Anesthesiology, 2002 Nov, 97(5), 1227 - 33 Reactive oxygen species scavengers attenuate endotoxin-induced impairment of hypoxic pulmonary vasoconstriction in mice; Baboolal HA et al.; BACKGROUND: Sepsis and endotoxemia attenuate hypoxic pulmonary vasoconstriction (HPV), thereby impairing systemic oxygenation . Reactive oxygen species (ROS) are implicated in the pathogenesis of sepsis-induced lung injury . The authors investigated whether treatment with scavengers of ROS prevents impairment of HPV in mice challenged with endotoxin . METHODS: The pulmonary vasoconstrictor response to left mainstem bronchus occlusion (LMBO) was studied in anesthetized mice 22 h after an intraperitoneal challenge with saline solution or 10 mg/kg Escherichia coli endotoxin . In some mice, challenge with saline solution or endotoxin was followed after 1 h with intraperitoneal or intratracheal administration of the ROS scavengers N-acetylcysteine or EUK-8 . Myeloperoxidase activity and nitric oxide synthase-2 gene expression were measured in lung tissues . RESULTS: The LMBO increased left pulmonary vascular resistance by 106 +/- 24% in saline-challenged control mice but by only 23 +/- 12% (P < 0.05) in endotoxin-challenged mice . Intraperitoneal administration of N-acetylcysteine or EUK-8 1 h after endotoxin challenge attenuated the endotoxin-induced impairment of HPV (58 +/- 6% and 68 +/- 10%, respectively; both P< 0.05 endotoxin-challenged mice) . Intratracheal administration of ROS scavengers 1 h after endotoxin challenge was equally effective but required lower doses than systemic treatment . Administration of the ROS scavengers 22 h after endotoxin challenge did not restore HPV . CONCLUSIONS: Administration of N-acetylcysteine or EUK-8 1 h after endotoxin challenge in mice prevented the impairment of HPV after LMBO . Early therapy with ROS scavengers, either systemically or by inhalation, may provide a means to preserve HPV in sepsis-associated acute lung injury. EMBO J, 2002 Nov 1, 21(21), 5673 - 81 Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition; Wang X et al.; In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7) . However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport . When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer . In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer . Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE . These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo . Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition. Mol Microbiol, 2002 Nov, 46(3), 813 - 26 Regulation and mode of action of the second small RNA activator of RpoS translation, RprA; Majdalani N et al.; Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA . DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA . Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA . This is the first example of two different small RNAs regulating a common target . Regulation of these RNAs differs . DsrA synthesis is increased at low temperature . We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC . An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter . An essential site with similarity to other RcsB-regulated promoters was defined in the rprA promoter . Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA-dependent fashion . This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ. Mol Microbiol, 2002 Nov, 46(3), 761 - 8 Selection of plasmid molecules for conjugative transfer and replacement strand synthesis in the donor; Parker C et al.; Plasmid selection and strand replacement synthesis in donor cells during conjugative transfer was examined by a procedure involving electroporation of test plasmid DNA, containing a base pair mismatch, into donor cells prior to mating . Multiple copies of the plasmid were transferred from a donor cell that allowed vegetative replication of the plasmid . Under conditions non-permissive for vegetative replication, there were further rounds of transfer after a lag period . Strand replacement in the donor did not depend solely on the initiation mechanism for vegetative replication, indicating a conjugation-specific mechanism was also available . The lag period between first and second rounds of transfer argues against the transfer of multiple copies into recipients by the spooling of copies generated on a master molecule by rolling-circle replication. Mol Microbiol, 2002 Nov, 46(3), 699 - 707 Knotting dynamics during DNA replication; Olavarrieta L et al.; The topology of plasmid DNA changes continuously as replication progresses . But the dynamics of the process remains to be fully understood . Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining . Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork . To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process . The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size. Scand J Immunol, 2002 Nov, 56(5), 484 - 91 CD14-mediated induction of interleukin-8 and monocyte chemoattractant protein-1 by a heat-resistant constituent of Porphyromonas gingivalis in endothelial cells; Mao S et al.; Viable and inactivated Porphyromonas gingivalis dose-dependently induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion in human umbilical vein endothelial cells (HUVECs) . The inactivated P . gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly . The production of MCP-1 protein began increasing immediately after stimulation by P . gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL-8 production showed a linear increase between 4 and 12 h of incubation . The IL-8 and MCP-1 mRNA expressions in HUVECs as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P . gingivalis . Furthermore, the time courses of IL-8 and MCP-1 mRNA expressions were in accordance with those of protein production . Addition of polymyxin B or boiling did not weaken the stimulatory effect of P . gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E . coli LPS) and tumour necrosis factor-alpha (TNF-alpha), respectively . In contrast, the induction of IL-8 and MCP-1 by P . gingivalis was significantly reduced by anti-CD14 antibody . Our results suggest that some heat-stable component of P . gingivalis, including LPS, may be responsible for the induction of IL-8 and MCP-1 in HUVECs by a CD14-dependent mechanism . These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis. Scand J Immunol, 2002 Nov, 56(5), 436 - 42 In vitro production and characterization of partly assembled human CD3 complexes; Kastrup J et al.; Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells . Subsequently, the CD3 heterodimers form complexes with Ti alpha and Tiss chains forming hexameric Ti alpha beta CD3 gamma epsilon delta epsilon complexes . Finally, association with the zeta 2 homodimer occurs in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface . To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3 gamma, CD3 delta and CD3 epsilon . Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro . CD3 gamma and CD3 epsilon folded as soluble monomers, whereas CD3 delta did not yield any soluble proteins . When folding the chains pairwise, soluble CD3 gamma epsilon and CD3 delta epsilon heterodimers could be isolated, whereas CD3 gamma delta heterodimers were not produced . Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization . Our data indicate that CD3 epsilon undergoes a conformational change after dimerization with CD3 gamma or CD3 delta . Furthermore, we demonstrated that the CD3 gamma epsilon heterodimer could be purified using immunoaffinity chromatography. Nucleic Acids Res, 2002 Nov 1, 30(21), 4583 - 91 Global genome removal of thymine glycol in Escherichia coli requires endonuclease III but the persistence of processed repair intermediates rather than thymine glycol correlates with cellular sensitivity to high doses of hydrogen peroxide; Alanazi M et al.; Using a monoclonal antibody that specifically recognizes thymine glycol (Tg) in DNA, we measured the kinetics of the removal of Tg from the genomes of wild-type and repair gene mutant strains of Escherichia coli treated with hydrogen peroxide . Tg is rapidly and efficiently removed from the total genomes of repair-proficient cells in vivo and the removal of Tg is completely dependent on the nth gene that encodes the endonuclease III glycosylase . Hence, it appears that little redundancy in the repair of Tg occurs in vivo, at least under the conditions used here . Moreover, previous studies have found that nth mutants are not sensitive to killing by hydrogen peroxide but xth mutant strains (deficient in the major AP endonuclease, exonuclease III) are sensitive . We find that cell death correlates with the persistence of single-strand breaks rather than the persistence of Tg . We attempted to measure transcription-coupled removal of Tg in the lactose operon using the Tg-specific monoclonal antibody in an immunoprecipitation approach but were not successful in achieving reproducible results . Furthermore, the analysis of transcription-coupled repair in the lactose operon is complicated by potent inhibition of beta-galactosidase expression by hydrogen peroxide. J Biol Chem, 2003 Jan 10, 278(2), 1259 - 67 Epub 2002 Oct 29. Dissociation of intact Escherichia coli ribosomes in a mass spectrometer . Evidence for conformational change in a ribosome elongation factor G complex; Hanson CL et al.; We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding . From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10) . Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA . Replacement of Mg(2+) by Li(+) ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins . Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation . By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton . Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate . By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA . These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions. J Biol Chem, 2003 Jan 10, 278(2), 1022 - 8 Epub 2002 Oct 29. Spectroscopic observations of ferric enterobactin transport; Cao Z et al.; We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology . The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein . FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C . These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods . Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer . These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport . We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons . Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion. Biochim Biophys Acta, 2002 Oct 11, 1565(2), 333 - 46 Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes; Zakharov SD et al.; The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells . The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation . The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed . Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes . Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate . The nature of the open-channel structure is discussed. Biochim Biophys Acta, 2002 Oct 11, 1565(2), 232 - 45 Structural model of the transmembrane Fo rotary sector of H+-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ; Fillingame RH et al.; H(+)-transporting, F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism . ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites . The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector . The gamma subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the gamma and epsilon subunits of F(1) . In this essay we will review recent studies on the Escherichia coli F(o) sector . The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp(61) centered in the second transmembrane helix (TMH) . A model for the structural organization of the c(10) oligomer in F(o) was deduced from extensive cross-linking studies and by molecular modeling . The model indicates that the H(+)-carrying carboxyl of subunit c is occluded between neighboring subunits of the c(10) oligomer and that two c subunits pack in a "front-to-back" manner to form the H(+) (cation) binding site . In order for protons to gain access to Asp(61) during the protonation/deprotonation cycle, we propose that the outer, Asp(61)-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp(61) protonation/deprotonation drives the rotation of the c-ring . The NMR structures of wild-type subunit c differs according to the protonation state of Asp(61) . The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2 . The structural information is considered in the context of the possible mechanism of rotary movement of the c(10) oligomer during coupled synthesis of ATP. Phytochemistry, 2002 Nov, 61(5), 523 - 9 A cDNA clone for beta-caryophyllene synthase from Artemisia annua; Cai Y et al.; An homology-based cloning strategy yielded a full-length cDNA from Artemisia annua that encoded a protein of 60.3 kDa which resembled a sesquiterpene synthase in sequence . Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of farnesyl diphosphate to beta-caryophyllene, a sesquiterpene olefin found in the essential oil of A . annua . In reaction parameters and kinetic properties, beta-caryophyllene synthase resembles other sesquiterpene synthases of angiosperms . The beta-caryophyllene synthase gene is expressed in most plant tissues during early development, and is induced in mature tissue in response to fungal elicitor thus suggesting a role for beta-caryophyllene in plant defense. Biophys Chem, 2002 Nov 6, 99(3), 281 - 95 Membrane topology modulates beta-galactosidase activity against soluble substrates; Sanchez JM et al.; The effect of bio-surfaces of contrasting curvature, on the kinetic parameters of ortho-nitrophenyl-beta-D-galactopiranoside hydrolysis catalyzed by E . coli beta-galactosidase, was investigated . The self-aggregating state and structure of the amphiphiles (Phosphatidylcholine, Lubrol-PX, Triton X-100, DocNa, SDS and CTAB) were inferred from their c.m.c . values and light-scattering measurements . Low curvature phosphatidylcholine or mixed phosphatidylcholine-detergent vesicles increased V(max) without affecting K(M) . High curvature micellar structures containing ionic detergents modulated negatively the enzyme activity (decreased or abolished V(max) and increased K(M)) . Neither micelles containing non-ionic detergents nor the amphiphiles in a monomeric form, affected enzyme activity . CTAB at a concentration below its c.m.c but incorporated into a bilayer, became an activator (K(M) decreased respect to the control) . Non-enzymatic interfacial hydrolysis of the substrate was discarded . Enzyme-membrane interaction and membrane elasticity, were evaluated using monomolecular layers at the air-water interface . Beyond particular molecular structures, topology affected the direction of the modulatory effects exerted by these amphiphiles on beta-galactosidase activity. Mol Cell, 2002 Sep, 10(3), 671 - 81 Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery; Yamada K et al.; We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration . Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB . The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring . The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode. Mol Cell, 2002 Sep, 10(3), 647 - 57 DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands; Kaplan DL et al.; DnaB is a ring-shaped, hexameric helicase that unwinds the E . coli DNA replication fork while encircling one DNA strand . This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex . With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding . Thus, DnaB may clear proteins from chromosomal DNA . Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo . DnaB binds to just one DNA strand during branch migration . T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases. Mar Environ Res, 2002 Sep-Dec, 54(3-5), 263 - 6 Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products; Martinez-Lara E et al.; cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica . GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)) . GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1) . Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates . Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure. Bioorg Khim, 2002 Sep-Oct, 28(5), 440 - 6 {Deletion mutants of human granulocyte-macrophage colony-stimulating factor}; Petrovskaia LE et al.; To study the structure-function relationship of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), genes were constructed that encode its three deletion mutants: D1, a mutant with the deletion of six amino acid residues (37-42) some of which are a part of a beta-structural region; D2, a mutant with the deletion of the unstructured six-aa sequence of a loop (45-50); and D3, a mutant with the deletion of 14 aa residues (37-50) corresponding to the A-B loop and encoded by the second exon of the gmcsf gene . The expression products of these genes in E . coli were accumulated in a fraction of insoluble proteins . The secondary structures of the mutant proteins were similar to that of the full-size GM-CSF, but the biological activity of the deletion mutants was 130 times lower than that of the GM-CSF: they stimulated the proliferation of the TF-1 cell line at 3 ng/ml concentration . The resulting proteins displayed antagonistic properties toward the full-size GM-CSF, with the inhibition degree of its colony-stimulating activity being 27% . A decrease in the mutant activity in the row D2 > D1 > D3 implies the importance of the conserved hydrophobic residues involved in the formation of the beta-structure for the formation of the GM-CSF functional conformation. Bioorg Khim, 2002 Sep-Oct, 28(5), 426 - 33 {Hexameric, trimeric, dimeric, and monomeric forms of inorganic pyrophosphatase from Escherichia coli}; Vainonen IuP et al.; The conditions were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia coli inorganic pyrophosphatase from its native hexameric form . Interconversions of the oligomers were studied, and rate constants for their dissociation and association were determined . All forms were found to be catalytically active, with the activity decreasing in the order: hexamer-trimer-dimer-monomer . The activity of trimeric and dimeric forms was high enough to study and to compare their catalytic properties . The monomeric form of the enzyme was unstable. Neoplasia, 