytokine production in rat acute pancreatitis, which was induced by cerulein and aggravated by subsequent lipopolysaccharide (LPS) injection . A priming effect by induction of mild pancreatitis with cerulein enhanced the subsequent cytokine production by LPS injection . Alternatively, after induction of severe pancreatitis with cerulein and LPS, cytokine production was markedly suppressed for > or = 90 hours . Production of interleukin-2 (IL-2) by splenocytes decreased, and mortality rate after cecal ligation and puncture (CLP) increased significantly after induction of severe acute pancreatitis . These results suggest that the suppression of a cytokine response in severe acute pancreatitis may alter the defense system and, as a result, increase mortality after CLP.

Vaccine, 2000 Dec 8, 19(9-10), 1008 - 12
The comparison of the effect of LTR72 and MF59 adjuvants on mouse humoral response to intranasal immunisation with human papillomavirus type 6b (HPV-6b) virus-like particles; Greer CE et al.; Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site . Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity . We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant . Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation . These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

Chem Biol Interact, 2000 Dec 15, 129(3), 249 - 61
Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: the formation of a lipophilic ternary complex; Zhu BZ et al.; When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation . Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes . The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells . The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.

J Clin Microbiol, 2001 Jan, 39(1), 370 - 4
Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes; Bellin T et al.; In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument . In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary . A complete analysis of up to 32 samples takes about 45 min.

J Clin Microbiol, 2001 Jan, 39(1), 1 - 7
Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins; Saijo M et al.; The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system . Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system . The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves . The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity . The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak . We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens . We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients . The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies . The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.

Hum Mol Genet, 2001 Jan 1, 10(1), 47 - 54
The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy; Newbold RJ et al.; Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state . In this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation . The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50 . In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L . The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability . Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1 . In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra . However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy . It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter {Ca2+} and result in death of cells.

Hum Mol Genet, 2001 Jan 1, 10(1), 1 - 8
The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death; Putaala H et al.; A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination . The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter . Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25% . Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h . The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients . In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/-) and (-/-) mice . In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb . In the cerebellum, the expression was seen in radial glial cells . Neither anatomical nor morphological abnormalities were observed in the brains of null mice.

Mol Genet Metab, 2000 Dec, 71(4), 623 - 32
Changes in the carboxyl terminus of the beta subunit of human propionyl-CoA carboxylase affect the oligomer assembly and catalysis: expression and characterization of seven patient-derived mutant forms of PCC in Escherichia coli; Chloupkova M et al.; Propionyl-CoA carboxylase (PCC) catalyzes the biotin-dependent carboxylation of propionyl-CoA to d-methylmalonyl-CoA in the mitochondrial matrix . Human PCC is a dodecamer composed of pairs of nonidentical alpha and beta subunits encoded by PCCA and PCCB genes, respectively . Deficiency of PCC results in propionic acidemia (PA), a metabolic disorder characterized by severe metabolic ketoacidosis, vomiting, lethargy, and hypotonia . To date, almost 60 mutations have been reported in both genes . Exon 15 of the beta subunit is one of the two sites where a number of mutations have been identified in PA patients . In the primary betaPCC sequence, these mutations lead to three substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X, and W531X), and one insertion (A51_R514insP) . We expressed these mutant proteins in Escherichia coli in which the GroESL complex was overexpressed . The only mutation that does not impact the stability of mutant betaPCC in bacteria is W531X . The remaining mutations lead to either complete (L519P, N536D) or partial (R499X, R512C, A513_R514insP, and R514X) degradation of the mutant subunits . Size-exclusion chromatography revealed that R512C and W531X do not affect the assembly of alphaPCC and betaPCC to active oligomers . Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively . Taken together, the carboxyl-terminal portion of 40 amino acid residues of the beta subunit affects the stability and the assembly of the alpha and beta subunits as well as the carboxylation of propionyl-CoA .

Mol Microbiol, 2001 Jan, 39(2), 502 - 11
CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA; Stoyanov JV et al.; We have shown that the open reading frame ybbI in the genomic sequence of Escherichia coli K-12 encodes the regulator of expression of the copper-exporting ATPase, CopA . In vivo studies showed that ybbI (designated cueR for copper export regulator gene) was required for copper tolerance during growth, that disruption of cueR caused loss of copA expression and that copA gene expression was regulated by cueR and by copper or silver ions . Expression of a lacZ reporter gene under the control of the copA promoter was approximately proportional to the concentration of cupric ions in the medium, but increased more rapidly in response to silver ion concentrations . The start of the copA transcript was located by primer extension mapping, and DNase I protection assays showed that the CueR protein binds in vitro to a dyad symmetrical sequence within a 19 bp spacer sequence in the copA promoter . CueR binding occurs in vitro in both the presence and the absence of RNA polymerase with or without copper ions present but, in the presence of CueR, RNA polymerase and copper ions, permanganate-sensitive transcription complexes were formed . CueR is predicted to have an N-terminal helix-turn-helix sequence and shows similarity to MerR family regulators.

Mol Microbiol, 2001 Jan, 39(2), 494 - 501
Single-stranded DNA intermediates in IS91 rolling-circle transposition; del Pilar Garcillan-Barcia M et al.; IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition . We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination . To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine . Mutation of either tyrosine resulted in complete loss of transposition activity in vivo . This result was taken as a first new line of evidence that TnpA is a functional analogue of phiX174 phage A protein . Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis . Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression . The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element . When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance . The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.

Mol Microbiol, 2001 Jan, 39(2), 416 - 28
The proteolytic control of restriction activity in Escherichia coli K-12; Doronina VA et al.; The endonuclease activity of EcoKI is regulated by the ClpXP-dependent degradation of the subunit that is essential for restriction, but not modification . We monitored proteolysis in mutants blocked at different steps in the restriction pathway . Mutations that prevent DNA translocation render EcoKI refractory to proteolysis, whereas those that permit DNA translocation, but block endonuclease activity, do not . Although proteolysis alleviates restriction in a mutant that lacks modification activity, some restriction activity remains; our evidence indicates residual EcoKI associated with the membrane fraction . ClpXP protects the bacterial chromosome, but little effect was detected on unmodified foreign DNA within the cytoplasm of a restriction-proficient cell . The molecular basis for the distinction between unmodified resident and foreign DNA remains to be determined.

Mol Microbiol, 2001 Jan, 39(2), 370 - 8
Two independent transcriptional units control the complex and simultaneous expression of the bmp paralogous chromosomal gene family in Borrelia burgdorferi; Dobrikova EY et al.; The chromosomal paralogous gene family 36 encodes for four lipoproteins with high amino acid homology that are expressed in vivo in humans and animals and are immunogenic . Transcriptional analysis of the bmp gene cluster indicated that all four genes of this cluster are expressed in vitro and constitute two transcriptional units with a complex pattern of transcription, including alternative monocistronic and polycistronic messages . One unit consists of bmpD, whose transcription is coupled to the transcription of the ribosomal protein genes, rpsG and rpsL . The second unit includes bmpC, bmpA and bmpB . The simultaneous expression of the four bmp genes in Borrelia burgdorferi suggests that their gene products may have either different or complementary functions . Primer extension experiments identified promoters for bmpD, bmpC and bmpA, but not for bmpB . The concentration of gene-specific mRNA paralleled its promoter homology to the Escherichia coli sigma70 promoter . The linkage of bmpD expression to rpsL and rpsG suggests that the expression of this gene may be controlled by growth-related global regulation mechanisms in B . burgdorferi . These results indicate that the bmp family constitutes a good model for the investigation of complex regulation of chromosomal gene expression in this bacteria.

Mol Microbiol, 2001 Jan, 39(2), 286 - 90
The Nudix hydrolases of Deinococcus radiodurans; Xu W et al.; All 21 of the Nudix hydrolase genes from the radiation-resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli . Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids) . Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases . Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified . In addition to representatives of known Nudix hydrolase subfamilies active on ADP-ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP-glucose pyrophosphatase and a CoA pyrophosphatase, were identified.

Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 81 - 6
Engineering of a functional human NADH-dependent cytochrome P450 system; Dohr O et al.; A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s . This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site . Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2 . Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH . The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively . In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant . Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity . Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.

Zhonghua Gan Zang Bing Za Zhi, 2000 Dec, 8(6), 364 - 6
{The construction of PQE-ARL-1 recombinant expression plasmid and the preparation and purification of ARL-1 protein}; Zheng C et al.; OBJECTIVE: To further understand the function and biological activity of aldose reductase like-1 (ARL-1), and to provide the basis for the preparation of the specific antibody against ARL-1 . METHODS: The ARL-1 cDNA was cloned into procaryotic expression vector PQE-30 and the recombinant ARL-1 expressed in E.coli M(15); then Ni(+)-NTA agarose columns were used to purify the ARL-1 protein . RESULTS: With the digestion of the enzymes, we identified that the ARL-1 gene was inserted into the procaryotic expression vector PQE-30 . The expression products of PQE-ARL-1 (human recombinant ARL-1) showed a single protein band on SDS-PAGE . The molecular weight of ARL-1 was approximately 37.7 x 10(3) and the expression level was about 25% of the total bacterial protein . The concentration of ARL-1 protein was about 100mg/L . CONCLUSION: The construction of the recombinant plasmid PQE-ARL-1 and the preparation of the protein ARL-1 have laid a solid foundation for further studying the function of ARL-1 and preparing the specific antibody against ARL-1.

Nat Struct Biol, 2001 Jan, 8(1), 62 - 7
Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I; Hadden JM et al.; We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 A resolution using the multiwavelength anomalous dispersion (MAD) technique . Endonuclease I exhibits strong structural specificity for four-way DNA junctions . The structure shows that it forms a symmetric homodimer arranged in two well-separated domains . Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site . While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases . T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.

Nat Struct Biol, 2001 Jan, 8(1), 52 - 6
Crystal structure of an activated response regulator bound to its target; Lee SY et al.; The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli . We have determined the crystal structure of BeF3--activated CheY from E . coli in complex with an N-terminal peptide derived from its target, FliM . The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face . The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Nat Struct Biol, 2001 Jan, 8(1), 32 - 6
Dissection of a (betaalpha)8-barrel enzyme into two folded halves; Hocker B et al.; The (betaalpha)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain . However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (betaalpha) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C) . HisF-N consists of the four N-terminal (betaalpha) units and HisF-C of the four C-terminal (betaalpha) units . It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel . To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized . Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive . Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex . These findings support the hypothesis that the (betaalpha)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.

Nat Biotechnol, 2001 Jan, 19(1), 75 - 8
A helper phage to improve single-chain antibody presentation in phage display; Rondot S et al.; We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage) . Hyperphage have a wild-type pIII phenotype and are therefore able to infect F(+) Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII-antibody fusion is the sole source of pIII in phage assembly . This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface . Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle . When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin . After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used . Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.

Biotechnol Bioeng, 2001 Feb 5, 72(3), 315 - 22
Kinetic model of in vivo folding and inclusion body formation in recombinant Escherichia coli; Hoffmann F et al.; Aggregation of misfolded proteins can reduce the yield in recombinant protein production . The underlying complex processes are additionally influenced by cellular physiology . Nevertheless, a lumped-parameter model of kinetic competition between folding and aggregation was sufficient to track properly the specific concentration of a human protein produced in E . coli and its partitioning into soluble and insoluble cell fractions . Accurate estimation of the protein-specific parameters required informative experiments, which were designed using the Fisher information matrix . The model was employed to calculate the influence of the specific glucose uptake rate in high-cell-density cultivation of E . coli on accumulation and aggregation of the recombinant protein . Despite its simplicity, the model was flexible and unbiased concerning unidentified mechanisms . Assuming an exponentially decreasing production rate, the irreversible aggregation step was found to follow first order kinetics, while assuming a constant production rate with simultaneous degradation, the model predicted transient aggregation only . Implications for strain and process development are discussed .

Plant J, 2000 Dec, 24(6), 797 - 804
Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase; Irmler S et al.; The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown . We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific . It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis . The early reactions in that pathway, i.e . from geraniol to strictosidine, contain several candidates for P450 activities . We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS) . The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant . We show for the first time that both enzyme activities are present in microsomes from C . roseus cell cultures . We then tested whether CYP72A1 expressed in E . coli as a translational fusion with the C . roseus P450 reductase (P450Red) has one or both of these activities . The results show that CYP72A1 converts loganin into secologanin.

Kidney Int, 2001 Jan, 59(1), 44 - 51
Effect of indomethacin on peritoneal protein loss in a rabbit model of peritonitis; Peng H et al.; BACKGROUND: Although various inflammatory mediators have been previously shown to be released into the peritoneal cavity during peritonitis in peritoneal dialysis patients, those that are involved in governing changes in peritoneal permeability to small solutes and protein remain incompletely defined . METHODS: We determined the importance of prostanoid production in the enhanced protein loss observed during acute peritonitis by inhibition experiments using indomethacin, an inhibitor of cyclooxygenase activity . The association between changes in peritoneal permeability and the generation of inflammatory mediators after adding Escherichia coli to peritoneal dialysate was first examined in series 1 experiments . Series 2 experiments then determined the effect of intraperitoneal administration of indomethacin (75 microg/mL) on changes in peritoneal permeability after adding E . coli to peritoneal dialysate . All experiments were performed in male New Zealand White rabbits (2.6 to 3.4 kg body weight) using an eight-hour dwell of dialysate containing 2.5% glucose . Peritoneal permeability to creatinine and protein was assessed by time-dependent changes in the dialysate to plasma concentration ratios of these solutes . RESULTS: Series 1 experiments showed enhanced leukocyte migration into the peritoneal cavity and increased peritoneal permeability to protein during bacterial challenge that was accompanied by an increase in the dialysate concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1alpha, and interleukin-8, but not nitrate + nitrite (a measure of local nitric oxide production) . Inhibition of prostanoid production by intraperitoneal administration of indomethacin in series 2 experiments resulted in lower dialysate concentrations of PGE2 and 6-keto-PGF1alpha and in lower peritoneal permeability to protein, both to control levels . No effect of indomethacin on transperitoneal migration of leukocytes or the generation of interleukin-8 was observed . CONCLUSIONS: Enhanced production of prostanoids likely plays an important role in governing the increase in peritoneal permeability to protein during acute, bacterial peritonitis in the rabbit.

J Biochem (Tokyo), 2001 Jan, 129(1), 119 - 24
The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases; Gaitatzis N et al.; Myxothiazol is synthesized by the myxobacterium Stigmatella aurantiaca DW4/3-1 via a combined polyketide synthase/polypeptide synthetase . The biosynthesis of this secondary metabolite is also dependent on the gene product of mtaA . The deduced amino acid sequence of mtaA shows similarity to 4'-phosphopantetheinyl transferases (4'-PP transferase) . This points to an enzyme activity that converts inactive forms of the acyl carrier protein domains of polyketide synthetases (PKSs) and/or the peptidyl carrier protein domains of nonribosomal polypeptide synthetases (NRPSs) of the myxothiazol synthetase complex to their corresponding holo-forms . Heterologous co-expression of MtaA with an acyl carrier protein domain of the myxothiazol synthetase was performed in Escherichia coli . The proposed function as a 4'-PP transferase was confirmed and emphasizes the significance of MtaA for the formation of a catalytically active myxothiazol synthetase complex . Additionally, it is shown that MtaA has a relaxed substrate specificity: it processes an aryl carrier protein domain derived from the enterobactin synthetase of E . coli (ArCP) as well as a peptidyl carrier protein domain from a polypeptide synthetase of yet unknown function from Sorangium cellulosum . Therefore, MtaA should be a useful tool for activating heterologously expressed PKS and NRPS systems.

J Biochem (Tokyo), 2001 Jan, 129(1), 101 - 5
Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase; Nakanishi M et al.; Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized . Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values . The values of both forward and reverse reactions of P . falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme . Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively . On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P . falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme . P . falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria . This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.

J Biochem (Tokyo), 2001 Jan, 129(1), 87 - 91
Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli; Nada S et al.; Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter {TetA(B)His} as an antigen . From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1 . Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein . First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed . Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C . Then, we constructed 13 cysteine-scanning mutants, F179C to T191C . Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding . These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B) . TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 159 - 61
Crystallization of the yeast elongation factor complex eEF1A-eEF1B alpha; Pedersen L et al.; Crystals of the Saccharomyces cerevisiae elongation factor eEF1A (formerly EF-1 alpha) in complex with a catalytic C-terminal fragment of the nucleotide-exchange factor eEF1B alpha (formerly EF-1 beta) were grown by the sitting-drop vapour-diffusion technique, using polyethylene glycol 2000 monomethyl ether as precipitant . Crystals diffract to better than 1.7 A and belong to the space group P2(1)2(1)2(1) . The unit-cell parameters of the crystals are sensitive to the choice of cryoprotectant . The structure of the 61 kDa complex was determined with the multiple anomalous dispersion technique using three selenomethionine residues in a 11 kDa eEF1B alpha fragment generated by limited proteolysis of full-length eEF1B alpha expressed in Escherichia coli.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 150 - 2
Expression, purification, crystallization and preliminary X-ray analysis of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase; Lee JE et al.; A recombinant form of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (E.C . 3.2.2.9) has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique . While several different crystallization conditions were obtained, only one set of conditions yielded crystals suitable for X-ray diffraction analysis . These crystals grow as diamond-shaped wedges, with unit-cell parameters a = 50.92, b = 133.99, c = 70.88 A, alpha = beta = gamma = 90 degrees . The crystals belong to space group P2(1)2(1)2 and diffract to a minimum d spacing of 2.3 A on a MAR345 image plate with a Rigaku RU-200 rotating-anode X-ray generator . On the basis of density calculations, two monomers are predicted per asymmetric unit (Matthews coefficient, V(M) = 2.37 A(3) Da(-1)), with a solvent content of 48%.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 148 - 9
Crystallization and preliminary X-ray crystallographic analysis of DFPase from Loligo vulgaris; Scharff EI et al.; 'Squid-type' diisopropylfluorophosphatases (DFPases), a subclass of the phosphotriesterases, are enzymes capable of hydrolysing organophosphorus nerve agents . To date, no three-dimensional structure of a 'squid-type' DFPase is known . Here, the crystallization of the DFPase originally isolated from head ganglion of the squid Loligo vulgaris is reported . The protein has been heterologously expressed in Escherichia coli, purified to homogeneity and subsequently crystallized . The protein crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.1, b = 82.1, c = 86.6 A and one monomer per asymmetric unit . Under cryoconditions (120 K) the crystals diffracted beyond 2.0 A using a Cu rotating-anode X-ray generator.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 140 - 2
Purification, crystallization and preliminary X-ray analysis of a maize cytokinin glucoside specific beta-glucosidase; Vevodova J et al.; Zm-p60.1, a cytokinin glucoside specific beta-glucosidase from maize, is a key enzyme involved in plant development and growth . It has been overexpressed in soluble form from Escherichia coli with a His tag at its N-terminus . The recombinant protein has been purified and crystallized at room temperature using PEG 4000 as the main precipitant . At least three crystal forms have been observed from very similar growth conditions . A flash-annealed monoclinic crystal diffracted to high resolution (beyond 2 A) with space group P2(1) and unit-cell parameters a = 55.66, b = 110.72, c = 72.94 A, beta = 92.10 degrees . The asymmetric unit is estimated and confirmed by molecular-replacement solution to contain one Zm-p60.1 dimer, giving a crystal volume per protein mass (V(M)) of 1.89 A(3) Da(-1) and a solvent content of 35%.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 137 - 9
Recombinant chemosensory protein (CSP2) from the moth Mamestra brassicae: crystallization and preliminary crystallographic study; Campanacci V et al.; Chemosensory proteins (CSPs) are small proteins (13 kDa on average) present in several sensory organs from a wide range of insect species . They are believed to be involved in chemoperception (olfaction or taste) and to play a role in chemical transport from air or water to chemosensitive receptors . Here, the first crystals of a CSP originating from the moth Mamestra brassicae (Mbra) proboscis and expressed as recombinant protein in Escherichia coli periplasm are reported . Crystals of MbraCSP2 were obtained by the hanging-drop vapour-diffusion method under the following conditions: 1 microl of a 46 mg ml(-1) protein solution in 50 mM Tris pH 8.0 containing cetyl alcohol as ligand (1:5 molar ratio) was mixed with 1 microl of well solution containing 30% PEG 4000, 0.2 M sodium acetate in 100 mM Tris at pH 8.4 . The protein-cetyl alcohol complex crystallizes in space group P2(1), with unit-cell parameters a = 47.9, b = 49.7, c = 50.3 A, beta = 110.1 degrees . With two molecules in the asymmetric unit, the V(M) is 2.15 A(3) Da(-1) and the solvent content is 42% . A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble) . Se-Met expression has been performed with a view to solving the CSP2 structure with MAD data collection using the Se absorption edge.

Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 63 - 8
RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E; Liou GG et al.; RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay . Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase . Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E . coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E . Whereas PNPase and enolase are present in E . coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages . Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E . coli, also suggest that RNA processing and decay may occur at specific sites within cells.

Pediatr Res, 2001 Jan, 49(1), 30 - 7
Transcytosis of gastrointestinal epithelial cells by Escherichia coli K1; Burns JL et al.; Escherichia coli K1 is an important neonatal pathogen that is usually transferred from maternal to infant gastrointestinal tract at the time of parturition . Approximately 20% of neonates are colonized, and a proportion of colonized infants goes on to have systemic infection . Entry into the bloodstream from the gastrointestinal tract is hypothesized to occur via epithelial cell invasion . Invasion of multiple epithelial cell lines was studied using gentamicin protection assays and transcytosis of polarized monolayers . Electron microscopy was used to confirm cellular invasion . Cell lines used include two human gastrointestinal lines, Caco-2 and T84; a human respiratory cell line, A549; a human laryngeal cell line, HEp-2; and a canine kidney cell line, MDCK . A virulent E . coli K1 strain, RS218, readily invaded HEp-2, A549, and T84 cell lines in gentamicin protection assays, but was less invasive into MDCK and Caco-2 cells . RS218 also demonstrated transcytosis of both T84 and Caco-2 cells . Four clinical isolates of E . coli K1 demonstrated levels of transcytosis of T84 cells similar to RS218 . Caco-2 invasiveness correlated with length of time in tissue culture with maximum invasiveness demonstrated at 11 d in culture, when cells were polarized and differentiated.

J Virol, 2001 Jan, 75(2), 979 - 87
Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat; Verhoef K et al.; Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic . We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox) . This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression . These designer "HIV-rtTA" viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration . These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the E . coli-derived Tet reagents in a eukaryotic background . Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.

J Virol, 2001 Jan, 75(2), 672 - 86
In vitro analysis of human immunodeficiency virus type 1 minus-strand strong-stop DNA synthesis and genomic RNA processing; Driscoll MD et al.; Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA . A detailed understanding of these activities is required to understand the process of reverse transcription . HIV-1 viral DNA is initiated from a tRNA(3)(Lys) primer bound to the viral genome at the primer binding site . The U3 and R regions of the RNA genome are the first sequences to be copied . The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation . Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR . In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization . If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities . After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization . Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion . RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5' end of the RNA that remained annealed to the 3' end of the minus-strand strong-stop DNA only if NC was present in the reaction.

J Bacteriol, 2001 Jan, 183(2), 671 - 9
Substitutions in the periplasmic domain of low-abundance chemoreceptor trg that induce or reduce transmembrane signaling: kinase activation and context effects; Beel BD et al.; We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor of Escherichia coli . Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand . We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors . Differential activation by the two mutant classes was not dependent on high-abundance receptors . Cellular context can affect the function of low-abundance receptors . Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors . In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors . Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites in trans in clusters of suppressing and suppressed receptors . As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.

J Bacteriol, 2001 Jan, 183(2), 644 - 53
Transcription of nhaA, the main Na(+)/H(+) antiporter of Escherichia coli, is regulated by Na(+) and growth phase; Dover N et al.; The transcription of nhaA, encoding the main Na(+)/H(+) antiporter of Escherichia coli, is induced by Na(+), regulated by NhaR, and affected by H-NS . In this work the roles of the two nhaA promoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations . We found that P1 is an NhaR-dependent, Na(+)-induced, and H-NS-affected promoter both in the exponential and stationary phases . An in vitro transcription assay demonstrated that P1 is activated by sigma(70)-RNA polymerase and both NhaR and H-NS increase the specificity of P1 . Remarkably, in marked contrast to P1, P2 exhibits very low activity during the exponential phase but is induced in the stationary phase to become the major promoter . Furthermore, P2 is activated by sigma(S) and is neither induced by Na(+) nor dependent on NhaR or affected by H-NS . Hence, this work establishes that nhaA has a dual mode of regulation, each involving a different promoter, and reveals that P2 and sigma(S) together are responsible for the survival of stationary-phase cells in the presence of high Na(+), alkaline pH, and the combination of high Na(+) and alkaline pH, the most stressful condition.

J Bacteriol, 2001 Jan, 183(2), 570 - 9
Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain; Gropp M et al.; The E . coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation . RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids {aa} 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity . We used mutational analysis to localize sites important for RelA activity and control in these two domains . We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp . When we caused the CTD in relA(+) cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected . Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD . When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation . In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD . Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G . Karimova, J . Pidoux, A . Ullmann, and D . Ladant, Proc . Natl . Acad . Sci . USA 95:5752-5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions . Our findings support a model in which RelA activation is regulated by its oligomerization state.

J Bacteriol, 2001 Jan, 183(2), 545 - 56
High-density microarray-mediated gene expression profiling of Escherichia coli; Wei Y et al.; A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides . To exploit this reagent, conditions for RNA isolation from E . coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established . A brief isopropyl-beta-D-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged . Distinct RNA expression patterns between E . coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media . The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor . This inventory provided a quantitative view of the steady-state level of each mRNA species . Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E . coli physiology.

J Bacteriol, 2001 Jan, 183(2), 520 - 7
Negative control of rpoS expression by phosphoenolpyruvate: carbohydrate phosphotransferase system in Escherichia coli; Ueguchi C et al.; The sigma(S) (or sigma(38)) subunit of RNA polymerase, encoded by the rpoS gene, is a crucial regulator in the transcriptional control of a set of genes under stressful conditions, such as nutrient starvation . The expression of rpoS is regulated in a complex manner at the levels of transcription, translation, and stability of the product . Although a number of factors involved in the regulation of rpoS expression have been identified, the underlying molecular mechanisms are not fully understood . In this study, we identified the Crr (or EIIA(Glc)) protein as a novel factor that plays an important role not only in the transcriptional control but also in the translational control of rpoS expression . Crr is an important component in glucose uptake through the well-characterized phosphoenolpyruvate:carbohydrate phosphotransferase system . The results of a series of genetic analyses revealed that Crr negatively controls rpoS translation and transcription . The observed transcriptional control by Crr appears to be mediated by cyclic AMP . However, it was found that Crr negatively controls rpoS translation rather directly . These results suggest a possible linkage between the control of rpoS expression and carbon metabolism.

J Bacteriol, 2001 Jan, 183(2), 512 - 9
The gaf fimbrial gene cluster of Escherichia coli expresses a full-size and a truncated soluble adhesin protein; Tanskanen J et al.; The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E . coli by affinity chromatography on GlcNAc-agarose . The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named DeltaGafD . Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of DeltaGafD . The peptide was also detected in the periplasm of the wild-type E . coli strain from which the gaf gene cluster originally was cloned . We expressed gafD fragments encoding C-terminally truncated peptides . Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of DeltaGafD . Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts . In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases . Pulse-chase assays using gafD indicated that DeltaGafD was processed from GafD and is not a primary translation product . The DeltaGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose . Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E . coli strain and in the binding of {(14)C}DeltaGafD to GlcNAc-agarose . DeltaGafD bound specifically to laminin, a previously described tissue target for the G fimbria . Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster.

J Bacteriol, 2001 Jan, 183(2), 461 - 7
Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity; Tseng CP et al.; Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle . Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time . Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively . There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source . However, these fumarase activities were still shown to be under growth rate control . Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently . Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent . Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels . While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2% . Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4% . The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants . Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.

J Bacteriol, 2001 Jan, 183(2), 426 - 34
Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity; Jubier-Maurin V et al.; Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel . DNA sequence analysis of the corresponding B . suis nik locus showed that it was highly similar to that of E . coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation . Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system . The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess . Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess . Moreover, the nikA mutant of B . suis was functionally complemented with the E . coli nik gene cluster, leading to the recovery of urease activity . Reciprocally, an E . coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B . suis nik locus . Taking into account these results, we propose that the nik locus of B . suis encodes a nickel transport system . The results further suggest that nickel could enter B . suis via other transport systems . Intracellular growth rates of the B . suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection . We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

Appl Environ Microbiol, 2001 Jan, 67(1), 363 - 70
Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544; Heyer AG et al.; We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein . With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library . The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no . AJ289046) is expressed in conidia, while no expression could be detected in mycelia by Northern blot analysis of mycelial RNA . The gene encodes a protein with a calculated molecular mass of 75 kDa that is different from all fructosyltransferases in the databases . The only homology that could be detected was to the invertase of Aspergillus niger (EMBL accession no . L06844) . The gene was functionally expressed in Escherichia coli, yeast, and potato plants . With protein extracts from transgenic bacteria and yeast, fructooligosaccharides could be produced in vitro . In transgenic potato plants, inulin molecules of up to 40 hexose units were synthesized in vivo . While in vitro experiments with protein extracts from conidia of Aspergillus sydowi yielded the same pattern of oligosaccharides as extracts from transformed bacteria and yeast, in vivo inulin synthesis with fungal conidia leads to the production of a high-molecular-weight polymer.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 421 - 6 Epub 2001 Jan 02.
A dietary source of coenzyme Q is essential for growth of long-lived Caenorhabditis elegans clk-1 mutants; Jonassen T et al.; Mutations in the clk-1 gene of the nematode Caenorhabditis elegans result in slowed development, sluggish adult behaviors, and an increased lifespan . CLK-1 is a mitochondrial polypeptide with sequence and functional conservation from human to yeast . Coq7p, the Saccharomyces cerevisiae homologue, is essential for ubiquinone (coenzyme Q or Q) synthesis and therefore respiration . However, based on assays of respiratory function, it has been reported that the primary defect in the C . elegans clk-1 mutants is not in Q biosynthesis . How do the clk-1 mutant worms have essentially normal rates of respiration, when biochemical studies in yeast suggest a Q deficiency? Nematodes are routinely fed Escherichia coli strains containing a rich supply of Q . To study the Q synthesized by C . elegans, we cultured worms on an E . coli mutant that lacks Q and found that clk-1 mutants display early developmental arrest from eggs, or sterility emerging from dauer stage . Provision of Q-replete E . coli rescues these defects . Lipid analysis showed that clk-1 worms lack the nematode Q(9) isoform and instead contain a large amount of a metabolite that is slightly more polar than Q(9) . The clk-1 mutants also have increased levels of Q(8), the E . coli isoform, and rhodoquinone-9 . These results show that the clk-1 mutations result in Q auxotrophy evident only when Q is removed from the diet, and that the aging and developmental phenotypes previously described are consistent with altered Q levels and distribution.

J Biol Chem, 2001 Apr 6, 276(14), 11230 - 6 Epub 2000 Dec 21.
The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding; Chu SY et al.; The x-ray crystal structure of the cAMP-ligated T127L/S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 A . Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain . In addition, water-mediated interactions replace the hydrogen bonding interactions between the N(6) of anti-cAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr(127) and Ser(128) residues in the C alpha-helix of wild type CRP . This replacement induces flexibility in the C alpha-helix at Ala(128), which swings the C-terminal domain of the open subunit more toward the N-terminal domain in the T127L/S128A double mutant of CRP (CRP*) than is observed in the open subunit of cAMP-ligated CRP . Isothermal titration calorimetry measurements on the binding of cAMP to CRP* show that the binding mechanism changes from an exothermic independent two-site binding mechanism at pH 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar to that observed for CRP at both pH levels . Differential scanning calorimetry measurements exhibit a broadening of the thermal denaturation transition of CRP* relative to that of CRP at pH 7.0 but similar to the multipeak transitions observed for cAMP-ligated CRP . These properties and the bound syn-cAMP ligand, which has only been previously observed in the DNA bound x-ray crystal structure of cAMP-ligated CRP by Passner and Steitz (Passner, J . M., and Steitz, T . A . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 2843-2847), imply that the cAMP-ligated CRP* structure is closer to the conformation of the allosterically activated structure than cAMP-ligated CRP . This may be induced by the unique flexibility at Ala(128) and/or by the bound syn-cAMP in the hinge region of CRP*.

J Biol Chem, 2001 Mar 30, 276(13), 9931 - 5 Epub 2000 Dec 21.
gamma-phosphate protonation and pH-dependent unfolding of the Ras.GTP.Mg2+ complex: a vibrational spectroscopy study; Cheng H et al.; The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis . Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified . Recent theoretical and experimental work suggest that the gamma-phosphate of GTP could be the general base . The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes . Isotope-edited IR studies of the Ras.GTP.Mg(2+) complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma-phosphate is not protonated at pH > or = 3.3, indicating that the active site decreases the gamma-phosphate pK(a) by at least 1.1 pK(a) units compared with solution . Amide I studies show that the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes partially unfold in what appear to be two transitions . The first occurs in the pH range 5.4-2.6 and is readily reversible . Differences in the pH-unfolding midpoints for the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes (3.7 and 4.8, respectively) reveal that the enzyme-gamma-phosphoryl interactions stabilize the structure . The second transition, pH 2.6-1.7, is not readily reversed . The pH-dependent unfolding of the Ras.GTP.Mg(2+) complex provides an alternative interpretation of the data that had been used to support the gamma-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.

J Biol Chem, 2001 Mar 23, 276(12), 9071 - 6 Epub 2000 Dec 21.
Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli; Yang IY et al.; Acrolein, a reactive alpha,beta-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(beta-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido{3,2-a}purine-9-one (gamma-OH-PdG) . The cellular processing and mutagenic potential of gamma-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA . Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair . The apparent level of inhibition of DNA synthesis is approximately 70% in Escherichia coli DeltarecA, uvrA . The block to DNA synthesis can be overcome partially by recA-dependent recombination repair . Targeted G --> T transversions were observed at a frequency of 7 x 10(-4)/translesion synthesis . Inactivation of polB, dinB, and umuD,C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis . In vitro primer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite gamma-OH-PdG . We conclude from this study that DNA polymerase III catalyzes translesion synthesis across gamma-OH-PdG in an error-free manner . Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E . coli from the potential genotoxicity of this DNA adduct.

J Biol Chem, 2001 Mar 30, 276(13), 10492 - 7 Epub 2000 Dec 21.
Mutational analysis of two putative catalytic motifs of the type IV restriction endonuclease Eco57I; Rimseliene R et al.; The role of two sequence motifs (SM) as putative cleavage catalytic centers (77)PDX(13)EAK (SM I) and (811)PDX(20)DQK (SM II) of type IV restriction endonuclease Eco57I was studied by site-directed mutational analysis . Substitutions within SM I; D78N, D78A, D78K, and E92Q reduced cleavage activity of Eco57I to a level undetectable both in vivo and in vitro . Residual endonucleolytic activity of the E92Q mutant was detected only when the Mg(2+) in the standard reaction mixture was replaced with Mn(2+) . The mutants D78N and E92Q retained the ability to interact with DNA specifically . The mutants also retained DNA methylation activity of Eco57I . The properties of the SM I mutants indicate that Asp(78) and Glu(92) residues are essential for cleavage activity of the Eco57I, suggesting that the sequence motif (77)PDX(13)EAK represents the cleavage active site of this endonuclease . Eco57I mutants containing single amino acid substitutions within SM II (D812A, D833N, D833A) revealed only a small or moderate decrease of cleavage activity as compared with wild-type Eco57I, indicating that the SM II motif does not represent the catalytic center of Eco57I . The results, taken together, allow us to conclude that the Eco57I restriction endonuclease has one catalytic center for cleavage of DNA.

J Biol Chem, 2001 Apr 13, 276(15), 12113 - 9 Epub 2000 Dec 21.
Mutational analysis of the MutH protein from Escherichia coli; Loh T et al.; Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models . The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL . No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding . Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis . These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro . The results indicate that the MutH signal for DNA binding and catalysis remains unknown . Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79) . Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

Bioorg Med Chem Lett, 2000 Dec 18, 10(24), 2795 - 8
Inhibition of Escherichia coli glucosamine synthetase by novel electrophilic analogues of glutamine--comparison with 6-diazo-5-oxo-norleucine; Walker B et al.; A series of electrophilic glutamine analogues based on 6-diazo-5-oxo-norleucine has been prepared, using novel synthetic routes, and evaluated as inhibitors of Escherichia coli glucosamine synthetase . The gamma-dimethylsulphonium salt analogue of glutamine was found to be one of the most potent inactivators of this enzyme yet reported, with an apparent second order rate constant (k2/Ki) of 3.5 x 10(5) M(-1) min(-1).

Biol Cell, 2000 Sep, 92(6), 397 - 407
Mouse peripherin isoforms; Landon F et al.; Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene . They generate three translation products, one major form, Pe-58, and two minor forms, Pe-56 which possess a shorter C-terminal sequence, and Pe-61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J . 8, 1719-1726) . In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing . Recombinant peripherin isoforms were purified from E . coli expressing full-length cDNAs . Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe-61 and were immunoadsorbed until they became monoreactive . By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis . In addition, each isoform was resolved into several charge variants . At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.

J Inorg Biochem, 2000 Nov, 82(1-4), 27 - 32
Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin; Nakayama M et al.; Plant sulfite reductase contains the siroheme and the {4Fe-4S} cluster as catalytically active redox centers and catalyzes the six-electron reductions of sulfite and nitrite using electrons donated from ferredoxin . A heterologous expression of a cDNA for maize sulfite reductase in E . coli has enabled us to produce the wild-type and mutant enzymes . Putative substrate-binding basic residues, located at the siroheme distal side, have been substituted for other residues with neutral or negatively charged side chains . Kinetic studies of the resulting mutant enzymes have demonstrated that substrate specificity for the two anions is remarkably changed by amino acid substitutions at a single site . We have also produced two classes of ferredoxin mutants with less ability to donate electrons to sulfite reductase: one with a defect in the recognition of the partner enzyme and the other with an unfavorable redox property . This article summarizes our knowledge about the structure function relationships of plant sulfite reductase.

J Inorg Biochem, 2000 Nov, 82(1-4), 215 - 9
Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58); Hosaka H et al.; The precursor of an RNA molecule from T4-infected E . coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions {(UpA (139-140) and CpA (170-171)}, within a putative loop and stem structure . This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents . We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents . It requires Mg2+ and is aided by a non-ionic detergent, Brij 58 . The cleavage reaction is time, temperature, and pH-dependent . The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1) . Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).

