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 l