2002 Nov-Dec, 4(6), 486 - 92 Ultraviolet radiation-induced apoptosis is mediated by Daxx; Wu S et al.; UV irradiation and other stress-activated signals activate the Jun N-terminal kinase (JNK, SAPK) pathway . The induction of JNK activity results in the activation of proto-oncogene c-Jun and activator protein-1 (AP-1) transcriptional activity . Data presented here show that UV mediated the activation of JNK correlated with UV-induced apoptosis and that overexpression of a dominant negative JNK blocked UV-induced apoptosis . However, the molecular events that lead to JNK activation in response to UV treatment are not clear . In this report, we provide evidence that a Fas receptor binding protein, Daxx, mediates UV-induced JNK activation and apoptosis . A dominant negative Daxx, coding for the C-terminal region (112 amino acids) of Daxx, was constructed and used in the experiments . Our data show that overexpression of the dominant negative Daxx partially inhibits UV-induced JNK phosphorylation in 293 cells . Inhibition of JNK phosphorylation resulted in the inhibition of c-Jun activation upon UV irradiation . Our data also show that the inhibition of JNK activation by dominant negative Daxx correlates with the reduced rate of apoptotic death of 293 cells after UV irradiation . Surprisingly, overexpression of wild-type Daxx also inhibited UV-induced apoptosis, suggesting that Daxx competes for Fas receptor binding sites with other proapoptotic factors such as FADD . In addition, overexpression of a dominant negative mutant of FADD did not affect UV-induced JNK activation but does inhibit UV-induced apoptosis . These results suggest that UV-induced JNK activation is not sufficient but required for induction of apoptosis. Invest Ophthalmol Vis Sci, 2002 Nov, 43(11), 3480 - 8 Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor; Wu WC et al.; PURPOSE: To examine the protective effect of glial cell line-derived neurotrophic factor (GDNF) on retinal detachment (RD)-induced photoreceptor damage by using gene delivery . METHODS: Gene delivery to photoreceptors was achieved by subretinal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Lewis rats . RD in bilateral eyes was induced with subretinal injection of high-density vitreous substitute in the temporal retina 3 weeks after gene delivery . The synthesis and accumulation of GDNF within the retina was monitored 3 weeks after RD by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively . The rescue of photoreceptors was evaluated by monitoring the preservation of the thickness of photoreceptor outer segment (OS) and outer nuclear layer (ONL) . Apoptosis in the photoreceptors was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method 2 days after RD . Muller cell activity was checked using the immunohistochemistry with glial fibrillary acidic protein (GFAP) antibody 28 days after RD . RESULTS: Gene delivery was demonstrated by immunohistochemical study . The results of ELISA confirmed that high levels of neurotrophic factors were produced in retinas . Photoreceptor OS degeneration and the gradual shortening of the ONL were noted after RD in all the eyes . However, rAAV-GDNF-treated eyes retained longer OS than rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD . ONL was also longer in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD . GDNF-treated eyes had statistically less apoptotic cells than control eyes in photoreceptor layer (P = 0.043) . Subretinal proliferation of Muller cells was suppressed in the GDNF-treated group, indicating less scar formation . CONCLUSIONS: GDNF is a potential factor that can protect photoreceptors from degeneration . In addition to preserving the OS and ONL structures, GDNF may exert its protective action by preventing the apoptosis of photoreceptors after RD . GDNF gene therapy may be a valuable adjuvant to current treatments in certain complicated forms of RD. Appl Environ Microbiol, 2002 Nov, 68(11), 5718 - 27 Function of native OmtA in vivo and expression and distribution of this protein in colonies of Aspergillus parasiticus; Lee LW et al.; The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro . Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis . We generated A . parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli . Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA . Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo . Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development . We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation . OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract . OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed . OmtA in older fractions of the colony (24 to 72 h) was partly degraded . Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores . These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age. Appl Environ Microbiol, 2002 Nov, 68(11), 5288 - 95 Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water; McElhiney J et al.; A naive (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR . Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA) . The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin . Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography . Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography . Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR . The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol . Large-scale, low-cost production of anti-microcystin-LR scAb in E . coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples. Protein Expr Purif, 2002 Nov, 26(2), 301 - 8 Rapid refolding and polishing of single-chain antibodies from Escherichia coli inclusion bodies; Sinacola JR et al.; An inexpensive and fast-folding strategy for single-chain antibody (scFv) recovered from Escherichia coli inclusion bodies has been developed . Two anti-fluorescein single-chain antibodies, 4-4-20 and 4M5.3, were expressed as inclusion bodies in E . coli for use in a comparative refolding study . Active protein yields as well as degree of aggregation were evaluated for scFv produced by stepwise dialysis, redox dialysis, and a newly developed controlled dilution and filtration strategy . Although all three methods produced active protein for both 4-4-20 and 4M5.3, the extent of aggregation differed greatly among the methods . For 4-4-20, the controlled dilution and filtration strategy reduced aggregation by half, allowed batch processing times of 8h (an 18-fold improvement), and significantly reduced denaturant usage while increasing active yields by 150% . A hydroxyapatite resin polishing step was used to remove completely the aggregate species and inactive monomeric scFv from active scFv. Protein Expr Purif, 2002 Nov, 26(2), 284 - 9 Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli; Subramanian S et al.; Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen . Most efforts to derive minimized proteins have employed synthetic peptide fragments . Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies . The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues . The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system . The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography . Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein . This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals. Protein Expr Purif, 2002 Nov, 26(2), 275 - 83 Purification and characterization of progenipoietins produced in Escherichia . coli; Violand BN et al.; The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor . These unique proteins have previously been shown to induce the proliferation of multiple cell lineages . The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described . These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity . Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains . ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different . Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable . Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation . This study describes the purification and physico-chemical properties of this class of proteins generated using an E . coli expression system. Protein Expr Purif, 2002 Nov, 26(2), 266 - 74 Chaperonin assisted overexpression, purification, and characterisation of human PP2A methyltransferase; George RR et al.; Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase found in many eukaryotic cell types and is involved in regulating a number of intracellular signalling pathways . Its activity, in turn, is regulated through covalent modification, involving phosphorylation and methylation reactions . The effect of phosphorylation on the activity of the protein is well known, but the effects of methylation have only recently been documented and the mechanistic details of methylation are lacking . Methylation, which occurs on the catalytic subunit of PP2A, is catalysed by PP2A methyltransferase (PP2Amt) . Here, we present a method for the large-scale purification of human PP2Amt using an Escherichia coli host, coexpressing the chaperonins GroEL and GroES . Purified PP2Amt was identified by peptide mass mapping using MALD-MS and peptide sequencing using ESI-LC-MS/MS . The CD spectrum indicated that purified PP2Amt was folded, with about one-third of the protein adopting an alpha-helical conformation . Analytical gel filtration estimated the molecular weight to be 34kDa, equivalent to the monomeric form of the protein . Further CD analysis showed that in the presence and absence of the ligand S-adenosylhomocysteine, the thermal denaturation profiles were biphasic . However, the transition midpoints shifted to a higher temperature in the presence of ligand, indicating stabilisation of ligand-bound PP2Amt compared to the apo-form . We also report on the progress made in determining the structure of PP2Amt, using both X-ray crystallography and NMR spectroscopy. Protein Expr Purif, 2002 Nov, 26(2), 229 - 34 Production of extracellular domain of human tissue factor using maltose-binding protein fusion system; Guan M et al.; Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases . Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available . In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli . The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene . The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG) . MBP-tTF was purified by amylose affinity chromatography . MBP can be removed by digestion with factor Xa . Expression could represent 21.5% of the total soluble protein in E . coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture . The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay . This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF. Protein Expr Purif, 2002 Nov, 26(2), 218 - 28 Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli; Zhang Z et al.; Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level . Recovery of functionally active products from inclusion bodies is onerous and ineffective . We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E . coli disulfide isomerases with general chaperone function, to scFvs . Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis . The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter . Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel . Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E . coli separately to a considerable portion of soluble CBD-scFv . Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens . We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E . coli . We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs. Protein Expr Purif, 2002 Nov, 26(2), 187 - 93 Recombinant human insulin IX . Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin; Tikhonov RV et al.; The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated . The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain . The kind and the size of leader peptide do not have essential influence on folding efficiency . However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography . In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents . A negative influence of nucleic acid and heavy metal ions on folding has been found . S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding . Folded fusion proteins are transformed into insulin by enzymatic cleavage. Vet Immunol Immunopathol, 2002 Nov, 90(1-2), 45 - 54 Sows intramammarily inoculated with Escherichia coli at parturition . II . Effects on the densities of MHC class II(+), CD4(+) and CD8(+) cells in the mammary gland; Loving M et al.; The aim of this study was to determine the density of MHC class II, CD4 and CD8 positive cells in mammary glands of sows around parturition, and whether the densities were altered following intramammary inoculation with Escherichia coli prior to parturition . Also, animals developing clinical disease after inoculation were compared with animals not developing clinical disease . Fourteen cross-bred primiparous sows were subject to intramammary inoculation with E . coli bacteria 24h before estimated parturition . Mammary gland biopsies were collected and clinical observations were made . Four sows were categorised as clinically ill based on general condition, body temperature and gross mammary affection . There were no changes in density of MHC class II, CD4 and CD8 positive cells in non-inoculated glands around parturition, while significant changes in densities were shown in inoculated glands . Here, the density of MHC class II, CD4 and CD8 positive cells reached a peak at 72 h post-inoculation (p<0.01) . In sows developing clinical disease, there was a tendency to an over all lower density (p=0.07) of MHC class II positive cells in inoculated glands compared with sows not developing clinical disease . When comparing the categories with respect to the density of CD4 and CD8 positive cells, the sows developing clinical disease showed a higher density (p=0.03) of CD4 and CD8 positive cells in inoculated glands than sows not developing disease . No differences were shown between categories in non-inoculated glands . It is concluded that the density of MHC class II, CD4 and CD8 positive cells seems to be unaltered around parturition . However, there is a rapid increase in density of these cells following intramammary inoculation with E . coli . Also, the data suggest that there is a difference between sows developing and sows not developing clinical disease after inoculation with respect to the increase in density of MHC class II, CD4 and CD8 positive cells in the mammary gland. Vet Immunol Immunopathol, 2002 Nov, 90(1-2), 35 - 44 Sows intramammarily inoculated with Escherichia coli at parturition: I . Functional capacity of granulocytes in sows affected or non-affected by clinical mastitis; Osterlundh I et al.; The objective of this study was to investigate if occurrence of clinical disease was related to granulocyte traits in sows . Functional capacity of granulocytes and plasma steroid hormone concentrations were assessed before inoculation with Escherichia coli in the mammary glands in sows at parturition . Blood samples were taken for 3 days approximately 1 week before parturition, and granulocyte migration, phagocytic capacity and expression of CD 18 adhesion molecules were determined . Inoculation was done within 36 h before partus . Thereafter, daily thorough clinical examinations were performed including udder health, habitus, appetite and rectal temperature, to assess the severity of disease . Based on the clinical findings four sows were classified as affected and eight as non-affected by clinical mastitis within 48 h after parturition.No difference (p>0.10) in pre-inoculation chemotaxis, phagocytosis or CD 18 expression was found between granulocytes from the sows resisting and developing clinical mastitis, respectively . However, there was an effect by the individual sow (p=0.001) on the numbers of granulocytes and white blood cells, and on plasma concentrations of estradiol-17beta and progesterone . In conclusion, these data does not suggest that impaired chemotaxis or phagocytosis by blood granulocytes contribute to the development of clinical coliform mastitis in the periparturient sow. J Invest Dermatol, 2002 Oct, 119(4), 820 - 9 Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen; Mossabeb R et al.; The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells . It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids . Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients . By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library . Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family . Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography . By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity . Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies . Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein . Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient . As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis. Res Microbiol, 2002 Sep, 153(7), 469 - 74 Expression and folding of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm; Laden JC et al.; In this review, we summarize some of our results on folding and directed evolution of an antibody fragment in Escherichia coli cytoplasm . We will also discuss some attempts to construct other antibodies active in this cellular compartment. Res Microbiol, 2002 Sep, 153(7), 435 - 40 SOS Chromotest methodology for fundamental genetic research; Vasilieva S; The present mini-review summarizes data in selected fields of basic genetics which were exclusively obtained in agreement with the principles of SOS Chromotest methodology and with Escherichia coli PQ37 sfiA::lacZ as a tester strain. Res Microbiol, 2002 Sep, 153(7), 425 - 6 From maltose to cell division; D'Ari R; Seeing connections between apparently unrelated areas is the hallmark of a deep thinker . Maurice Hofnung showed that his interest in the maltose regulon and my own interest in the regulation of cell division in Escherichia coli could lead to fruitful collaboration . From the maltose regulon to the LamB receptor to phage A to the SOS response to the Mutatest to induction of expression of the SOS-inducible division inhibitor SfiA to the SOS Chromotest based on sfiA::lacZ induction to the development of a commercial kit for measuring the genotoxicity of environmental substances...this was but one of the original trails that Maurice Hofnung blazed and exploited successfully. Res Microbiol, 2002 Sep, 153(7), 399 - 404 Folding and aggregation of export-defective mutants of the maltose-binding protein; Betton JM et al.; We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31 . To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences . Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation. Res Microbiol, 2002 Sep, 153(7), 395 - 8 Harnessing malE for the study of antigen/antibody recognitions; Bedouelle H et al.; The construction of hybrids between the variable fragment (Fv) of antibodies and protein MalE of Escherichia coli at the genetic level makes possible their preparation in a functional state, independently of any interaction with the antigen . We used such hybrids and a mutagenesis approach to study the recognition between antibody D1.3 and its antigen lysozyme, and its maturation . We subsequently transformed D1.3 into a reagentless fluorescent biosensor by knowledge-based design. J Biol Chem, 2002 Dec 27, 277(52), 51077 - 83 Epub 2002 Oct 25. DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli; Qi HY et al.; Consistent with many other results indicating that SecA plays an essential role in the translocation of presecretory proteins across the Escherichia coli inner membrane, we previously found that a approximately 95% depletion of SecA completely blocks the export of periplasmic proteins in vivo . Surprisingly, we found that about 25% of the outer membrane protein (OMP) OmpA synthesized after SecA depletion was gradually translocated across the inner membrane . In this study we analyzed the export of several other OMPs after SecA depletion . We found that 25-50% of each OMP as well as an OmpA-alkaline phosphatase fusion protein was exported from SecA-deficient cells . This partial export was completely abolished by the SecA inhibitor sodium azide and therefore still required the participation of SecA . Examination of a variety of OmpA derivatives, however, ruled out the possibility that OMPs are selectively translocated in SecA-deficient cells because SecA binds to their N termini with unusually high affinity . Export after SecA depletion was observed in cells that lack SecB, the primary targeting factor for OMPs, but was abolished by partial inactivation of DnaK . Furthermore, OmpA could be isolated in a stable complex with DnaK . The data strongly suggest that OMPs require only a relatively low level of translocase activity to cross the inner membrane because they can be preserved in a prolonged export-competent state by DnaK. Biochemistry, 2002 Nov 5, 41(44), 13318 - 27 A circularly permuted myoglobin possesses a folded structure and ligand binding similar to those of the wild-type protein but with a reduced thermodynamic stability; Fishburn AL et al.; A circular permutein of sperm whale myoglobin in which the G helix is C-terminal, the H helix is N-terminal, and 16 amino acids link the H helix to the A helix has been expressed in Escherichia coli . The permutein sequence begins with Gly121 (using the numbering scheme for the wild-type protein) and terminates with Pro120 . The ligand binding function of the permutein was assayed using stopped-flow methods and shown to be essentially identical to that of the wild-type protein . In addition, one- and two-dimensional NMR studies of the cyanomet isoform of the permutein show a nativelike structure with a heme binding pocket very similar to that of the wild-type myoglobin . Although the structure and function of the permutein resemble those of the wild-type myoglobin, the permutein is less stable to chemical denaturation by 5.2 kcal/mol. Biochemistry, 2002 Nov 5, 41(44), 13198 - 206 Site-specific labeling of supercoiled DNA at the A+T rich sequences; Potaman VN et al.; Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules . For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension . Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA . Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region . The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend . Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time . Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy. Anal Chem, 2002 Oct 15, 74(20), 5350 - 7 Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy; Kunst BH et al.; Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool . The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow . The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule . It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam. RNA, 2002 Oct, 8(10), 1294 - 307 The dispersal of five group II introns among natural populations of Escherichia coli; Dai L et al.; Group II introns are self-splicing RNAs that also act as retroelements in bacteria, mitochondria, and chloroplasts . Group II introns were identified in Escherichia coli in 1994, but have not been characterized since, and, instead, other bacterial group II introns have been studied for splicing and mobility properties . Despite their apparent intractability, at least five distinct group II introns exist naturally in E . coli strains . To illuminate their function and learn how the introns have dispersed in their natural host, we have investigated their distribution in the ECOR reference collection . Two introns were cloned and sequenced to complete their partial sequences . Unexpectedly, southern blots showed all ECOR strains to contain fragments and/or full-length copies of group II introns, with some strains containing up to 15 intron copies . One intron, E.c.14, has two natural homing sites in IS629 and IS911 elements, and the intron can be present in one, both, or neither homing site in a given strain . Nearly all strains that contain full-length introns also contain unfilled homing sites, suggesting either that mobility is highly inefficient or that most full-length copies are nonfunctional . The data indicate independent mobility of the introns, as well as mobility via the host DNA elements, and overall, the pattern of intron distribution resembles that of IS elements. Int J Artif Organs, 2002 Sep, 