J Inorg Biochem, 2000 Nov, 82(1-4), 163 - 70
Roles of the heme proximal side residues tryptophan409 and tryptophan421 of neuronal nitric oxide synthase in the electron transfer reaction; Yumoto T et al.; Nitric oxide synthase (NOS) has an oxygenase domain with a thiol-coordinated heme active side similar to cytochrome P450 . In contrast to cytochrome P450, however, conserved aromatic amino acids are situated in the heme proximal side of NOS . For example, in endothelial NOS (eNOS), the indole-ring nitrogen of Trp180 hydrogen-binds to the thiol of Cys186, the internal axial ligand to the heme . And, the aromatic side chain of Trp192 forms a bridge between this residue and the protein . Trp180 and Trp192 of eNOS correspond to Trp409 and Trp421 of neuronal NOS (nNOS), respectively . In order to understand the roles of the aromatic amino acids in catalysis, we generated Trp409His, Trp409Leu, Trp421His and Trp421Leu mutants of nNOS and determined their catalytic parameters . The Trp409Leu mutant was very poorly expressed in E . coli and was easily denatured during purification procedures . The NO formation activities of the Trp409His and Trp421Leu mutants were 11 and 25 micromol/min per micromol heme, respectively, and are lower than that (44 micromol/min per micromol heme) of the wild type . The activity (46 micromol/min per micromol heme) of the Trp421His mutant was comparable to that of the wild-type enzyme . However, NADPH oxidation rates of Trp421His (230 micromol/min per micromol heme) and Trp421Leu (104 micromol/min per microol heme) in the presence of L-Arg were much larger than those observed for the wild type (65 micromol/min per micromol heme) and the Trp409His mutant (43 micromol/min per micromol heme) . The cytochrome c reduction rate of the Trp421His mutant was 6-fold larger than that of the wild type . The heme reduction rate with NADPH for the Trp421His mutant (0.09 min(-1)) was much lower than that (1.0 min(-1)) of the wild type . Taken together, it appears that Trp421 may be involved in inter-domain/inter-subunit electron transfer reactions.

J Biol Chem, 2001 Apr 6, 276(14), 10999 - 1006 Epub 2000 Dec 20.
Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-diaminopimelate ligase from Escherichia coli; Gordon E et al.; UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan . The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, has been solved to 2.0 A resolution . Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium . The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor . The refined model reveals the binding site for UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism . Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site . In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified.

J Biol Chem, 2001 Mar 23, 276(12), 9009 - 15 Epub 2000 Dec 20.
Kinetic analysis of adenovirus fiber binding to its receptor reveals an avidity mechanism for trimeric receptor-ligand interactions; Lortat-Jacob H et al.; Most adenoviruses bind to the N-terminal immunoglobulin domain D1 of the coxsackievirus and adenovirus receptor via the head part of their fiber proteins . Three receptor molecules can bind per fiber head . We expressed the D1 domain and the adenovirus type 2 fiber head in bacteria and studied binding interactions by surface plasmon resonance measurements . When receptor domains bind adenovirus fiber independently of each other, the dissociation constant is 20-25 nm . However, when adenovirus fiber binds to receptors immobilized on the sensor chip, a situation better mimicking adenovirus binding to receptors on the cell surface, the dissociation constant was around 1 nm . Kinetic analysis shows that this happens via an avidity mechanism; three identical interactions with high on and off rate constants lead to tight binding of one fiber head to three receptor molecules with a very low overall off rate . The avidity mechanism could be used by other viruses that have multimeric adhesion proteins to attach to target cells . It could also be more general to trimeric receptor-ligand interactions, including those involved in intracellular signaling.

J Biol Chem, 2001 Mar 23, 276(12), 9158 - 65 Epub 2000 Dec 20.
Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution . Implication for a substrate specificity; Le Du MH et al.; Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors . The crystal structure, determined at 1.8-A resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli . The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved . Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M . F., Manes, T., and Millan, J . L . (1997) J . Biol . Chem . 272, 22781-22787) . Allostery is probably favored by the quality of the dimer interface, by a long N-terminal alpha-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other . In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by l-amino acids, consistent with the acquisition of substrate specificity . The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.

J Biol Chem, 2001 Mar 30, 276(13), 9679 - 87 Epub 2000 Dec 19.
Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes; Schuck P et al.; The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds single-stranded RNA cooperatively, with high affinity, and independent of sequence . Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear . In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy . We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP . In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed . This was reversed by binding of ADP and RNA . We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation . The secondary structure of NSP2 showed a high fraction of beta-sheet, with small changes induced by magnesium that were reversed in the presence of RNA . That NSP2 can exist in different conformations lends support to the previously proposed hypothesis (Taraporewala, Z., Chen, D., and Patton, J . T . (1999) J . Virol . 73, 9934-9943) of its function as a molecular motor involved in the packaging of viral mRNA.

J Biol Chem, 2001 Mar 30, 276(13), 10134 - 44 Epub 2000 Dec 19.
Epilysin, a novel human matrix metalloproteinase (MMP-28) expressed in testis and keratinocytes and in response to injury; Lohi J et al.; We have cloned a new human matrix metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis cDNA libraries . Like most MMPs, epilysin contains a signal sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain . In addition, epilysin has a furin activation sequence (RRKKR) but has no transmembrane sequence . The exon-intron organization and splicing pattern of epilysin differ from that of other MMP genes . It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs . Another novel feature of epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal half of the catalytic domain . Northern hybridization of tissue RNA indicated that epilysin is expressed at high levels in testis and at lower levels in lungs, heart, colon, intestine, and brain . RNase protection assay with various cell lines indicated that epilysin was selectively expressed in keratinocytes . Recombinant epilysin degraded casein in a zymography assay, and its proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor . Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal epidermis of intact skin . In injured skin, prominent staining for epilysin was seen in basal keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from that of other MMPs expressed during tissue repair . These findings suggest that this new MMP functions in several tissues both in tissue homeostasis and in repair.

J Biol Chem, 2001 Apr 20, 276(16), 13264 - 72 Epub 2000 Dec 15.
Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB; Lebars I et al.; The T4 endoribonuclease RegB is involved in the inactivation of the phage early messengers . It cuts specifically in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers . In vitro RegB activity is very low but is enhanced by a factor up to 100 by the ribosomal protein S1 . In the absence of clear sequence motif distinguishing substrate and non-substrate GGAG-containing RNAs, we postulated the existence of a structural determinant . To test this hypothesis, we correlated the structure, probed by NMR spectroscopy, with the cleavage propensity of short RNA molecules derived from an artificial substrate . A kinetic analysis of the cleavage was performed in the presence and absence of S1 . In the absence of S1, RegB efficiently hydrolyses substrates in which the last G of the GGAG motif is located in a short stem between two loops . Both strengthening and weakening of this structure strongly decrease the cleavage rate, indicating that this structure constitutes a positive cleavage determinant . Based on our results and those of others, we speculate that S1 favors the formation of the structure recognized by RegB and can thus be considered a "presentation protein."

J Biol Chem, 2001 Apr 27, 276(17), 13524 - 9 Epub 2000 Dec 15.
Functional replacement of the essential ESS1 in yeast by the plant parvulin DlPar13; Metzner M et al.; A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity . Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases . Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone . Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain . Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells . Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed . When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1 . In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures . These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Immunogenetics, 2000 Nov, 52(1-2), 129 - 36
A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion; Meijerink E et al.; The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions . In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine . FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0 . The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu) . Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa . ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme . Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia . In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs . Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position . Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.

Mol Biotechnol, 2000 Oct, 16(2), 151 - 60
Overview of vector design for mammalian gene expression; Kaufman RJ; The expression of cloned genes in mammalian cells is a basic tool for understanding gene expression, protein structure, and function, and biological regulatory mechanisms . The level of protein expression from heterologous genes introduced into mammalian cells depends upon multiple factors including DNA copy number, efficiency of transportation, mRNA processing, mRNA transport, mRNA stability, and translational efficiency, and protein processing, transport, and stability . Different genes exhibit different rate limiting steps for efficient expression . Multiple strategies are available to obtain high level expression in mammalian cells . This article reviews vector design for expression of foreign genes in mammalian cells.

Anticancer Res, 2000 Sep- Oct, 20(5B), 3615 - 8
Radiation induced effect of the vitamins C, E and beta-carotene on sanazole efficiency . A study in vitro; Heinrich E et al.; The effect of the vitamins C, E-acetate and beta-carotene on the sanazole (AK-2123) efficiency was investigated in vitro (E . coli bacteria AB 1157) under the influence of gamma-rays . The vitamins were applied with sanazole individually or in appropriate mixtures in airfree media as well as in media saturated with nitrous oxide (N2O) or air . The addition of vitamin C led in all cases to a very strong enhancement of the sanazole efficiency, namely: in airfree media to 2-fold, in the presence of N2O to 1.5-fold and in the presence of air to 2-fold . The influence of vitamin E-acetate and beta-carotene and mixtures of them depended on the respective gas used for saturation of the medium . An attempt was made to estimate the role of the reducing (e-aq, H-atoms) and oxidizing radicals (OH, O2.-) in respect to the influence of the sanazole activity . The results are of interest for the chemoradiation therapy of cancer.

Nature, 2000 Dec 7, 408(6813), 735 - 40
InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase; Hermosura MC et al.; Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx . InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively . Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result . Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive . Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref . 9) . Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation . At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.

Nature, 2000 Dec 7, 408(6813), 682 - 8
Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1; Worthylake DK et al.; The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins . Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1 . The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction . The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent . The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1 . The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.

Mol Cell Biochem, 2000 Oct, 213(1-2), 127 - 35
Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones; Wang H et al.; The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein . The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5 . P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA) . No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions . Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6beta-, 16alpha-, and 21-hydroxyprogesterone . Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6beta, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450 . Based on the Vmax/Km values for P450 3A9 using either 21-hydroxprogesterone or 6beta-hydroxyprogesterone as a substrate, 4-pregnen-6beta, 21-diol-3, 20-dione may be formed either by 6beta-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6beta-hydroxyprogesterone . As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.

Virus Genes, 2000 Oct, 21(3), 223 - 6
Expression and membrane association of hepatitis C virus envelope 1 protein; Ciccaglione AR et al.; The expression of hepatitis C virus (HCV) E1 protein is toxic for Escherichia coli cells . For this reason, we have cloned the E1 gene in the pET3a vector and analyzed the inducible expression of the protein in two strains of E . coli characterised by a different level of reduction of basal synthesis . The results indicated that synthesis of E1 was supported only by the BL21(DE3)pLysS strain which provides a tightest control of protein expression before the induction . The BL21(DE3)pLysS cells were then used for the expression of E1 gene, varying at its carboxy terminus in order to retain (E1, aa 192-383) or delete (Elt, aa 192-340) a C-terminal hydrophobic region that may be involved in membrane association . Following cell fractionation, E1 protein was found associated with the membrane fraction . By contrast, the truncated mutant E1t, was identified in the soluble phase suggesting a direct role for the C-terminal domain in E1 membrane association.

Virus Genes, 2000 Oct, 21(3), 193 - 5
The vaccinia virus E3L protein interacts with SUMO-1 and ribosomal protein L23a in a yeast two hybrid assay; Rogan S et al.; We report the results of a two-hybrid study which identified clones from a HeLa cDNA library that interact with the vaccinia virus protein E3L . These clones encode the nuclear protein SUMO-1 (also known as PIC-1, sentrin or GMP-1); the cytoplasmic ribosomal protein L23a; and a small peptide sequence of unknown significance.

Virus Genes, 2000 Oct, 21(3), 157 - 65
Genomic analysis of matrix gene and antigenic studies of its gene product (M1) of a swine influenza virus (H1N1) causing chronic respiratory disease in pigs; Welman M et al.; The nucleotide sequence of gene coding for the matrix protein (M1 and M2) of swine influenza (H1N1) virus, A/Sw/Quebec/5393/91 (SwQc91), associated with chronic respiratory disease in pigs, was determined . The deduced amino acid (aa) sequence was compared with the other North American swine strains including the A/Sw/Quebec/192/81 (SwQc81) strain associated with the chronic and acute respiratory disease in pigs . Separate analysis of the M1 and M2 gene products showed different evolutions . M1 had 2 aas changes among 252 aas and these were at positions 4 and 205 . The mutation rate was 0.08%, aa changes per residue per year, and its homology with other strains was 99.2% . The M2 protein (97 aas) was relatively more variable than M1 with 5 substitutions . Differences observed were at positions 4, 16, 21, 54 and 95 . The mutation rate was 0.51% and its homology with other strains was 94.8% . The M1 gene was cloned in the procaryotic plasmid pET21a and the recombinant plasmid was expressed in Escherichia coli under pre-determined optimal conditions . The recombinant M1 protein (RM1P) (approximately 28 kDa) comigrated as a single band on SDS-PAGE . RM1P was antigenic and reacted with polyclonal sera and 5 monoclonal antibodies (MAbs) spanning 4 epitopes including the membrane binding site and the transcription inhibition activity site . RM1P was immunogenic . The mouse anti-RM1P ELISA antibodies reacted with the purified viral M1 protein and the whole virus.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2128 - 37
Characterization of human recombinant transglutaminase 1 purified from baculovirus-infected insect cells; Hitomi K et al.; Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia . Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa . Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases . Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced . Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2121 - 7
Role of the non-essential region encompassing the N-terminal two transmembrane stretches of Escherichia coli SecE; Nishiyama K et al.; SecE is an essential component of the protein translocation machinery of Escherichia coli and has three transmembrane stretches . An N-terminal region (SecE-N) encompassing the first two transmembrane stretches is dispensable for protein translocation but a SecE derivative (SecE-C) lacking this region is very unstable . We show here that FtsH, the AAA (ATPases associated with diverse cellular activities) family protease, causes the instability of SecE-C . SecE-C became stable when SecE-N was co-expressed . Deletion of the N-terminal region of SecE also rendered the SecE-SecY-SecG complex unstable . In spite of these alterations, the N-terminal region of SecE had little stimulatory effect on protein translocation in vivo or in vitro.

Biometrics, 2000 Dec, 56(4), 1222 - 6
A generalized nonparametric test for lattice-ordered means; Strand M; Treatment means in factorial experiments are lattice ordered when there is an increase in mean response as the level of any factor is increased while holding the other factors fixed . Such means occur naturally in many experiments . A nonparametric test for lattice-ordered means involving a Kendall-type statistic will be summarized for k-factor factorial experiments . Specifically, the form of the test statistic and variance under the null hypothesis will be presented . In addition, a normalized version of the test statistic will be discussed and applied to relevant data.

J Muscle Res Cell Motil, 2000, 21(5), 415 - 22
Primary structure of myosin from the striated adductor muscle of the Atlantic scallop, Pecten maximus, and expression of the regulatory domain; Janes DP et al.; We have determined the complete cDNA and deduced amino acid sequences of the heavy chain, regulatory light chain and essential light chain which constitute the molecular structure of myosin from the striated adductor muscle of the scallop, Pecten maximus . The deduced amino acid sequences of P . maximus regulatory light chain, essential light chain and heavy chain comprise 156, 156 and 1940 amino acids, respectively . These myosin peptide sequences, obtained from the most common of the eastern Atlantic scallops, are compared with those from three other molluscan myosins: the striated adductor muscles of Argopecten irradians and Placopecten magellanicus, and myosin from the siphon retractor muscle of the squid, Loligo pealei . The Pecten heavy chain sequence resembles those of the other two scallop sequences to a much greater extent as compared with the squid sequence, amino acid identities being 97.5% (A . irradians), 95.6% (P . magellanicus) and 73.6% (L . pealei), respectively . Myosin heavy chain residues that are known to be important for regulation are conserved in Pecten maximus . Using these Pecten sequences, we have overexpressed the regulatory light chain, and a combination of essential light chain and myosin heavy chain fragment, separately, in E . coli BL21 (DE3) prior to recombination, thereby producing Pecten regulatory domains without recourse to proteolytic digestion . The expressed regulatory domain was shown to undergo a calcium-dependent increase (approximately 7%) in intrinsic tryptophan fluorescence with a mid-point at a pCa of 6.6.

J Biol Inorg Chem, 2000 Dec, 5(6), 720 - 9
Desulfoferrodoxin: a modular protein; Ascenso C et al.; The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure . These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized . Both recombinant fragments behaved as independent metal-binding domains . The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif . The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide . The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c . The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

Biometals, 2000 Sep, 13(3), 223 - 9
Escherichia coli beta-galactosidase is heterogeneous with respect to a requirement for magnesium; Craig DB et al.; Commercially obtained E . coli beta-galactosidase was stored at 25 degrees C in buffer containing 1 mM MgCl2 and in buffer containing no added MgCl2 . Samples were removed at set times and the activity of individual enzyme molecules assayed . When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period . When stored in the absence of added MgCl2, over half the enzyme molecules became inactive within the first hour . However, those molecules which retained activity remained active for the duration of the experiment . This indicates that there may exist two populations of E . coli beta-galactosidase, one which requires storage in the presence of the higher concentration of Mg2+ in order to remain active . There was no observed correlation between this requirement for magnesium and reaction rate . Additionally, the presence of the 1 mM MgCl2 was found to decrease the average activity of the beta-galactosidase molecules under the conditions employed.

Curr Genet, 2000 Nov, 38(4), 218 - 25
Engineering of the rpl23 gene cluster to replace the plastid RNA polymerase alpha subunit with the Escherichia coli homologue; Suzuki JY et al.; The Escherichia coli RNA polymerase (RNAP) alpha, beta, and beta' core subunits are evolutionarily conserved among bacteria and plastids, and the plastid specificity factors form a functional holoenzyme with the E . coli core . To investigate whether the E . coli core subunits may form a functional hybrid enzyme with the plastid core subunits, we replaced the tobacco plastid RNAP alpha subunit gene (rpoA) with the E . coli alpha subunit gene by targeted gene insertion . The transplastomic tobacco plants look similar to tobacco rpoA deletion mutants in that they are chlorophyll-deficient and nonphotoautotrophic . In addition, they lack transcripts from promoters recognized by the E . coli-like plastid RNA polymerase . These results indicate that evolutionary conservation between the E . coli and plastid RNA polymerase alpha subunits is insufficient to allow substitution of the tobacco alpha subunit with its bacterial counterpart . Interestingly, the cyanobacterial alpha subunits are as different as the E . coli alpha subunits; and therefore it is unlikely that replacement of the tobacco alpha subunit with cyanobacterial alpha subunits would yield a functional enzyme . Replacement of plastid rpoA with the E . coli RNA polymerase alpha subunit gene represents the first engineering of a plastid operon in higher plants.

J Med Entomol, 2000 Nov, 37(6), 885 - 92
Influence of repeated infestations with pathogen-free Ixodes scapularis (Acari: Ixodidae) on in vitro lymphocyte proliferation responses of C3H/HeN mice; Schoeler GB et al.; In the United States, Ixodes scapularis Say has been implicated as the vector of at least three human pathogens . Tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens . This study was conducted to determine the effects of repeated infestations with pathogen-free I . scapularis nymphs on in vitro proliferative responses of splenic lymphocytes from C3H/HeN mice . Lymphocytes from repeatedly infested and uninfested mice were exposed to concanavalin A (Con A), Escherichia coli Castellini & Chalmers lipopolysaccharide (LPS), or I . scapularis salivary gland soluble proteins (SGSP), to determine if lymphocyte responses differed between tick-exposed and nonexposed mice . Female C3H/HeN mice were infested one to four times with pathogen-free I . scapularis nymphs, with a 14-d tick-free period between each exposure . After each infestation, tick biology parameters were measured and lymphocyte proliferative responses assessed . Acquired resistance to I . scapularis was not evident in mice subjected to tick feeding . Significant differences in the responses of lymphocytes exposed to I . scapularis SGSP were observed between infested and noninfested mice . In contrast, few differences between infested and noninfested mice were evident for lymphocytes exposed to Con A or LPS . Our results suggest that repeated exposure to I . scapularis nymphs does not affect Con A or LPS-induced proliferation of splenic lymphocytes, but significantly effects lymphocyte responses to tick salivary gland antigens.

J Nat Toxins, 2000 Nov, 9(4), 363 - 79
Interaction of tetanus toxin derived hybrid proteins with neuronal cells; Figueiredo DM et al.; The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons . There is little information on the cellular processing of proteins delivered by linkage to TTC . We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E . coli . Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry . Hybrid proteins were bound and internalized in a manner very similar to TTC . Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types . Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization . Proteins coupled to TTC share its long-term stability after cellular internalization . After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol . TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

J Comb Chem, 2000 Nov-Dec, 2(6), 736 - 48
Preparation of fluorinated linkers: use of 19F NMR spectroscopy to establish conditions for solid-phase synthesis of pilicide libraries; Svensson A et al.; Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared . One of the linkers was used in combination with gel-phase 19F NMR spectroscopy to develop conditions for solid-phase synthesis of two libraries of pilicides, i.e . compounds designed to inhibit assembly of adhesive pili in uropathogenic Escherichia coli . Attachment to and cleavage from the linker could be monitored based on the chemical shift of the fluorine atom of the linker . In addition, use of the linker as internal standard allowed quantification and optimization of reactions occurring further away from the linker when fluorinated building blocks were employed . Importantly, high-quality 19F NMR spectra were obtained for compounds linked to a TentaGel resin in a standard NMR tube using an ordinary NMR instrument.

Intensive Care Med, 2000 Oct, 26(10), 1531 - 9
Hepatic O2 exchange and liver energy metabolism in hyperdynamic porcine endotoxemia: effects of iloprost; Trager K et al.; OBJECTIVE: To compare the effects of a 12 h continuous infusion of iloprost, a stable prostacyclin analogue, on hepatic blood flow (Qliv), O2 exchange, and energy metabolism during a 24 h hyperdynamic, porcine endotoxemia with volume resuscitation alone . DESIGN: Prospective, randomized, experimental study with repeated measures . Setting: Investigational animal laboratory . SUBJECTS: Twenty-eight domestic pigs: 16 animals during endotoxemia with volume resuscitation alone (ETX), 12 with endotoxemia, volume resuscitation, and treatment with iloprost (ILO) . INTERVENTIONS: Endotoxemia was initiated by continuous infusion of E . coli lipopolysaccharide . Animals were resuscitated with hetastarch, aimed at maintaining a MAP of > 60 mmHg . After 12 h of endotoxemia, iloprost was administered for 12 h in the treatment group, titrated to avoid pharmacologically induced hypotension (MAP < 60 mmHg) . MEASUREMENTS AND RESULTS: Iloprost significantly increased Qliv, with no effect on hepatic O2 delivery . Mean capillary hemoglobin O2 saturation (HbScO2) on the liver surface, as well as HbScO2 frequency distributions--a measure of microcirculatory O2 availability--remained unchanged . Treatment with iloprost, however, significantly attenuated the endotoxin-induced derangements of cellular energy metabolism as reflected by the diminished progressive decrease in hepatic lactate uptake rate and a blunted increase in hepatic venous lactate/pyruvate ratios . While endotoxin significantly increased endogenous glucose production (EGP) rate, iloprost restored EGP to normal at the end of the experiment . CONCLUSIONS: Thus, in a clinically relevant model of human sepsis, iloprost did not produce potential adverse effects but rather ameliorated hepatic metabolic disturbances and, thereby, hepatic energy balance.

Carbohydr Res, 2000 Dec 1, 329(4), 873 - 8
Synthesis of alpha-Gal epitope derivatives with a galactosyltransferase-epimerase fusion enzyme; Fang J et al.; Alpha-Gal epitopes are carbohydrate structures bearing an alpha-D-Galp-(1-->3)-beta-D-Galp terminus and are the main cause of antibody-mediated hyperacute rejection in xenotransplantation . Nine monosaccharides and ten disaccharides were evaluated as substrates for a fusion protein, which contains both alpha-(1-->3)-galactosyltransferase and uridine-5'-diphosphogalactose 4-epimerase . Four disaccharide and six trisaccharide alpha-Gal epitope derivatives were synthesized utilizing this novel fusion enzyme.

J Gen Virol, 2001 Jan, 82(Pt 1), 191 - 9
The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability; Dorsch S et al.; The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity . Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30-42 . The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children . The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins . To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E . coli as intein-fusion proteins . The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions . After purification via affinity chromatography, the dissociation constants K(D) of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor . A value of 5.4 x 10(-8) M was determined for the prototype isolate pJB; the affinity constants for the variant VP1-unique regions were similar . Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid . It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.

J Gen Virol, 2001 Jan, 82(Pt 1), 183 - 90
Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene; Tuboly T et al.; Five recombinant porcine adenoviruses of serotype 5 (PAdV-5) carrying the full-length or the 5' 2.2 kb half of the transmissible gastroenteritis virus (TGEV) spike (S) gene were generated by homologous recombination in E . coli strain BJ5183 cells and subsequent transfection of swine testicle cells . The foreign genes were inserted into the E3 region of PAdV-5 . One recombinant virus had no deletion in the E3 region, whereas a 1.2 kb fragment was removed from the E3 region in the remainder of the recombinant viruses . One stable construct with a 4.4 kb insertion had a genome size of 109.6% of the wild-type genome, the largest reported for any recombinant adenovirus . Only those viruses that carried the S gene in the left to right orientation expressed the S gene . Three recombinant viruses were tested by oral immunization of pigs and both antibody response and virus shedding were monitored . None of the pigs showed clinical signs and the virus was recovered from rectal swabs until 6-7 days post-infection . Viruses expressing the S gene induced TGEV- and PAdV-5-specific virus-neutralizing antibodies . Moreover, TGEV-specific secretory IgA was detected in the small intestine and in the lungs of the immunized animals.

J Gen Virol, 2001 Jan, 82(Pt 1), 139 - 48
Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli; Berger N et al.; In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1) . The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates . Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction . Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme . The N terminus appears to have no considerable influence . Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN . Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments . Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.

J Gen Virol, 2001 Jan, 82(Pt 1), 129 - 38
T-helper and humoral responses to Puumala hantavirus nucleocapsid protein: identification of T-helper epitopes in a mouse model; de Carvalho Nicacio C et al.; Puumala hantavirus (PUUV) is a rodent-borne agent causing nephropathia epidemica in humans, a milder form of haemorrhagic fever with renal syndrome occurring in Fennoscandia, central Europe and western Russia . In this study we characterized the immunogenicity of an E . coli-expressed nucleocapsid (N) protein of PUUV (strain Kazan-E6) in inbred mice (BALB/c, CBA and C57/BL6) . The recombinant N (rN) protein raised PUUV-specific antibodies in all three tested murine haplotypes, and all IgG subclasses were detected . Epitope mapping using peptides spanning the N protein revealed that the B-cell recognition sites were mainly located at the amino-terminal part of the protein . Proliferative T-helper (Th) lymphocyte responses were detected in all haplotypes after a single immunization with rN . Several Th-recognition sites, spanning amino acids 6-27, 96-117, 211-232 and 256-277, were identified using overlapping peptides . Peptides representing the identified sites could also prime Th-lymphocytes to proliferate in response to recall with rN protein, thereby confirming the authenticity of the identified sites . The rN-primed Th-lymphocytes produced predominantly interleukin (IL)-2 and gamma interferon, together with lower levels of IL-4 and IL-6, indicating a mixed Th1/Th2 response.

J Gen Virol, 2001 Jan, 82(Pt 1), 121 - 8
Zinc-binding properties of Junín virus nucleocapsid protein; Tortorici MA et al.; The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle . First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid . We have expressed the N protein of Junin virus in E . coli and shown that it binds zinc in vitro . This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX(2)HX(23)CX(4)C sequence, which resembles a classical zinc-finger motif . The specificity for zinc binding was demonstrated by competition with other divalent metal ions . The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions . In addition, alternative zinc-binding sites were found.

J Gen Virol, 2001 Jan, 82(Pt 1), 35 - 44
Interaction of the movement and coat proteins of Maize streak virus: implications for the transport of viral DNA; Liu H et al.; We have shown previously that the movement protein (MP) and coat protein (CP) of Maize streak virus (MSV) are both required for systemic infection . Towards understanding the roles of these two proteins in virus movement, each was expressed in E: coli and interactions of the MP with viral DNA or CP were investigated using south-western, gel overlay and immunoprecipitation assays . Unlike the CP, the MP did not bind to viral DNA but it interacted with the CP in vitro and an MP-CP complex was detected in extracts from MSV-infected maize, indicating the potential for an interaction in vivo . Microinjection showed that the MP could prevent the nuclear transport of an MSV CP-DNA complex in maize and tobacco cells . These results are consistent with a model in which the MP diverts a CP-DNA complex from the nucleus (where viral DNA replication takes place) to the cell periphery, and in co-operation with the CP, mediates the cell-to-cell movement of the viral DNA . In this respect, the MSV MP and CP have functional analogy with the BC1 and BV1 proteins, respectively, of the BEGOMOVIRUS: genus of the GEMINIVIRIDAE:

Mol Pharmacol, 2001 Jan, 59(1), 30 - 7
SCH-202676: An allosteric modulator of both agonist and antagonist binding to G protein-coupled receptors; Fawzi AB et al.; A novel thiadiazole compound, SCH-202676 (N-(2,3-diphenyl-1,2, 4-thiadiazol-5-(2H)-ylidene)methanamine), has been identified as an inhibitor of both agonist and antagonist binding to G protein-coupled receptors (GPCRs) . SCH-202676 inhibited radioligand binding to a number of structurally distinct, heterologously expressed GPCRs, including the human mu-, delta-, and kappa-opioid, alpha- and beta-adrenergic, muscarinic M1 and M2, and dopaminergic D1 and D2 receptors, but not to the tyrosine kinase epidermal growth factor receptor . SCH-202676 had no direct effect on G protein activity as assessed by {35S}guanosine-5'-O-(gamma-thio)triphosphate binding to purified recombinant G(oalpha)- or G(betagamma)-stimulated ADP-ribosylation of G(oalpha) by pertussis toxin . In addition, SCH-202676 inhibited antagonist binding to the beta2-adrenergic receptor expressed in Escherichia coli, a system devoid of classical heterotrimeric G proteins . SCH-202676 inhibited radiolabeled agonist and antagonist binding to the alpha2a-adrenergic receptor with an IC50 value of 0.5 microM, decreased the Bmax value of the binding sites with a slight increase in the KD value, and inhibited agonist-induced activation of the receptor . The effects of SCH-202676 were reversible . Incubation of plasma membranes with 10 microM SCH-202676 did not alter subsequent radioligand binding to the alpha2a-adrenergic receptor and the dopaminergic D1 receptor . Taken together, our data suggest that SCH-202676 has the unique ability to allosterically regulate agonist and antagonist binding to GPCRs in a manner that is both selective and reversible . The scope of the data presented suggests this occurs by direct interaction with a structural motif common to a large number of GPCRs or by activation/inhibition of an unidentified accessory protein that regulates GPCR function.

J Mol Biol, 2001 Jan 12, 305(2), 331 - 9
In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates; Matsumura I et al.; The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity . The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site . The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes . The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic . An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides . These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity .

J Mol Biol, 2001 Jan 12, 305(2), 181 - 9
NMR spectroscopic evidence for Mn(2+)(Mg(2+)) binding to a precursor-tRNA microhelix near the potential RNase P cleavage site; Zuleeg T et al.; The binding of Mg(2+)/Mn(2+) to acceptor stem microhelices as minimal models for precursor-tRNA(Gly) is demonstrated by NMR spectroscopy . From the evaluation of COSY and NOESY spectra, binding sites for Mg(2+)/Mn(2+) can be inferred . In particular, one binding site exists near the ribose moiety of nucleotide -1 at the position of cleavage by RNase P . From comparison with a variant possessing a deoxynucleotide at this position, it is concluded that the 2'-OH group of this nucleotide is indispensable for coordinating the divalent metal ion . Hence, this catalytically important metal ion is "pre-bound" to the precursor-tRNA before complexation with RNase P.

Cytogenet Cell Genet, 2000, 90(3-4), 227 - 30
Genomic organization and chromosome location of the murine Rpl23 gene; Kleiter N et al.; The mouse gene coding for ribosomal protein L23 (Rpl23) has been fully sequenced, including 580 bp of the 5' upstream region . The 5-kb gene comprises 5 exons and contains an unusually long (3,153 bp) third intron . The gene was mapped to the distal region of mouse chromosome 11, homologous to human chromosome 17q21-->q22 .

Drug Metab Dispos, 2001 Jan, 29(1), 23 - 9
A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry; Dierks EA et al.; A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s (CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2C19, CYP2A6, and CYP2C8) was developed . This method uses an in vitro cocktail of specific substrates (midazolam, bufuralol, diclofenac, ethoxyresorufin, S-mephenytoin, coumarin, and paclitaxel) and fast gradient liquid chromatography tandem mass spectrometry . The assay incubation time is 20 min, which is in the linear range for all of the substrates, and the analysis time is 4 min/sample . Substrate specificity was confirmed by incubating Escherichia coli-expressed enzymes with the cocktail . Potent specific inhibitors of the seven enzymes (ketoconazole, quinidine, sulfaphenazole, tranylcypromine, quercetin, furafylline, and 8-methoxypsoralen) were evaluated in cocktail and individual substrate incubations . Five of these inhibitors were further studied to determine more precise IC(50) values for inhibition of the seven enzymes . The IC(50) values obtained in both cocktail and individual incubations were in good agreement with published values . This cocktail method offers an efficient, robust way to determine the cytochrome P450 inhibition profile of large numbers of compounds . The enhanced throughput of this method greatly facilitates its use to assess CYP inhibition as a drug candidate selection criterion.

Mol Ther, 2000 Dec, 2(6), 649 - 56
Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo; Hackett NR et al.; To assess the biodistribution and pharmacokinetics of gene transfer vectors, real-time PCR with fluorescent TaqMan chemistry was used to quantify tissue levels of adenovirus gene transfer vectors (Ad) following myocardial administration . After optimizing the detection of the genome of Ad vectors expressing human vascular endothelial growth factor (Ad(GV)VEGF121.10) and Escherichia coli cytosine deaminase (Ad(GV)CD.10), a comparison was made of intramyocardial injection versus intracoronary delivery to the left ventricle of the pig . One hour post-intramyocardial administration, the left ventricular Ad genome level was 6.2 copies per cellular genome, 26-fold higher than the level of 0.24 copies per cellular genome following intracoronary administration . Relative to the vector levels after 1 h, the amount dropped 14- and 5.5-fold by 24 h following intramyocardial and intracoronary administration, respectively . Interestingly, the vector that escaped the left ventricle after intracoronary or intramyocardial administration to pigs was found primarily within the lung, an observation in marked variance to the biodistribution of Ad vector in rodents . In this regard, after intravenous injection to the pig, 90% of the recovered vector was found in the lung, and even after intrahepatic portal vein injection, 55% of the recovered vector was in the lung . These data have important implications regarding the use of experimental animals for safety studies on administration of Ad to humans.

J Mol Biol, 2000 Dec 15, 304(5), 983 - 94
Macromolecular assemblage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein interactions and mechanism of complex assembly; Robinson JC et al.; The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches . This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins . The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a His-tagged protein . The different components of the complex were shown to associate in vitro with p38 immobilized on Ni(2+)-coated plates . Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between peripheral enzymes . Kinetic parameters of the interactions were determined by the means of a biosensor-based approach . The two dimeric proteins LysRS and AspRS were found to tightly bind to p38, with a K(d) value of 0.3 and 4.7 nM, respectively . These interactions involved the catalytic core of the enzymes . By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5 microM) . ArgRS and p43, two peripheral components, were shown to interact with moderate affinity (K(d)=93 nM) . Since all the components of the complex are tightly associated within this particle, lateral interactions were believed to contribute to the stabilization of this assemblage . Using an in vitro binding assay, concomitant association of several components of the complex on immobilized p38 could be demonstrated, and revealed the involvement of synergistic effects for association of weakly interacting proteins . Taking into account the possible synergy between central and lateral contributions, a sub-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro . These data provide compelling evidence for an ordered and concerted mechanism of complex assembly .

J Mol Biol, 2000 Dec 15, 304(5), 897 - 910
From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL; Chatellier J et al.; The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer . A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings . That heptameric complex is functionally inactive because it is unable to release GroES . We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the overall structure of GroEL44 mutant . We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active . The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency . Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E . coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL . We envisage the notional evolution of the structure and properties of GroEL . The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation . The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity . The acquisition of binding sites for ATP then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites . This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites . Finally, dimerization of the heptamer enhances its biological activity .

J Mol Biol, 2000 Dec 15, 304(5), 847 - 59
Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 A resolution; Passner JM et al.; After an allosteric transition produced by the binding of cyclic AMP (cAMP), the Escherichia coli catabolite gene activator protein (CAP) binds DNA specifically and activates transcription . The three-dimensional crystal structure of the CAP-cAMP complex has been refined at 2.1 A resolution, thus enabling a better evaluation of the structural basis for CAP phenotypes, the interactions of cAMP with CAP and the roles played by water structure . A review of mutational analysis of CAP together with the additional structural information presented here suggests a possible mechanism for the cAMP-induced allostery required for DNA binding and transcriptional activation . We hypothesize that cAMP binding may reorient the coiled-coil C-helices, which provide most of the dimer interface, thereby altering the relative positions of the DNA-binding domains of the CAP dimer . Additionally, cAMP binding may cause a further rearrangement of the DNA-binding and cAMP-binding domains of CAP via a flap consisting of beta-strands 4 and 5 which lies over the cAMP .

J Mol Biol, 2000 Dec 15, 304(5), 835 - 45
Crystal structure of Hsc20, a J-type Co-chaperone from Escherichia coli; Cupp-Vickery JR et al.; Hsc20 is a 20 kDa J-protein that regulates the ATPase activity and peptide-binding specificity of Hsc66, an hsp70-class molecular chaperone . We report herein the crystal structure of Hsc20 from Escherichia coli determined to a resolution of 1.8 A using a combination of single isomorphous replacement (SIR) and multi-wavelength anomalous diffraction (MAD) . The overall structure of Hsc20 consists of two distinct domains, an N-terminal J-domain containing residues 1-75 connected by a short loop to a C-terminal domain containing residues 84-171 . The structure of the J-domain, involved in interactions with Hsc66, resembles the alpha-topology of J-domain fragments of Escherichia coli DnaJ and human Hdj1 previously determined by solution NMR methods . The C-terminal domain, implicated in binding and targeting proteins to Hsc66, consists of a three-helix bundle in which two helices comprise an anti-parallel coiled-coil . The two domains make contact through an extensive hydrophobic interface ( approximately 650 A(2)) suggesting that their relative orientations are fixed . Thus, Hsc20, in addition to its role in the regulation of the ATPase activity of Hsc66, may also function as a rigid scaffold to facilitate positioning of the protein substrates targeted to Hsc66 .

J Mol Biol, 2000 Dec 15, 304(5), 821 - 33
Multiple disordered loops function in corepressor-induced dimerization of the biotin repressor; Kwon K et al.; Cooperative association of the Escherichia coli biotin repressor with the biotin operator is allosterically activated by binding of the corepressor, bio-5'-AMP . The corepressor function of the adenylate is due, in part, to its ability to induce repressor dimerization . Since a high-resolution structure of only the apo or unliganded repressor is currently available, the location of the dimerization interface on the protein structure is not known . Here, five mutants in the corepressor-binding domain of the repressor have been analyzed with respect to their DNA-binding and self-assembly properties . Results of these studies reveal that four of the mutant proteins exhibit defects in DNA binding . These same proteins are compromised in self-assembly . Furthermore, in the three-dimensional structure of the apo protein the mutations all lie in partially disordered surface loops, one of which is known to participate directly in corepressor binding . These results suggest that multiple disordered surface loops function in the corepressor-induced dimerization required for sequence-specific DNA binding by the biotin repressor .

J Mol Biol, 2000 Dec 15, 304(5), 715 - 21
Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin; Xiao W et al.; Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR) . Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation . The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle . The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin . The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins . Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base .

J Mol Biol, 2000 Dec 15, 304(5), 699 - 705
Kinetic analysis of the M1 RNA folding pathway; Kent O et al.; The biological activity of large RNAs is dependent on the formation of complex folded structures that determine function . Typically the creation of such structures requires divalent magnesium and in many cases the folding process takes place over the course of several minutes . It has been proposed that the folding paths of large RNAs proceed through discrete intermediates but the nature of these intermediates is not known in most cases . Here, we describe our studies on the folding of the M1 RNA sub-unit of Escherichia coli RNase P . We performed kinetic footprinting studies of M1 RNA folding with the chemical footprinting reagent peroxynitrous acid to provide a detailed description of the folding pathway of RNase P RNA . Our results indicate that, in contrast to the Group I ribozyme, the M1 RNA folds into its catalytically active structure through the formation of two separately folded domains and that the folding of each proceeds through a discrete series of intermediates . Similar rates of folding were observed for regions believed to form the interface between the two domains . This observation is consistent with a kinetic trap which occurs by interaction of the domains during folding .

Biochemistry, 2000 Dec 26, 39(51), 16213 - 9
Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori; Olson JW et al.; The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori . Obtaining viable H . pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron . When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency . This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H . pylori viability . H . pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized . Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer . This iron was determined to be in the form of a redox-active {2Fe-2S}(2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra . The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein . Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme . NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters . The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H . pylori lacks other Fe-S cluster assembly proteins, that the H . pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.

Biochemistry, 2000 Dec 26, 39(51), 16125 - 34
Ensuring productive resolution by the junction-resolving enzyme RuvC: large enhancement of the second-strand cleavage rate; Fogg JM et al.; RuvC is the principal junction-resolving enzyme of Escherichia coli, cleaving four-way DNA junctions created in homologous recombination . It binds with structural specificity to DNA junctions as a dimer, whereupon each subunit cleaves a phosphodiester bond of diametrically disposed strands . To generate a productive resolution event, these cleavages must be symmetrically located with respect to the point of strand exchange, and in the context of a branch-migrating junction, this requires near-simultaneous cleavage by the two subunits . Using a supercoil-stabilized cruciform as a substrate, we have analyzed the kinetics of strand cleavage . Coordinated bilateral cleavage is not essential in RuvC action, because a heterodimer comprising active and inactive subunits is active in unilateral cleavage . However, in operational terms, fully active RuvC appears to introduce simultaneous cleavages of two strands, because the rate of second-strand cleavage is accelerated by a factor of almost 150 relative to the first . We suggest that relief of strain following the first cleavage could lead to acceleration of subsequent cleavage, and show that DNA junctions rendered more flexible by the presence of strand breaks or bulges are subject to faster cleavage by RuvC . Cleavage of one strand of a junction generated in situ by the action of RuvC can accelerate cleavage at an intrinsically poor site by a factor of 500 . Very large rate enhancement of second-strand cleavage by RuvC is likely to be essential to ensure productive resolution of a junction that is being actively branch migrated by the RuvAB machinery.

Biochemistry, 2000 Dec 26, 39(51), 16000 - 7
Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1); Chen G et al.; The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1) . SULT1A1 was purified using the pMAL-c2 expression system in E . coli . WK inactivated SULT1A1 activity in a time- and concentration-dependent manner . The inactivation followed first-order kinetics relative to both SULT1A1 and WK . Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme . With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly . A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure . According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK . According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant) . Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity . E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly . This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity . Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.

Biochemistry, 2000 Dec 26, 39(51), 15961 - 70
Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1; Micaloni C et al.; We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively . These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation . Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized . The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene . Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product . However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult . Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate . With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event . Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.

Biochemistry, 2000 Dec 26, 39(51), 15932 - 43
The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein; Kamen DE et al.; Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized . The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding . In this paper, we report studies of the unfolding equilibrium of pelC . PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence . The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5 . No evidence was found for a loosely folded intermediate in the transition region at pH 5 . At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M) . Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins . Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD . Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition . DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5 . At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH . Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH . The crystal structure of pelC shows no obvious domain structure, however.

Biochemistry, 2000 Dec 26, 39(51), 15879 - 86
The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase; Biswas EE et al.; The rod outer segment ATP binding cassette (ABC) transporter protein (ABCR) plays an important role in retinal rod cells presumably transporting retinal . Genetic studies in humans have linked mutations in the ABCR gene to a number of inherited retinal diseases particularly Stargardt macular degeneration and age-related macular degeneration (ARMD) . The ABCR protein is characterized by two nucleotide binding domains and two transmembrane domains, each consisting of six membrane-spanning helices . We have cloned and expressed the 376 amino acid (aa) C-terminal end of this protein (amino acid residues 1898-2273) containing the second nucleotide binding domain (NBD2) with a purification tag at its amino terminus . The expressed protein was found to be soluble and was purified using a rapid and high-yield single-step procedure . The purified protein was monomeric and migrated as a 43 kDa protein in SDS-PAGE . The purified NBD2 protein had strong ATPase activity with a K(m) of 631 microM and V(max) of 144 nmol min(-1) mg(-1) . This ATPase activity on normalization was kinetically comparable to that observed for purified and reconstituted native ABCR . Nucleotide inhibition studies suggest that the binding of NBD2 is specific for ATP/dATP, and that none of the other ribonucleotides appeared to compete for binding at this site . These studies demonstrate that cloned and expressed NBD2 protein is a fully functional ATPase in the absence of the remainder of the molecule . The level of ATPase activity was comparable to that of trans-retinal-stimulated ABCR ATPase . The NBD2 expression plasmid was used to generate a Leu2027Phe mutation associated with Stargardt disease . Analysis of the ATPase activity of the mutant protein demonstrated that it had a 14-fold increase in binding affinity (K(m) = 46 microM) with a corresponding 9-fold decrease in the rate of hydrolysis (V(max) = 16.6 nmol min(-1) mg(-1)), indicating a significant alteration of the ATPase function . It also provided a molecular basis of Stargardt disease involving this mutation.

Biochemistry, 2000 Dec 26, 39(51), 15870 - 8
Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: time-resolved fluorescence of 3'-anthraniloyl-2'-deoxy-ADP and molecular modeling; Li de La Sierra IM et al.; The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme . The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution . These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents . These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP . The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding . The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ) . The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring . Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins . In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data . These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.

Biochemistry, 2000 Dec 26, 39(51), 15721 - 9
Role of the beta-strand insert in the central domain of the fibrinogen gamma-module; Yakovlev S et al.; The crystal structure of the fibrinogen gamma-module (residues gamma143-411) {Yee, V . C., et al . (1997) Structure 5, 125-138} revealed an unusual feature . Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible . To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli . It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region . A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized . This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286 . They all were folded, but those devoid of the beta-strand insert were destabilized . The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter . On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Biochemistry, 2000 Dec 26, 39(51), 15686 - 94
Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29; Hoke DE et al.; Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell growth in the cancerous state, and the modulation of blood coagulation . These activities are consistent with the proposed function of HIP/L29 as a heparin/heparan sulfate (Hp/HS) binding growth factor that has a preference for anticoagulantly active Hp/HS . Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion . However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the ability to bind Hp, suggesting that there may be additional Hp/HS binding sites outside of the HIP peptide-1 domain . To test this hypothesis, a systematic study of the domains within human and murine HIP/L29 responsible for Hp/HS binding activity was undertaken . Using deletion mutants, proteolytic fragments, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins . Furthermore, a conformational change is induced in human HIP/L29 upon Hp binding as detected by circular dichroism spectroscopy . These studies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.

Mol Microbiol, 2001 Jan, 39(1), 199 - 210
Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli; Arie JP et al.; The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined . Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31 . This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein . In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase . We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.

Mol Microbiol, 2001 Jan, 39(1), 191 - 8
DNA supercoiling-dependent transcriptional coupling between the divergently transcribed promoters of the ilvYC operon of Escherichia coli is proportional to promoter strengths and transcript lengths; Opel ML et al.; The twin-domain model of Liu and Wang suggested that high levels of DNA supercoiling generated in the region between closely spaced divergently transcribed promoters could serve to couple the activities of these promoters transcriptionally . In this report, we use topoisomer sets of defined superhelical densities as DNA templates in a purified in vitro transcription system to demonstrate transcriptional coupling between the divergently transcribed ilvY and ilvC promoters of the ilvYC operon of Escherichia coli . Current evidence for this type of DNA supercoiling-dependent transcriptional coupling, based largely on the in vivo activities of promoters contained in engineered DNA constructs, suggests that the transcription complex must be physically hindered to generate DNA supercoils and to prevent their diffusion throughout the DNA duplex . However, the in vitro results presented here demonstrate that (i) transcriptional coupling is observed between the divergent promoters of the ilvYC operon in the absence of transcript anchoring; (ii) the magnitude of the negative DNA supercoiling generated in the divergent promoter region is proportional to the sum of the global and transcription-induced superhelicity; and (iii) the magnitude of this transcription-induced superhelicity is proportional to promoter strengths and transcript lengths.

Mol Microbiol, 2001 Jan, 39(1), 158 - 65
Identification of a segment of DsbB essential for its respiration-coupled oxidation; Kobayashi T et al.; In the Escherichia coli protein disulphide bond formation pathway, membrane-bound DsbB oxidizes periplasmic DsbA, the disulphide bond-introducing enzyme . The Cys-41-Val-Leu-Cys-44 motif in the first periplasmic domain of DsbB is kept strongly oxidized by the respiratory function of the cell . We now show that the characteristic dithiothreitol resistance of the Cys-41-Cys-44 bond was retained even when the flanked Val-Leu combination was replaced by XX sequences from other oxidoreductases . Results of insertion mutagenesis showed that only the insertions (1-31 amino acids) in the region C-terminally adjacent to the CXXC motif impaired the oxidized state of DsbB . Deletion of a single amino acid from this region also rendered DsbB reduced and inactive . However, single amino acid substitutions of the four residues flanked by CXXC and the transmembrane segment did not abolish the oxidation of DsbB . These results suggest that some physical property, such as distance of the CXXC motif from the membrane, is important for the respiration-coupled oxidation of DsbB.

Mol Microbiol, 2001 Jan, 39(1), 112 - 25
Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation; Beran RK et al.; When Escherichia coli cells are shifted to low temperatures (e.g . 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes . One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome . At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism . Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader . The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader . We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed . This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C . However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates . Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.

Mol Microbiol, 2001 Jan, 39(1), 89 - 99
Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin; Pethe K et al.; Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence . To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs) . A cross-reactive protein was detected by immunoblotting of M . smegmatis whole-cell lysates . However, the M . tuberculosis HBHA-encoding gene failed to hybridize with M . smegmatis chromosomal DNA in Southern blot analyses . The M . smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M . smegmatis does not produce HBHA . Biochemical analysis of the M . smegmatis histone-like protein shows that it is glycosylated like HBHA . Immunoelectron microscopy demonstrated that the M . smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity . In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes . Therefore, the protein was called M . smegmatis laminin-binding protein (MS-LBP) . MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages . Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M . tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

Mol Microbiol, 2001 Jan, 39(1), 47 - 53
Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli; Thomas JD et al.; The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif . In this study, we have analysed the mechanism and capabilities of the E . coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA) . Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active . Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway . Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant . Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state . These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.

Mol Microbiol, 2000 Dec, 38(5), 1093 - 103
The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressordagger; Bucca G et al.; The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp . Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel-shift assays . This requirement is independent of the co-chaperones, DnaJ and GrpE, and it is ATP independent . Furthermore the retarded protein-DNA complex can be 'supershifted' by anti-DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex . It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK . We conclude that the formation of the stable DnaK-HspR-DNA ternary complex does not depend on the chaperoning activity of DnaK . In affinity chromatography experiments using whole-cell extracts, DnaK was shown to co-purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used . The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter . In addition, it is shown that DnaK confers enhanced HspR-mediated repression of transcription in vitro . Taken together, these results suggest that DnaK functions as a transcriptional co-repressor by binding to HspR at its operator sites . In this model, the DnaK-HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK-sigma32 and Hsp70-HSF systems of other organisms).

Mol Microbiol, 2000 Dec, 38(5), 1086 - 92
Modulation of KdpD phosphatase implicated in the physiological expression of the kdp ATPase of Escherichia coli; Brandon L et al.; The KdpD sensor kinase and the KdpE response regulator control the expression of the kdpFABC operon, encoding the KdpFABC high-affinity K+ transport system of Escherichia coli . Low turgor pressure has been postulated to be the environmental stimulus to express KdpFABC . KdpD has autokinase, phosphotransferase and, like many sensor kinases, response regulator (phospho-KdpE) specific phosphatase activity . To determine which of these activities are altered in response to the environmental stimulus, we isolated and analysed six kdpD mutants that cause constitutive expression of KdpFABC . In three of the mutants, phosphatase activity was undetectable and, in two, phosphatase was reduced . Kinase activity was unaffected in four of the mutants, but elevated in one . In one mutant, a pseudorevertant of a kdpD null mutation, kinase and phosphatase were both reduced to 20% of the wild-type level . These findings suggest that initiation of signal transduction by KdpD is mediated by the inhibition of the phospho-KdpE-specific phosphatase activity of KdpD, leading to an accumulation of phospho-KdpE, which in turn activates the expression of the KdpFABC system . The data also suggest that levels of activity in vitro may differ from what occurs in vivo, because in vitro conditions cannot replicate those in vivo.

Mol Microbiol, 2000 Dec, 38(5), 1074 - 85
N-mediated transcription antitermination in lambdoid phage H-19B is characterized by alternative NUT RNA structures and a reduced requirement for host factors; Neely MN et al.; Gene expression in lambdoid phages in part is controlled by transcription antitermination . For most lambdoid phages, maximal expression of delayed early genes requires an RNA polymerase modified by the phage N and host Nus proteins at RNA NUT sites . The NUT sites (NUTL and NUTR) are made up of three elements: BOXA, BOXB and an intervening spacer sequence . We report on N antitermination in H-19B, a lambdoid phage carrying shiga toxin 1 genes . H-19B N requires NusA, but not two other host factors required by lambda N, NusB and ribosomal protein S10 . The H-19B NUT site BOXA is not required, whereas the BOXB is required for N action . H-19B nut sites have dyad symmetries in the spacer regions that are not in other nut sites . Changes in one arm of the dyad symmetry inactivate the NUT RNA . Compensating changes increasing the number of mutant nucleotides but restoring dyad symmetry restore activity . Deletion of the sequences encoding the dyad symmetry has little effect . Thus, the specific nucleotides composing the dyad symmetry seem relatively unimportant . We propose that the RNA stem-loop structure, called the 'reducer', by sequestering nucleotides from the linear RNA brings into proximity sites on either side of the dyad symmetry that contribute to forming an active NUT site.

Mol Microbiol, 2000 Dec, 38(5), 1061 - 73
Interplay between three global regulatory proteins mediates oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon; Govantes F et al.; The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA . Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression . ArcA is shown to antagonize H-NS action to render cydAB expression insensitive to H-NS repression in anaerobiosis . The targets for H-NS-mediated aerobic repression are the four oxygen-regulated promoters, designated P1, P2, P3 and P4 . H-NS control is the result of H-NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters . We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein-DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions . According to this model, ArcA-P plays a central role in cydAB regulation by antagonizing H-NS repression of cydAB transcription when oxygen becomes limiting . This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.

J Appl Microbiol, 2000 Dec, 89(6), 1048 - 58
An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1; Chart H et al.; AIMS: To examine Escherichia coli strains EQ1, DH5alpha, BLR and BL21 for known pathogenic mechanisms . METHODS AND RESULTS: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins . The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement . Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut . Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut . In Merino sheep, only strain EQ1 was detected 6 d post-infection . CONCLUSIONS: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E . coli causing the majority of enteric infections . SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

J Immunol, 2001 Jan 1, 166(1), 559 - 65
A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway; Kishore U et al.; The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM . The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain . The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen . In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure . Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein . Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes . The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer . The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.

Eur J Neurosci, 2000 Dec, 12(12), 4434 - 46
The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune signals; a functional anatomical analysis; Konsman JP et al.; Cytokines act on the brain to induce fever and behavioural depression after infection . Although several mechanisms of cytokine-to-brain communication have been proposed, their physiological significance is unclear . We propose that behavioural depression is mediated by the vagus nerve activating limbic structures, while fever would primarily be due to humoral mechanisms affecting the preoptic area, including interleukin-6 (IL-6) action on the organum vasculosum of the laminae terminalis (OVLT) and induction of prostaglandins . This study assessed the effects of subdiaphragmatic vagotomy in rats on fever, behavioural depression, as measured by the social interaction test, and Fos expression in the brain . These responses were compared with induction of the prostaglandin-producing enzyme cyclooxygenase-2 and the transcription factor Stat3 that translocates after binding of IL-6 . Vagotomy blocked behavioural depression after intraperitoneal injection of recombinant rat IL-1beta (25 microg/kg) or lipopolysaccharide (250 microg/kg; LPS) and prevented Fos expression in limbic structures and ventromedial preoptic area, but not in the OVLT . Fever was not affected by vagotomy, but associated with translocation of Stat3 in the OVLT and cyclooxygenase-2 induction around blood vessels . These results indicate that the recently proposed vagal link between the immune system and the brain activates limbic structures to induce behavioural depression after abdominal inflammation . Although the vagus might play a role in fever in response to low doses of LPS by activating the ventromedial preoptic area, it is likely to be overridden during more severe infection by action of circulating IL-6 on the OVLT or prostaglandins induced along blood vessels of the preoptic area.

Clin Exp Allergy, 2000 Dec, 30(12), 1750 - 8
Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1; Aceituno E et al.; The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies . Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients . To obtain a full-length complementary DNA for Cup a 1, the major allergen of Cupressus arizonica pollen . It was cloned and sequenced and the recombinant protein was expressed . Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3'-RACE system and primers based on the N-terminal amino acid sequence . Recombinant Cup a 1 was cloned in pBluescript and expressed in a glycosylated form in rabbit reticulocytes . The cDNA was subcloned in pGEX-5X-1 and expressed in Escherichia coli as a fusion protein with GST . Recombinant Cup a 1 is highly homologous with the major allergens of mountain cedar (Jun a 1), Japanese cypress (Cha o 1) and Japanese cedar (Cry j 1) . Cup a 1 contains three potential N-glycosylation sites that are different from those found in Jun a 1 and Cry j 1 . The cloned protein contains a pectate lyase active site identical to those of Cry j 1 and Jun a 1 . The IgE from patients' sera recognizes recombinant Cup a 1, and this reactivity is higher with the glycosylated protein . Cup a 1 has been cloned and sequenced . As expected, the high degree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross-reactivity of conifer pollens . Different IgE reactivity with the glycosylated and non-glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.

Cancer Lett, 2001 Jan 10, 162(1), 117 - 22
Tamoxifen mutagenesis and carcinogenesis in livers of lambda/lacI transgenic rats: selective influence of phenobarbital promotion; Styles JA et al.; Administration of tamoxifen (TAM) (20 mg/kg per day p.o.) for 6 weeks to female lambda/lacI transgenic rats caused a 4-fold increase in mutation frequency (MF) at the lacI gene locus in the livers of dosed animals compared with controls . After cessation of dosing, the MF showed a further increase with time at 2, 12 and 24 weeks, respectively . Phenobarbital promotion of similarly treated animals resulted in no increase in mutation frequency compared with TAM alone . Treatment with phenobarbital or TAM+phenobarbital resulted in time-dependent increases in liver weight compared with the corresponding controls . There was an increase in cell proliferation in the phenobarbital and TAM+phenobarbital groups, and at 24 weeks in the TAM dosed animals compared with controls . There was also a progressive increase in the number of GST-P expressing foci in the livers of TAM and TAM + phenobarbital rats compared with controls . The induction of cell proliferation and GSTP foci in the rat liver by phenobarbital is consistent with its ability to promote tamoxifen-initiated liver tumours in the rat . If the lacI gene is regarded as being representative of the rat genome in general (albeit that the gene is bacterial) the above observations suggest that promotion by tamoxifen confers selective advantage on mutated genes at loci that contribute to the tumour phenotype and that promotion of rat liver tumours by tamoxifen is not dependent simply upon the enhancement of cellular proliferation.

J Microbiol Methods, 2000 Dec 15, 43(2), 91 - 6
An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract; Kang SH et al.; An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP . S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation . The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.

Biochim Biophys Acta, 2000 Dec 1, 1494(3), 277 - 81
Molecular characterization of Drosophila melanogaster myo-inositol-1-phosphate synthase; Park D et al.; We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS) . The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene . The putative active site residues are well conserved in Drosophila INOS protein . Southern blot analysis shows that Drosophila INOS gene is a single copy gene . Northern blot analysis reveals that Drosophila INOS gene expresses a 2.0-kb transcript that is more abundant in the head than the body, suggesting that it may be involved in brain function . The recombinant Drosophila INOS protein was expressed in Escherichia coli and the purified protein has proved to have a myo-inositol-1-phosphate synthase activity.

Biochim Biophys Acta, 2000 Dec 1, 1494(3), 248 - 55
Regulation of a recombinant pea nuclear apyrase by calmodulin and casein kinase II; Hsieh HL et al.; A cDNA encoding a pea nuclear apyrase was previously cloned . Overexpressions of a full-length and a truncated cDNA have been successfully expressed in Escherichia coli BL21(DE3) . The resulting fusion proteins, apyrase and the C-terminus (residues 315-453) of apyrase, were used for calmodulin (CaM) binding and phosphorylation studies . Fusion protein apyrase but not the C-terminus of apyrase can be recognized by polyclonal antibody pc480 . This suggested that the motif recognized by pc480 was located in the N-terminal region of apyrase . The recombinant apyrase protein also showed an activity 70 times higher than that of endogenous apyrase using ATP as a substrate . The recombinant apyrase has a preference for ATP more than other nucleoside triphosphate substrates . CaM can bind to recombinant apyrase, but not to the C-terminus of apyrase . This implies that the CaM-binding domain must be in the first 315 amino acids of the N-terminal region of apyrase . We found that one segment from residue 293 to 308 was a good candidate for the CaM-binding domain . This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-helical structure, which shows the predominance of basic residues on one side and hydrophobic residues on the other when displayed on a helical wheel plot . Using the gel mobility shift binding assay, this synthetic peptide was shown to bind to CaM, indicating that it is the CaM-binding domain . Both recombinant apyrase and the C-terminus of apyrase can be phosphorylated by a recombinant human protein kinase CKII . Phosphorylation does not affect CaM binding to recombinant apyrase . However, CaM does inhibit CKII phosphorylation of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA.

Biochim Biophys Acta, 2000 Dec 1, 1494(3), 236 - 41
Differential binding and transcriptional behaviour of two highly related orphan receptors, ROR alpha(4) and ROR beta(1); Gawlas K et al.; Nuclear receptors are ligand-inducible transcription factors that can be classified into two major groups according to their DNA-binding properties . Members of the first group bind to DNA as dimers, either homo- or heterodimers; members of the second group are also able to bind as monomers . While the first group has been extensively studied biochemically, very little is known about nuclear receptors that bind and act as monomers . In this study, we compared the binding and transcriptional behaviour of ROR alpha (NR1F1) and ROR beta (NR1F2), two representatives of the subgroup of monomer-binding receptors . We show that although they are highly related in their amino acid structures, they display remarkably different binding behaviours . Furthermore, we provide evidence that ROR beta can efficiently activate transcription in vitro as a monomer.

J Struct Biol, 2000 Oct, 132(1), 46 - 62
Multiple restraints to the unfolding of spermidine nucleoids from Escherichia coli; Murphy LD et al.; Bacterial DNA is largely localized in compact bodies known as nucleoids . The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown . In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction . Isolated nucleoids unfolded at approximately 3.2 M urea . Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction . The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively . Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure . The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea . To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged .

Eur J Biochem, 2001 Jan, 268(1), 179 - 85
Additional PKA phosphorylation sites in human cardiac troponin I; Ward DG et al.; We used mass spectrometry to monitor cAMP-dependent protein kinase catalysed phosphorylation of human cardiac troponin I in vitro . Phosphorylation of isolated troponin I by cAMP-dependent protein kinase resulted in the covalent incorporation of phosphate on at least five different sites on troponin I, and a S22/23A troponin I mutant incorporated phosphates on at least three sites . In addition to the established phosphorylation sites (S22 and S23) we found that S38 and S165 were the other two main sites of phosphorylation . These 'overphosphorylation' sites were not phosphorylated sufficiently slower than S22 and S23 that we could isolate pure S22/23 bisphosphorylated troponin I . Overphosphorylation of troponin I reduced its affinity for troponin C, as measured by isothermal titration microcalorimetry . Phosphorylation of S22/23A also decreased its affinity for troponin C indicating that phosphorylation of S38 and/or S165 impedes binding of troponin I to troponin C . Formation of a troponin I/troponin C complex prior to cAMP-dependent protein kinase treatment did not prevent overphosphorylation . When whole troponin was phosphorylated by cAMP-dependent protein kinase, however, {(32)P}phosphate was incorporated only into troponin I and only at S22 and S23 . Mass spectrometry confirmed that overphosphorylation is abolished in the ternary complex . Troponin I bisphosphorylated exclusively at S22 and S23 troponin I showed reduced affinity for troponin C but the effect was diminished with respect to overphosphorylated troponin I . These results show that care should be exercised when interpreting data obtained with troponin I phosphorylated in vitro.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14674 - 9
Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation; Zimmer DP et al.; Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions . To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes {glnL(Up)} to those in a strain with an ntrC (glnG) null allele by using DNA microarrays . Both strains could be grown under conditions of nitrogen excess . Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se . Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones . Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation . In all, approximately 2% of the E . coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Am J Physiol Endocrinol Metab, 2001 Jan, 280(1), E75 - 82
Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells; Li H et al.; Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II . To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC) . Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC . For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus {1-(14)C}arginine . Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC . Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC . Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC . Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC . Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC . Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC . Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.

Metab Eng, 2000 Oct, 2(4), 293 - 9
Co-overexpression of RspAB improves recombinant protein production in Escherichia coli; Weikert C et al.; The Escherichia coli mutant CWML2 was previously reported to exhibit improved physiological characteristics, including recombinant protein production . Here we investigate the molecular basis of this phenotype by comparing the cellular level of three RNA polymerase sigma subunits by immunoblot analysis . While the level of housekeeping sigma(D) was similar in parent and mutant, the levels of the flagella synthesis regulator sigma(F) and the stationary phase regulator sigma(S) were higher in the mutant strain, indicating a different motility and stationary phase phenotype . Evidence for this conclusion was provided by the significantly higher motility of CWML2, compared to its parent . Based on these results, we hypothesized that alterations in ppGpp regulation via a homoserine lactone-dependent mechanism may be relevant for the mutant phenotype . Indeed, transcription of the rspAB operon, which was previously described to be involved in the degradation of homoserine lactone, was found to be deregulated in CWML2 in a plasmid-based reporter protein assay . By overexpression of the E . coli rspAB operon, we could partly mimic the mutant phenotype and demonstrate that co-overexpression of RspAB is a pertinent metabolic engineering strategy to improve recombinant protein production.

J Biosci, 2000 Dec, 25(4), 339 - 46
Tyrosine phosphorylation of the human guanylyl cyclase C receptor; Bhandari R et al.; Tyrosine phosphorylation events are key components of several cellular signal transduction pathways . This study describes a novel method for identification of substrates for tyrosine kinases . Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases . Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling . This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

FEBS Lett, 2000 Dec 15, 486(3), 297 - 9
The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli; Carrozzo R et al.; A new mutation in human F(1)F(0) ATPase6, T9176G, which changes Leu 217 to an Arg, has been described in two siblings with Leigh syndrome {Carrozzo et al . (2000) Neurology, in press} . This mutation was modeled in Escherichia coli by changing Leu 259 (the equivalent residue) to Arg and the properties of the altered ECF(1)F(0) were compared to those of previously characterized ATPase6 mutants also modeled in the E . coli enzyme . The L259R change produced a fully assembled ECF(1)F(0) which had no significant ATP hydrolysis, ATP synthesis or proton pumping functions . This is very different from previously described human ATPase6 mutations . The presence of Arg at position 259 in subunit a did not make membranes permeable to protons . We conclude that the mutation inhibits functioning by blocking the rotary motor action of the enzyme.

FEBS Lett, 2000 Dec 15, 486(3), 267 - 9
Consensus predictions of membrane protein topology; Nilsson J et al.; We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology . Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E . coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority-vote approach.

FEBS Lett, 2000 Dec 15, 486(3), 200 - 2
Epigenes: design and construction of new hereditary units; Tchuraev RN et al.; A plasmid digene construction designed before {Tchuraev, R.N . (1982) J . Gen . Biol . 43, 79-87} has been realised, including feedback by repressing proteins with given trigger regime of gene functioning . Experimental tests of the expected epigene properties of the obtained pECPI recombinant plasmid involving lacI and cI(857) regulatory genes have shown a phenomenon of steady inheritance of two alternative epigenotypes lacI(1)cI(0) and lacI(0)cI(1), as well as an external toggle switch through metabolitic and temperature signals from one inherited functional state of the cyclic digene system into another . Thus, we have constructed a hereditary unit of a specific kind, namely, a two-component stationary epigene with preset properties.

FEBS Lett, 2000 Dec 15, 486(3), 195 - 9
5S rRNA binding proteins from the hyperthermophilic archaeon, Pyrococcus furiosus; Furumoto H et al.; A determination was made of the nucleotide sequence of the 2719 bp region of a ribosomal protein gene cluster (PfeL32-PfeL19-PfL18-PfS5-PfL30) containing a 5S rRNA binding protein L18 homolog of hyperthermophilic archaea Pyrococcus furiosus . The organization of the archaeal ribosomal protein gene cluster is similar to that in the spc-operon of Escherichia coli (L6-L18-S5-L30-L15) but has two additional genes, namely those encoding PfeL32 and PfeL19, which were identified as extra proteins that are apparently not present in bacterial E . coli . Using an inducible expression system, P . furiosus mature PfL18 protein and a mutant PfL18 with the basic N-terminal amino acid region deleted were produced in large amounts in E . coli and Northwestern analysis showed the N-terminal region of PfL18, including the conserved arginine-rich region, to have a significant role in 5S rRNA-PfL18 interaction.

Proteins, 2001 Feb 1, 42(2), 230 - 6
The C-terminal domain of HPII catalase is a member of the type I glutamine amidotransferase superfamily; Horvath MM et al.; Discovering distant evolutionary relationships between proteins requires detecting subtle similarities . Here we use a combination of sequence and structure analysis to show that the C-terminal domain of Escherichia coli HPII catalase with available spatial structure is a divergent member of the type I glutamine amidotransferase (GAT) superfamily . GAT-containing proteins include many biosynthetic enzymes such as E . coli carbamoyl phosphate synthetase and anthranilate synthase . Typical GAT domains have Rossmann fold-like topology and possess a catalytic triad similar to that of proteases . The C-terminal domain of HPII catalase has the GAT Rossmann fold but lacks the triad and therefore loses enzymatic activity . In addition, we detect significant sequence similarity between thiJ domains, some of which are known to have protease activity, and typical GAT proteins . Evolutionary tree analysis of the entire GAT superfamily indicates that the HPII catalase is more closely related to thiJ domains than to classical GAT domains and is likely to have evolved from a thiJ-like protein . This work illustrates the strength of sequence-based profile analysis techniques coupled with structural superpositions in developing an evolutionarily relevant classification of protein structures . Proteins 2001;42:230-236 .

Proteins, 2001 Feb 1, 42(2), 177 - 81
Stabilizing C-terminal tails on AraC; Ghosh M et al.; We examined the effects of the metabolic stability of random sequences appended to the C-terminus of the dimerization domain of the regulatory protein of the Escherichia coli arabinose operon, AraC . Genetic scoring utilized the trans dominant negative effect of the dimerization domain on the activity of intact AraC, and physical scoring used sodium dodecyl sulfate (SDS) gel electrophoresis . We confirmed previous results obtained with Arc and lambda repressors that C-terminal charged residues tend to be stabilizing and that hydrophobic residues are destabilizing . Additionally, we found that the provision of a single, charged C-terminal residue conferred significant stability that was independent of interior sequence . Hence, it appears that in the engineering of proteins, flexible tails may be freely added, with only the identity of the C-terminal amino acid being restricted . Proteins 2001;42:177-181 .

Infect Immun, 2001 Jan, 69(1), 579 - 83
Mouse toxicity and cytokine release by verotoxin 1 B subunit mutants; Wolski VM et al.; The crystal structure of the verotoxin 1 (VT1) B subunit complexed with a globotriaosylceramide (Gb(3)) analogue showed the presence of three receptor binding sites per monomer . We wished to study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro . We found that while the site 1 and 2 mutants were modestly (two- to sevenfold) reduced in their ability to cause disease in BALB/c mice, the site 3 mutant, W34A, was as toxic as VT1 . However, all the double-mutant proteins, irrespective of which two sites were mutated, exhibited approximately a 100-fold reduction in their 50% lethal doses for mice . These results suggest that multivalent receptor binding is important in vivo and that all three binding sites make a similar contribution to the latter process . The triple-mutant holotoxin, F30A G62T W34A, administered intraperitoneally without adjuvant, stimulated a strong antibody response in BALB/c mice, and the immune sera neutralized the activity of VT1 in vitro . Induction of tumor neurosis factor alpha release from differentiated human monocytes (THP-1 cells) was relatively impaired for site 1 and site 2 but not site 3 mutants, suggesting an auxiliary role for the latter site in mediation of cytokine release in vitro . Cytotoxicity assays on undifferentiated THP-1 cells have also demonstrated the importance of sites 1 and 2 and the relatively small role played by site 3 in causing cell death . These data suggest an association between the cytotoxicity of the protein and its ability to induce cytokine release.

Infect Immun, 2001 Jan, 69(1), 559 - 63
Tir tyrosine phosphorylation and pedestal formation are delayed in enteropathogenic Escherichia coli sepZ::TnphoA mutant 30-5-1(3); Devinney R et al.; Enteropathogenic Escherichia coli (EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation . In this study, we show that 30-5-1(3), which has a transposon insertion within the sepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate . A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.

Infect Immun, 2001 Jan, 69(1), 386 - 91
Vaccination with calpain induces a Th1-biased protective immune response against Schistosoma japonicum; Zhang R et al.; A large subunit of calpain, a calcium-activated neutral proteinase, from Schistosoma japonicum was cloned and expressed in Escherichia coli . When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed (P<0.01) . Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-gamma) by activated CD4(+) T cells . Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-gamma seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung . On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promoting S . japonicum infection . Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number . Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects . Taken together, these results point to S . japonicum calpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.

Infect Immun, 2001 Jan, 69(1), 315 - 24
espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin; Mellies JL et al.; At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries . However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island . EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa . Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E . coli . We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers . In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E . coli eliminated EspC enterotoxin activity . Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

Infect Immun, 2001 Jan, 69(1), 237 - 44
Human T- and B-cell responses to Schistosoma mansoni recombinant glyceraldehyde 3-phosphate dehydrogenase correlate with resistance to reinfection with S . mansoni or Schistosoma haematobium after chemotherapy; El Ridi R et al.; Recently we reported that human T- and B-cell recognition of a 42-kDa protein (p42) in soluble extracts of adult Schistosoma mansoni worms correlates with resistance to reinfection with S . mansoni or S . haematobium . Amino acid microsequencing of p42 revealed that it consists predominantly of schistosome glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) . We have expressed SG3PDH in Escherichia coli and purified the recombinant protein in a soluble and enzymatically active form . Recombinant SG3PDH (rSG3PDH) reacted with human monospecific antibodies to p42 . Lymphoproliferation and production of interleukin-4 and gamma interferon (IFN-gamma) after in vitro stimulation with rSG3PDH and serum isotype responses to rSG3PDH were examined in individuals with extremes of resistance and susceptibility to reinfection after treatment of previous S . mansoni or S . haematobium infection . Lymphoproliferation and IFN-gamma production in response to rSG3PDH and the presence of serum immunoglobulin G1 (IgG1), IgG3, and IgA antibodies to rSG3PDH generally characterized individuals who are resistant to reinfection after chemotherapy . The data indicate that T- and B-cell immune reactivity to rSG3PDH correlates with resistance to reinfection, confirming previous studies identifying SG3PDH as a target of protective immunity in humans, and suggest that SG3PDH should be investigated as a possible vaccine for human schistosomiasis.

Infect Immun, 2001 Jan, 69(1), 52 - 7
Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells; Lindenthal C et al.; Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon . Two separate invasion loci (tia and tib) that direct noninvasive E . coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407 . The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein . Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin . Here we report the purification of TibA and characterization of its biological activity . TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner . Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells . These results show that TibA acts as an adhesin . Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E . coli bearing tib locus clones, suggesting that TibA also acts as an invasin . The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis.

Infect Immun, 2001 Jan, 69(1), 9 - 14
Mutator natural Escherichia coli isolates have an unusual virulence phenotype; Picard B et al.; A small percentage of natural Escherichia coli isolates (both commensal and pathogenic) have a mutator phenotype related to defects in methyl-directed mismatch repair (MR) genes . We investigated whether there was a direct link between the mutator phenotype and virulence by (i) studying the relationships between mutation rate and virulence in a mouse model of extraintestinal virulence for 88 commensal and extraintestinal pathogenic E . coli isolates and (ii) comparing the virulence in mice of MR-deficient and MR-proficient strains that were otherwise isogenic . The results provide no support for the hypothesis that the mutator phenotype has a direct role in virulence or is associated with increased virulence . Most of the natural mutator strains studied displayed an unusual virulence phenotype with (i) a lack of correspondence between the number of virulence determinants and pathogenicity in mice and (ii) an intermediate level of virulence . On a large evolutionary scale, the mutator phenotype may help parasites to achieve an intermediate rate of virulence which mathematical models predict to be selected for during long-term parasite-host interactions.

Can J Urol, 2000 Apr, 7(2), 983 - 5
Perinephric abscess presenting as chronic diarrhea; McLellan RA et al.; Perinephric abscess is an uncommon diagnosis with a variable presentation and high mortality . We report an unusual case of a patient with a perinephric abscess who presented with chronic diarrhea and weight loss.

Yeast, 2000 Dec, 17(4), 283 - 93
Accurate prediction of protein functional class from sequence in the Mycobacterium tuberculosis and Escherichia coli genomes using data mining; King RD et al.; The analysis of genomics data needs to become as automated as its generation . Here we present a novel data-mining approach to predicting protein functional class from sequence . This method is based on a combination of inductive logic programming clustering and rule learning . We demonstrate the effectiveness of this approach on the M . tuberculosis and E . coli genomes, and identify biologically interpretable rules which predict protein functional class from information only available from the sequence . These rules predict 65% of the ORFs with no assigned function in M . tuberculosis and 24% of those in E . coli, with an estimated accuracy of 60-80% (depending on the level of functional assignment) . The rules are founded on a combination of detection of remote homology, convergent evolution and horizontal gene transfer . We identify rules that predict protein functional class even in the absence of detectable sequence or structural homology . These rules give insight into the evolutionary history of M . tuberculosis and E . coli .

FEMS Immunol Med Microbiol, 2000 Dec, 29(4), 295 - 301
A high molecular mass constituent of cranberry juice inhibits helicobacter pylori adhesion to human gastric mucus; Burger O et al.; Because previous studies have shown that a high molecular mass constituent of cranberry juice inhibited adhesion of Escherichia coli to epithelial cells and coaggregation of oral bacteria, we have examined its effect on the adhesion of Helicobacter pylori to immobilized human mucus and to erythrocytes . We employed three strains of H . pylori all of which bound to the mucus and agglutinated human erythrocytes via a sialic acid-specific adhesin . The results showed that a high molecular mass constituent derived from cranberry juice inhibits the sialic acid-specific adhesion of H . pylori to human gastric mucus and to human erythrocytes.

Vet Microbiol, 2000 Dec 20, 77(3-4), 513 - 8
In situ hybridization method for studies of cell wall deficient M . paratuberculosis in tissue samples; Hulten K et al.; Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis . However, no method has been available to localize this type of organisms in tissue sections . We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M . paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme . Pieces of beef were injected with the prepared spheroplasts . The samples were fixed in buffered formalin and paraffin embedded . A M . paratuberculosis-specific probe derived from the IS900 gene was used . Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria.Beef samples injected with M . paratuberculosis spheroplasts were the only samples that hybridized with the probe . Beef samples containing acid-fast or spheroplast forms of M . smegmatis and M . tuberculosis as well as the acid-fast forms of M . paratuberculosis did not hybridize with the probe . Unrelated bacterial controls, i.e . Helicobacter pylori and Escherichia coli were also negative in the assay . In situ hybridization with the IS900 probe provides a specific way to localize M . paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M . paratuberculosis and Crohn's disease and sarcoidosis . The assay may also be valuable for studies on Johne's diseased animals.

Biochim Biophys Acta, 2000 Dec 15, 1517(1), 159 - 63
Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus; Veau B et al.; Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-PCR strategy based on the design of degenerated oligonucleotides . The deduced amino acid sequence of crdxr is homologue to 1-deoxy-D-xylulose 5-phosphate reductoisomerases . Crmecs represents the first plant cDNA encoding a protein similar to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Escherichia coli . Expression of crdxr and crmecs genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids . Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

Biochim Biophys Acta, 2000 Dec 15, 1517(1), 153 - 8
Sequence analysis of the first complete cDNA clone encoding an American cockroach Per a 1 allergen; Yang CY et al.; C3, designated as Per a 1.0103 according to WHO/IUIS nomenclature, encoding a novel American cockroach allergen of 395 aa (44.6 kDa), is the first complete cDNA clone with a translatable immunoreactive protein among the reported group 1 cockroach allergens . Its deduced amino acid sequence possesses a signal sequence, phosphorylation sites, mitochondrial energy transfer protein signatures, and four repeats each sharing striking similarity with the corresponding repeats of the other cockroach allergens . The latter similarity suggests that the group 1 cockroach allergens might have evolved from a primordial mitochondrial sequence by exon duplication.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 335 - 45
Influence of transmembrane peptides on bilayers of phosphatidylcholines with different acyl chain lengths studied by solid-state NMR; Bystrom T et al.; The molecular orientation in a lipid membrane of the peptide fragment VEYAGIALFFVAAVLTLWSMLQYLSAAR (phosphatidylglycerophosphate synthase (Pgs) peptide E) of an integral membrane protein, Pgs, in Escherichia coli has been investigated by solid-state 15N nuclear magnetic resonance (NMR) on macroscopically aligned lipid bilayers . The secondary structure of the peptide in lipid vesicles was determined by circular dichroism spectroscopy . Furthermore, the phase behaviour of the Pgs peptide E/dierucoylphosphatidylcholine (DEruPC)/water system was determined by (2)H, (31)P and 15N solid-state NMR spectroscopy . The phase behaviour obtained was then compared to that of the Pgs peptide E solubilised in dioleoylphosphatidylcholine and water that was previously studied by Morein et al . {Biophys . J . 73 (1997) 3078-3088} . This was aimed to answer the question whether a difference in the length of the hydrophobic part of this peptide and the hydrophobic thickness of the lipid bilayer (hydrophobic mismatch) will affect the phase behaviour . The peptide mostly has a transmembrane orientation and is in an alpha-helical conformation . An isotropic phase is formed in DEruPC with high peptide content (peptide/lipid molar ratio (p/l) > or =1:15) and high water content (> or =50%, w/w) at 35 degrees C . At 55 and 65 degrees C an isotropic phase is induced at high water content (> or =50%, w/w) at all peptide contents studied (no isotropic phase forms in the lipid/water system under the conditions in this study) . At high peptide contents (p/l> or =1:15) an isotropic phase forms at 20 and 40% (w/w) of water at 55 and 65 degrees C . A comparison of the phase behaviour of the two homologous lipid systems reveals striking similarities, although the thicknesses of the two lipid bilayers differ by 7 A . This suggests that the rationalisation of the phase behaviour in terms of the hydrophobic mismatch is not applicable to these systems . The C-terminus of Pgs peptide E is amphiphilic and a considerable part of the peptide is situated outside the hydrophobic part of the bilayer, a property of the peptide that to a large extent will affect the lipid/peptide phase behaviour.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 123 - 30
Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site; Ding PZ et al.; Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities . Most of the mutants show good transport of melibiose but have lost the recognition of TMG . In addition, most mutants show little or no transport of lactose . Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose . The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid . The mutations were located on helices I, IV, VII, X and XI . We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 55 - 64
Selective NMR observation of inhibitor and sugar binding to the galactose-H(+) symport protein GalP, of Escherichia coli; Appleyard AN et al.; The binding of the transport inhibitor forskolin, synthetically labelled with (13)C, to the galactose-H(+) symport protein GalP, overexpressed in its native inner membranes from Escherichia coli, was studied using cross-polarization magic angle spinning (13)C NMR . (13)C-Labelled D-galactose and D-glucose were displaced from GalP with the singly labelled {7-OCO(13)CH(3)}forskolin and were not bound to any alternative site within the protein, demonstrating that any multiple sugar binding sites are not simultaneously accessible to these sugars and the inhibitor within GalP . The observation of singly (13)C-labelled forskolin was hampered by interference from natural abundance (13)C in the membranes and so the effectiveness of double-quantum filtration was assessed for the exclusive detection of (13)C spin pairs in sugar (D-{1,2-(13)C(2)}glucose) and inhibitor ({7-O(13)CO(13)CH(3)}forskolin) bound to the GalP protein . The solid state NMR methodology was not effective in creating double-quantum selection of ligand bound with membranes in the 'fluid' state (approx . 2 degrees C) but could be applied in a straightforward way to systems that were kept frozen . At -35 degrees C, double-quantum filtration detected unbound sugar that was incorporated into ice structure within the sample, and was not distinguished from protein-bound sugar . However, the method detected doubly labelled forskolin that is selectively bound only to the transport system under these conditions and provided very effective suppression of interference from natural abundance (13)C background . These results indicate that solid state NMR methods can be used to resolve selectively the interactions of more hydrophobic ligands in the binding sites of target proteins.

Virology, 2000 Dec 20, 278(2), 562 - 9
In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase; Willer DO et al.; During poxvirus infection, both viral genomes and transfected DNAs are converted into high-molecular-weight concatemers by the replicative machinery . However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unknown . Here we show that vaccinia virus DNA polymerase can catalyze single-stranded annealing reactions in vitro, converting linear duplex substrates into linear or circular concatemers, in a manner directed by sequences located at the DNA ends . The reaction required > or =12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpI3L) . Varying the structures at the cleaved ends of the molecules had no effect on efficiency . These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3'-to-5' proofreading exonuclease, as judged by the fact that only a 5'-(32)P-end label is retained in the joint molecules and the reaction is inhibited by dNTPs . The resulting concatemers are joined only through noncovalent bonds, but can be processed into stable molecules in E . coli, if the homologies permit formation of circular molecules . This reaction provides a starting point for investigating the mechanism of viral concatemer formation and can be used to clone PCR-amplified DNA .

Virology, 2000 Dec 20, 278(2), 490 - 500
Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli; Someya Y et al.; We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence . The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract . Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV . The ChV genome contains three open reading frames (ORFs) . A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase . ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids . The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV . When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease . Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase . Therefore, the ChV protease expressed in E . coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 708 - 11
Cofactor recycling with immobilized heterologous cytochrome P450 105D1 (CYP105D1); Taylor M et al.; Immobilisation of cells and enzymes can be a convenient and rapid way for testing and transforming substances . Cytochromes P450 may be useful in numerous biotransformations of varied lipophilic substrates, performing both regio- and stereo-specific monooxygenation reactions . However, one limitation of their use in vitro is the requirement of cofactor for the supply of electrons in the catalytic cycle . Here we report CYP105D1 from Streptomyces griseus expressed in Escherichia coli can be immobilised from cell-free extracts using DE52, that the immobilised protein is active in bioconversions and that a requirement for cofactor can be sustained by a recycling system for NADH regeneration .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 548 - 51
A family of compact plasmid vectors for DNA immunization in humans; Herrera AM et al.; DNA immunization technology is based on the availability of adequate vectors for cloning and expression of heterologous immunoactive proteins in mammalian cells . We have developed a family of DNA plasmid vectors suitable to manipulate antigen expression and location . Their in vitro and in vivo functionality and application are also reported . The developed immune response, the aspects considered for vector design, and the possible independent manipulation of both blocks for the generation of bicistronic constructs, make of the pAEC family of plasmid vectors a source for DNA vaccine candidate's development for further evaluation in human clinical trials, and for potential use in the gene therapy approach .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 534 - 42
Altered subunit communication in subfamilies of trimeric dUTPases; Fiser A et al.; The enzyme dUTPase is essential in preventing uracil incorporation into DNA . Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme . This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses . The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions . In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic . The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches . The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling . The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure . Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 427 - 32
Characterization of spontaneous mutation in the oxyR strain of Escherichia coli; Yamamura E et al.; Escherichia coli K-12 strain EY5, deficient in oxyR, was constructed to assess the role of oxyR and oxyR-regulated regulon in spontaneous mutagenesis . Mutagenesis was monitored by selecting two forward mutations of colicin B-sensitive to resistance and valine-sensitive to resistance, one base substitution mutation of rifampicin-sensitive to resistance and one reversion of argE3 his-4 to Arg(+) His(+) . Deficiency of oxyR did not lead to the enhancement of spontaneous mutation frequencies of the four markers tested . By DNA sequence analysis, we determined 49 colicin B-resistant mutants derived from EY5 and found that 37% were base substitutions, 29% IS element insertions, 20% deletions, and 14% single base frameshifts . Among the base substitutions, G:C-->T:A transversions predominated followed by G:C-->A:T transitions and A:T-->T:A transversions . These spectra were essentially the same as those from oxyR(+) strains . The results indicate that oxyR and oxyR-regulated genes do not play a significant role in the defense against spontaneous mutagenesis .

Enzyme Microb Technol, 2001 Jan 2, 28(1), 25 - 34
Display of green fluorescent protein on Escherichia coli cell surface; Shi H et al.; In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2 . The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E . coli cell surface . Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa) . Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane . The GFP displayed on the E . coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP . Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures . Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.

Enzyme Microb Technol, 2000 Dec, 27(10), 755 - 760
Construction and expression of antibody targeted plasminogen activator*
Jiang P, Changgeng R, Ru B.
It has been known that antibody-mediated plasminogen activator will be much more specific than its parent molecular . To get a cheaper and more effective medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoclonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator . PCR was used to amplify the region of VL and VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu-PA-32KD(leu144-leu411) from urokinase gene, respectively . Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via NdeI site . The recombinant protein was expressed in BL21 (DE3) plyS, a kind of E . coli . It was shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase . After partial purification: dialysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate . The specific activity was about 47,000 IU/mg, corresponding to 80,000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody . Activation of plasminogen by the chimera followed Michaelis-Menten kinetics, and the K(m) was 1.08 uM.

J Biol Chem, 2001 Mar 16, 276(11), 8436 - 44 Epub 2000 Dec 15.
Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function; Wang X et al.; Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin . Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown . Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin . Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1) . The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells . The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1) . The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures . These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.

J Biol Chem, 2001 Mar 30, 276(13), 9590 - 8 Epub 2000 Dec 05.
Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis; Dorus S et al.; The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K(+) with high affinity and specificity . Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K(+) binding from the analysis of mutants with reduced affinity for K(+) (Buurman, E., Kim, K.-T., and Epstein, W . (1995) J . Biol . Chem . 270, 6678-6685) . K(+) binding by this pump has been analyzed in detail by site-directed mutagenesis . We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K(+) binding . Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes . Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites . This study delineates the four clusters and confirms that they are important for K(+) affinity but have little effect on the rate of transport . At only 21 of the residues studied did at least three substitutions alter affinity for K(+), an indication that a residue is in or very near a K(+) binding site . At many residues lysine was the only substitution that altered its affinity . The effect of lysine is most likely a repulsive effect of this cationic residue on K(+) and thus reflects the effective distance between a residue and the site of binding or passage of K(+) in KdpA . Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K(+) as it moves through the KdpA subunit to cross the membrane.

J Biol Chem, 2001 Mar 23, 276(12), 9413 - 20 Epub 2000 Dec 05.
Escherichia coli MutS,L modulate RuvAB-dependent branch migration between diverged DNA; Fabisiewicz A et al.; This study examines the interaction between Escherichia coli MutS,L and E . coli RuvAB during E . coli RecA-promoted strand exchange . RuvAB is a branch migration complex that stimulates heterologous strand exchange . Previous studies indicate that RuvAB increases the rate at which heteroduplex products are formed by RecA, that RuvA and RuvB are required for this stimulation, and that RuvAB does not stimulate homologous strand exchange . This study indicates that MutS,L inhibit the formation of full-length heteroduplex DNA between M13-fd DNA in the presence of RuvAB, such that less than 2% of the linear substrate is converted to product . Inhibition depends on the time at which MutS,L are added to the reaction and is strongest when MutS,L are added during initiation . The kinetics of the strand exchange reaction suggest that MutS,L directly inhibit RuvAB-dependent branch migration in the absence of RecA . The inhibition requires the formation of base-base mismatches and ATP utilization; no effect on RuvAB-promoted strand exchange is seen if an ATP-deficient mutant of MutS (MutS501) is included in the reaction instead of wild-type MutS . These results are consistent with a role for MutS,L in maintaining genomic stability and replication fidelity.

EMBO J, 2000 Dec 15, 19(24), 6900 - 7
A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation; Dincbas-Renqvist V et al.; A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule . It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated . Overexpression of RF2 yields protein lacking the methylation . RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine . Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2 . Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs . Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246 . We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.

EMBO J, 2000 Dec 15, 19(24), 6853 - 9
GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming; Toulme F et al.; The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood . Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock . When RNAP is stalled within an (ATC/TAG)(n) sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3' terminus and its re-synthesis . Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming . We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced 'cleavage-and-restart' process to become catalytically competent . Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.

EMBO J, 2000 Dec 15, 19(24), 6833 - 44
Escherichia coli RNA polymerase core and holoenzyme structures; Finn RD et al.; Multisubunit RNA polymerase is an essential enzyme for regulated gene expression . Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme . Both structures were obtained by cryo-electron microscopy and angular reconstitution . Core RNA polymerase exists in an open conformation . Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss' . All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.

EMBO J, 2000 Dec 15, 19(24), 6637 - 43
Evolutionary dynamics of full genome content in Escherichia coli; Ochman H et al.; The evolutionary history of the entire Escherichia coli chromosome was traced by examining the distribution of the approximately 4300 open reading frames (ORFs) from E.coli MG1655 among strains of known genealogical relationships . Using this framework to deduce the incidence of gene transfer and gene loss, a total of 67 events-37 additions and 30 deletions-were required to account for the distribution of all genes now present in the MG1655 chromosome . Nearly 90% of the ORFs were common to all strains examined, but, given the variation in gene content and chromosome size, strains can contain well over a megabase of unique DNA, conferring traits that distinguish them from other members of the species . Moreover, strains vary widely in their frequencies of deletions, which probably accounts for the variation in genome size within the species.

Phytochemistry, 2000 Oct, 55(4), 305 - 9
Cloning and expression of homospermidine synthase from Senecio vulgaris: a revision; Ober D et al.; Homospermidine synthase . which catalyses the first pathway-specific reaction in pyrrolizidine alkaloid biosynthesis, was cloned from root cultures of Senecio vulgaris and expressed in E . coli . The open reading frame encodes a protein of 370 amino acids with a molecular mass of 40,740 Da . The enzyme is strictly dependent on spermidine as aminobutyl donor since it cannot be substituted by putrescine . The homospermidine synthase from S . vulgaris showed 97.9 and 99.3% nucleic acid identity with two HSS sequences from the closely related species Senecio vernalis . This report also revises data from a previous publication (Kaiser, A., 1999 . Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli . Plant J . 19 . 195 201.) that is incorrect.

Nature, 2000 Nov 30, 408(6812), 609 - 13
Coenzyme Q is an obligatory cofactor for uncoupling protein function; Echtay KS et al.; Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP . They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family . Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers . However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial . The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor . Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor . On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1 . The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ . H+ transport of native UCP1 correlated with the endogenous CoQ content.

Allergy, 2000 Dec, 55(12), 1179 - 83
Expression of the American cockroach Per a 1 allergen in mammalian cells; Wu CH et al.; BACKGROUND: Cockroach allergens are one of the major etiologic risk factors for developing IgE-mediated allergic respiratory illness throughout the world . Per a 1 is a cross-reactive allergen of American and German cockroaches . This study aimed to investigate the expression of a recombinant American cockroach (Periplaneta americana) Per a 1, C42, allergen in mammalian COS-1 cells . METHODS: The COS-1 cells and Escherichia coli were used to express the P . americana C42 allergen . Recombinant proteins were purified with hydroxylapatite and DE52 chromatography . Biologic reactivities of recombinant proteins were examined by direct IgE binding and IgE inhibition studies with the enzyme-linked immunosorbent assay (ELISA) . RESULTS: C42 was successfully expressed in the mammalian COS-1 cell as a 50-kDa secreted protein, and purified from the culture medium . The specific human IgE antibodies against recombinant C42 from either E . coli (C42-E . coli) or COS-1 (C42-COS-1) were compared by ELISA with 12 sera from Per a 1 and C42 skin-test-positive patients . All atopic sera contained specific IgE antibodies to C42 from either E . coli or COS-1 . Moreover, recombinant C42-COS-1 bound higher levels of serum IgE than recombinant C42-E . coli among C42-sensitive atopic patients, and a statistically significant difference (P<0.01) was found between them . In addition, recombinant C42-COS-1 as an inhibitor revealed higher inhibition of IgE binding to natural Per a 1 than recombinant C42-E . coli . CONCLUSIONS: The biologically highly reactive recombinant C42 produced in the COS-1 cell provides an alternative expression system and will facilitate studies on the immune response of asthma patients to cockroach allergens.

Plant Physiol, 2000 Dec, 124(4), 1775 - 85
Adenosine kinase of Arabidopsis . Kinetic properties and gene expression; Moffatt BA et al.; To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis . The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively . Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined . Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively . The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms . In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root . ADK1 transcript levels were generally higher than those of ADK2 . ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds . The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis . They may also contribute to cytokinin interconversion.

Vaccine, 2000 Nov 22, 19(7-8), 931 - 6
Adjuvant action of Escherichia coli enterotoxin for delayed-type hypersensitivity to Oka vaccine virus on pernasal co-administration in mice; Sasaki K et al.; The usefulness of a mutant of Escherichia coli enterotoxin for the induction of cellular immunity to varicella-zoster virus as a mucosal adjuvant is assessed in mice . When a commercially available live varicella vaccine (the Oka strain) and toxin were once administered simultaneously via the nasal route, delayed-type hypersensitivity to Oka vaccine virus was significantly induced and detected by footpad test in mice . Moreover, when spleen cells from mice immunized with the vaccine and toxin were re-stimulated with live vaccine in vitro, they showed more thymidine uptake and produced more IL-2 than those from mice immunized with the vaccine alone . These results suggest that mutant enterotoxin has adjuvant action to induce a specific delayed-type hypersensitivity to Oka vaccine virus on nasal co-administration with live vaccine virus.

Vaccine, 2000 Nov 22, 19(7-8), 788 - 95
DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC); Alves AM et al.; The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes . The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests . An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC . pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits . BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass . pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I . Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells . These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.

Vaccine, 2000 Nov 22, 19(7-8), 706 - 15
Microencapsulated enterotoxigenic Escherichia coli and detached fimbriae for peroral vaccination of pigs; Felder CB et al.; The feasibility of peroral immunisation with microencapsulated Escherichia coli and detached fimbriae to prevent enterotoxigenic E . coli infections in pigs was examined . For this E . coli and fimbriae were microencapsulated into poly(lactide-co-glycolide) microspheres by spray-drying . Various microsphere formulations designed to deliver priming and booster doses were fed to new-born and weaned pigs . The pigs were challenged 19 days after the booster dose by peroral administration of an infective dose of the homologous E . coli . Serum IgA antibody titres and excretion of challenge E . coli, as indicators for colonisation, were determined . The data showed that no significant serum antibodies were induced, and E . coli colonisation was not reduced by the peroral administration of the various antigen-loaded microspheres . These results are in contradiction to some of the previously published experiments typically in rats or rabbits, where model antigens or unpractical immunisation procedures have frequently been used.

Vaccine, 2000 Nov 22, 19(7-8), 684 - 93
Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein; Coeffier E et al.; The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system . We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope . Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope . This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity . Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity . However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies . This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Biochim Biophys Acta, 2001 Jan 19, 1503(3), 261 - 70
Kinetics of electron and proton transfer during O(2) reduction in cytochrome aa(3) from A . ambivalens: an enzyme lacking Glu(I-286); Gilderson G et al.; Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optimally at approximately 80 degrees C and pH 2.5 . The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily . One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E . coli ubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen . In this study we have investigated the dynamics of the reaction of the reduced A . ambivalens quinol oxidase with O(2) . With the purified enzyme, two kinetic phases were observed with rate constants of 1.8&z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3 . 7x10(3) s(-1), respectively . The first phase is attributed to binding of O(2) to heme a(3) and oxidation of both hemes forming the 'peroxy' intermediate . The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol . In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1) . Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center . Moreover, the overall electron and proton-transfer kinetics in the A . ambivalens quinol oxidase are the same as those in the E . coli ubiquinol oxidase, which indicates that in the A . ambivalens enzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved . Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A . ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E . coli cytochrome bo(3)).

Int J Oncol, 2001 Jan, 18(1), 117 - 20
Bystander effect in uracil phosphoribosyltransferase/5-fluorouracil-mediated suicide gene therapy is correlated with the level of intercellular communication; Kawamura K et al.; We examined whether a suicide gene/prodrug system using the uracil phosphoribosyltransferase (UPRT) of E . coli origin and 5-fluorouracil (5-FU) could achieve a bystander effect in two rodent tumor cell lines, murine colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells . Cytotoxicity tests of mixed populations consisting of parent and transduced cells showed that the bystander effect was not produced in Colon 26 cells in either the UPRT/5-FU system or the herpes simplex virus-thymidine kinase/ganciclovir system but a strong bystander effect was evidenced by both suicide gene systems in 9L cells . The expression level of connexin 43, a protein that constitutes gap junctions, was high in 9L but low in Colon 26 cells . A gap junction-permeable fluorescein dye could be transferred among 9L cells but hardly at all among Colon 26 cells . Taken together, the efficacy of the bystander effect in the UPRT/5-FU system can be affected by gap junction-mediated intercellular communication.

Mol Microbiol, 2000 Nov, 38(4), 854 - 66
Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly; Ow MC et al.; We demonstrate here that the assembly of the RNase E-based degradosome of Escherichia coli is not required for normal mRNA decay in vivo . In contrast, deletion of the arginine-rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay . When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal . An extensive RNase E truncation mutation (rnedelta610) had a more pronounced mRNA decay defect at 37 degrees C than the temperature-sensitive rne-1 allele at 44 degrees C . Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.

Mol Microbiol, 2000 Nov, 38(4), 817 - 27
FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3; Lamberg KE et al.; In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR . The ability of FNR to activate transcription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR-AR1 and FNR-AR3 . To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA . All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-AR1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from PnarG and PdmsA in vivo . Transcription assays of PdmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro . Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA-binding properties . The function of FNR-S187P and FNR-G85A was also measured in strains containing sigma70 mutants (sigma70-K593A, sigma70-R596A and sigma70-K597A) known to be impaired in FNR-dependent transcription activation . Of all of the combinations analysed, only FNR-G85 and sigma70-K597 showed a genetic interaction, supporting the notion that FNR-AR3 and sigma70 interact functionally in the process of transcription activation . Lastly, the transcription activation defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate . As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.

Mol Microbiol, 2000 Nov, 38(4), 781 - 93
Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler; Sperandio V et al.; Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells . The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE) . The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir . The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler) . The LEE2 and LEE3 operons are divergently transcribed with overlapping -10 promoter regions, and gene fusion studies have shown that they are both activated by Ler . Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than -247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation . We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3 . We observed that Ler bound to a DNA fragment containing the -300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the -300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2 . The Ler-activatable LEE3:lacZ fusions extended to what would be -246 bp of the LEE2 operon . A lacZ fusion from the -300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site . DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2 . Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region . These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.

Mol Microbiol, 2000 Nov, 38(4), 667 - 72
Riboregulation by DsrA RNA: trans-actions for global economy; Lease RA et al.; DsrA is an 87 nucleotide Escherichia coli RNA with extraordinary regulatory properties . The profound impact of its actions stems from DsrA regulating translation of two global transcription regulators, H-NS and RpoS (sigmas), by sequence-specific RNA-RNA interactions . H-NS is a major nucleoid-structuring and global repressor protein, and RpoS is the stationary phase and stress response sigma factor of RNA polymerase . DsrA changes its conformation to bind to these two different mRNA targets and thereby inhibits H-NS translation, while stimulating that of RpoS in a mechanistically distinct fashion . DsrA apparently binds both the start and the stop codons of hns mRNA and sharply decreases the mRNA half-life . DsrA also binds sequences in the 5'-untranslated leader region of rpoS mRNA, enhancing rpoS mRNA stability and RpoS translation . A cohort of genes, governed by H-NS repression and RpoS activation, are thus regulated . Low temperatures increase the levels of DsrA, with differential effects on H-NS and RpoS . Additionally, the RNA chaperone protein Hfq is involved with DsrA regulation, as well as with other small RNAs that also act on RpoS to co-ordinate stress responses . We address the possible functions of this genetic regulatory mechanism, as well as the advantages of using small RNAs as global regulators to orchestrate gene expression.

Immunol Cell Biol, 2000 Dec, 78(6), 603 - 7
Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats; Morton H et al.; Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties . In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared . Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system . Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF) . However, the half-life of activity (50% decrease in the log value) in serum, following i.p . injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days) . This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins . Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated . Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted . Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.

J Bacteriol, 2001 Jan, 183(1), 401 - 4
Cloning, expression, and characterization of cis-polyprenyl diphosphate synthase from the thermoacidophilic archaeon Sulfolobus acidocaldarius; Hemmi H et al.; cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms . However, only little is known about this enzyme of archaea . In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme.

J Bacteriol, 2001 Jan, 183(1), 375 - 81
Characterization of transmembrane segments 3, 4, and 5 of MalF by mutational analysis; Steinke A et al.; MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in Escherichia coli . They are thought to form the transport channel and are thus of critical importance for the mechanism of transport . To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5 . These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8 . While most of the isolated mutations appear to cause assembly defects, L(323)Q in helix 5 could interfere more directly with substrate specificity . The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.

J Bacteriol, 2001 Jan, 183(1), 347 - 57
Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage; Sutton MD et al.; Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites . The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD' . We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102 . These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins . Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop . Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS . These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.

J Bacteriol, 2001 Jan, 183(1), 301 - 8
Proteome analysis of metabolically engineered Escherichia coli producing Poly(3-hydroxybutyrate); Han MJ et al.; Recombinant Escherichia coli strains harboring heterologous polyhydroxyalkanoate (PHA) biosynthesis genes were shown to accumulate unusually large amounts of PHA . In the present study, integrated cellular responses of metabolically engineered E . coli to the accumulation of poly(3-hydroxybutyrate) (PHB) in the early stationary phase were analyzed at the protein level by two-dimensional gel electrophoresis . Out of 20 proteins showing altered expression levels with the accumulation of PHB, 13 proteins were identified with the aid of mass spectrometry . Three heat shock proteins, GroEL, GroES, and DnaK, were significantly up-regulated in PHB-accumulating cells . Proteins which play essential roles in protein biosynthesis were unfavorably influenced by the accumulation of PHB . Cellular demand for the large amount of acetyl coenzyme A and NADPH for the PHB biosynthesis resulted in the increased synthesis of two enzymes of the glycolytic pathway and one enzyme of the Entner-Doudoroff pathway . The expression of the yfiD gene encoding a 14.3-kDa protein, which is known to be produced at low pH, was greatly induced with the accumulation of PHB . Therefore, it could be concluded that the accumulation of PHB in E . coli acted as a stress on the cells, which reduced the cells' ability to synthesize proteins and induced the expression of various protective proteins.

J Bacteriol, 2001 Jan, 183(1), 292 - 300
Archaeal shikimate kinase, a new member of the GHMP-kinase family; Daugherty M et al.; Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds . Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts . In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes . The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily . The latter functionality in M . jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis . Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase . No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates . The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.

J Bacteriol, 2001 Jan, 183(1), 221 - 8
A long T . A tract in the upp initially transcribed region is required for regulation of upp expression by UTP-dependent reiterative transcription in Escherichia coli; Cheng Y et al.; In Escherichia coli, pyrimidine-mediated regulation of upp expression occurs by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be elongated . The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand) . Initiation can occur at either the first or the second base in this sequence (designated G6 and A7, with numbering from the promoter -10 region) . High intracellular UTP levels favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive UMP addition) within the 8-bp T . A tract in the initially transcribed region and are aborted . In contrast, low intracellular UTP levels favor initiation at position G6, which results in transcripts that can, in part, avoid reiterative transcription and be elongated normally . In this study, we examined the regulatory requirement for the long T . A tract in the upp initially transcribed region . We constructed upp promoter mutations that shorten the T . A tract to 7, 6, 5, 4, 3, or 2 bp and examined the effects of these mutations on upp expression and regulation . The results indicate that pyrimidine-mediated regulation is gradually reduced as the T . A tract is shortened from 7 to 3 bp; at which point regulation ceases . This reduction in regulation is due to large-percentage increases in upp expression in cells grown under conditions of pyrimidine excess . Quantitation of cellular transcripts and in vitro transcription studies indicate that the observed effects of a shortened T . A tract on upp expression and regulation are due to increases in the fraction of both G6- and A7-initiated transcripts that avoid reiterative transcription and are elongated normally.

J Bacteriol, 2001 Jan, 183(1), 139 - 44
Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope; Stanley NR et al.; The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane . Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident . Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect . Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene . The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.

J Bacteriol, 2001 Jan, 183(1), 94 - 100
Cloning of an intracellular Poly{D(-)-3-Hydroxybutyrate} depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product; Saegusa H et al.; An intracellular poly{D(-)-3-hydroxybutyrate} (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized . Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da . The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer . The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules . The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly . The gene product was expressed in R . eutropha cells concomitant with the synthesis of PHB and localized in PHB granules . Although a mutant of R . eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium . These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R . eutropha.

J Bacteriol, 2001 Jan, 183(1), 86 - 93
trans-acting mutations in loci other than kdpDE that affect kdp operon regulation in Escherichia coli: effects of cytoplasmic thiol oxidation status and nucleoid protein H-NS on kdp expression; Sardesai AA et al.; Transcription of the K(+) transport operon kdp in Escherichia coli is induced during K(+)-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE . In this study, we screened for new dke (named dke for decreased kdp expression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression . One dke mutation was shown to be in hns, encoding the nucleoid protein H-NS . Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also for trxA mutants that are deficient in thioredoxin 1 . Exogenously provided dithiothreitol rescued the kdp expression defect in trxB but not trxA mutants . Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR . Mutations in genes dsbC and dsbD did not affect kdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm . Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues . A trxB hns double mutant was even more severely affected for kdp expression than either single mutant . The dke mutations themselves had no effect on strength of the signal controlling kdp expression, and constitutive mutations in kdpDE were epistatic to hns and trxB . These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for kdp transcription.

Cell, 2000 Nov 22, 103(5), 781 - 92
X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding; Roll-Mecak A et al.; X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP . The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life . The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV) . Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV . Mechanisms of GTPase function and ribosome binding are discussed.

Cell, 2000 Nov 22, 103(5), 769 - 79
Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade; Katzen F et al.; The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of E . coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced state . We split DsbD into three structural domains, each containing two essential cysteines . Remarkably, when coexpressed, these truncated proteins restore DsbD function . Utilizing this three piece system, we were able to determine a pathway of the electrons through DsbD . Our findings strongly suggest that the pathway is based on a series of multistep redox reactions that include direct interactions between thioredoxin and DsbD, and between DsbD and its periplasmic substrates . A thioredoxin-fold domain in DsbD appears to have the novel role of intramolecular electron shuttle.

Cell, 2000 Nov 22, 103(5), 723 - 31
Adaptive amplification: an inducible chromosomal instability mechanism; Hastings PJ et al.; Adaptive mutation is an induced response to environmental stress in which mutation rates rise, producing permanent genetic changes that can adapt cells to stress . This contrasts with neo-Darwinian views of genetic change rates blind to environmental conditions . DNA amplification is a flexible, reversible genomic change that has long been postulated to be adaptive . We report the discovery of adaptive amplification at the lac operon in Escherichia coli . Additionally, we find that adaptive amplification is separate from, and does not lead to, adaptive point mutation . This contradicts a prevailing alternative hypothesis whereby adaptive mutation is normal mutability in amplified DNA . Instead, adaptive mutation and amplification are parallel routes of inducible genetic instability allowing rapid evolution under stress, and escape from growth inhibition.

Cell, 2000 Nov 22, 103(5), 711 - 21
Evolutionary implications of the frequent horizontal transfer of mismatch repair genes; Denamur E et al.; Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection . Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations . Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes . Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages . This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators . The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.

J Mol Biol, 2001 Jan 5, 305(1), 71 - 7
Projection structure of the monomeric porin OmpG at 6 A resolution; Behlau M et al.; The Escherichia coli porin OmpG, which acts as an efficient unspecific channel for mono-, di- and trisaccharides, has been purified and crystallized in two dimensions . Projection maps of two different crystal forms of OmpG at 6 A resolution show that the protein has a beta-barrel structure characteristic for outer membrane proteins, and that it does not form trimers, unlike most other porins such as OmpF and OmpC, but appears in monomeric form . The size of the barrel is approximately 2.5 nm, indicating that OmpG may consist of 14 beta-strands . The projection map suggests that the channel is restricted by internal loops .

J Mol Biol, 2001 Jan 5, 305(1), 49 - 60
Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex; Jones DD et al.; Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex . Residues in the lipoyl-lysine beta-turn region of the unlipoylated E2plip domain (E2plip(apo)) undergo significant changes in both chemical shift and transverse relaxation time (T(2)) in the presence of E1p but not E1o . Residue Gly11, in a prominent surface loop between beta-strands 1 and 2 in the E2plip domain, was also observed to undergo a significant change in chemical shift . Addition of pyruvate to the mixture of E2plip(apo) and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction . Residues in both beta-strands 4 and 5, together with those in the prominent surface loop and the following beta-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31 . The values of T(2) across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip(apo) binds more tightly after the addition of pyruvate . The lipoylated domain (E2plip(holo)) also exhibited significant changes in chemical shift and decreases in the overall T(2) relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue . In addition, small chemical shift changes and a general drop in T(2) times in the presence of E1o were observed, indicating that E2plip(holo) can interact, weakly but non-productively, with E1o . It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp .

J Mol Biol, 2001 Jan 5, 305(1), 23 - 31
The efficiency of strand invasion by Escherichia coli RecA is dependent upon the length and polarity of ssDNA tails; McIlwraith MJ et al.; RecA protein is essential for homologous recombination and the repair of DNA double-strand breaks in Escherichia coli . The protein binds DNA to form nucleoprotein filaments that promote joint molecule formation and strand exchange in vitro . RecA polymerises on ssDNA in the 5'-3' direction and catalyses strand exchange and branch migration with a 5'-3' polarity . It has been reported previously, using D-loop assays, in which ssDNA (containing a heterologous block at one end) invades supercoiled duplex DNA that 3'-homologous ends are reactive, whereas 5'-ends are inactive . This polarity bias was thought to be due to the polarity of RecA filament formation, which results in the 3'-ends being coated in RecA, whereas 5'-ends remain naked . Using a range of duplex substrates containing ssDNA tails of various lengths and polarities, we now demonstrate that when no heterologous block is imposed, 5'-ends are just as reactive as 3'-ends . Moreover, using short-tailed substrates, we find that 5'-ends form more stable D-loops than 3'-ends . This bias may be a consequence of the instability of short 3'-joints . With more physiological substrates containing long ssDNA tails, we find that RecA shows no intrinsic preference for 5' or 3'-ends and that both form D-loop complexes with high efficiency . Copyright 2001 Academic Press.

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