25(9), 832 - 7 Is steam sterilization really making any difference in dialysis-induced cytokine release? Aucella F, Tetta C, Tessore V, De Nitti C, Vigilante M, Gatta G, Grandone E, Margaglione M, Colaizzo D, Cappucci G, Modoni S, Stallone C. Ethylene oxide (ETO) is presently the most commonly used sterilization method for medical devices . Although alternative sterilization modes such as steam sterilization have been suggested, the effect of steam on dialysis-induced cytokine release is unknown . We enrolled 9 patients on chronic hemodialysis and evaluated at different intervals IL-1beta production while treated with ETO (NC 1785-Bellco) and steam sterilized NC 1785S-Bellco) Synthetically Modified Cellulose (SMC) . A basal test during treatment with NC 1785 was performed (A); the same test was set up 4 weeks after treatment with NC 1785S (B) and, lastly, 4 weeks after returning to NC 1785 (C) . Peripheral blood mononuclear cells (PBMC) were purified before and after the dialysis session, were isolated on a Ficoll/Hypaque gradient and incubated for 24 h . Spontaneous IL-1beta release was evaluated in the supernatant and in the lysate . In A, IL-1beta levels were (in pg/ml/10(6) cells, in supematant and lysate, respectively): 5.8 +/- 4.8 and 7.6+/-5.2 in pre-HD and 4.68 +/- 3.6 and 9.7 +/- 6.65 in post-HD . These levels showed a clear reduction in B: 2.5 +/- 2.2 and 4.4 +/- 3.1 in pre-HD, and 4.35+/- 6.6 and 7.52 +/- 7.22 in post-HD . In the C test, 4 weeks after the return to the ETO membrane, IL-1beta levels remained unchanged: 2.9 +/- 1.8 and 4.5 +/- 3.1 in pre-HD; and 2.6 +/- 3 and 5.7 +/- 6.6 in post-HD . Statistical analysis showed significant changes in the pre-HD levels both in supematant (p < 0.04) and in lysate (p < 0.04) . Steam sterilization of SMC induced a lower spontaneous IL-1beta release, but this effect was not statistically significant due to the large inter-individual variation . Hence, contrary to claims of better biocompatibility, steam sterilization does not result in a reduced production of pro-inflammatory IL-1beta. Genetica, 2002 Jun, 115(2), 233 - 41 Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity; Kholodii G et al.; Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30 degrees C but sensitive to this compound at 37-41.5 degrees C . We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures . Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures . However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent . These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion. Consum Rep, 2002 Nov, 67(11), 29 - 35 How safe is that burger? {Emphysematous perinephric abscess with diabetes mellitus: a case report} Suzuki K, Hagisawa S, Mitsuzuka K, Takeuchi M. Department of Urology, Iwaki Kyoritsu General HospitalA 48-year-old woman was referred to our hospital with high fever and left flank pain . She was diagnosed with diabetes mellitus (DM), and abdominal computed tomography (CT) revealed left perinephric abscess with much emphysema . She underwent drainage of the abscess by left flank incision after treatment with antibiotics and insulin . The pus culture revealed Escherichia coli . Immediately after drainage, the symptoms began to subside . At three months after drainage, abdominal CT revealed no emphysema around the left kidney . At 18 months after the discharge, left perinephric abscess was not seen and DM was well controlled with insulin. Proteins, 2002 Dec 1, 49(4), 446 - 56 Nonatomic solvent-driven Voronoi tessellation of proteins: an open tool to analyze protein folds; Angelov B et al.; A three-dimensional Voronoi tessellation of folded proteins is used to analyze geometrical and topological properties of a set of proteins . To each amino acid is associated a central point surrounded by a Voronoi cell . Voronoi cells describe the packing of the amino acids . Special attention is given to reproduction of the protein surface . Once the Voronoi cells are built, a lot of tools from geometrical analysis can be applied to investigate the protein structure; volume of cells, number of faces per cell, and number of sides per face are the usual signatures of the protein structure . A distinct difference between faces related to primary, secondary, and tertiary structures has been observed . Faces threaded by the main-chain have on average more than six edges, whereas those related to helical packing of the amino acid chain have less than five edges . The faces on the protein surface have on average five edges within 1% error . The average number of faces on the protein surface for a given type of amino acid brings a new point of view in the characterization of the exposition to the solvent and the classification of amino acid as hydrophilic or hydrophobic . It may be a convenient tool for model validation . Biotechnol Bioeng, 2002 Dec 30, 80(7), 762 - 76 Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon; Smolke CD et al.; The effects of endoribonuclease sites, secondary structures in mRNA, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing the genes encoding beta-galactosidase (lacZ) from Escherichia coli and green fluorescent protein (gfp) from Aequorea victoria . Two previously identified RNase E sites were placed separately between the coding regions to direct cleavage in this area and produce two secondary transcripts, each containing a single-gene coding region . Novel secondary structures were engineered into the 3' and 5' ends of each of the coding regions to protect the transcript from inactivation by endoribonucleases (5' hairpins) and degradation by exoribonucleases (3' hairpins) . In addition, the effects of relative gene placement were examined by switching the locations of the two coding regions . Depending on the particular secondary structures and RNase E sites placed between the genes the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 2.5-fold and 4-fold, respectively . By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively . Curr Microbiol, 2002 Dec, 45(6), 440 - 5 Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli; Dutkiewicz R et al.; GTPases belonging to the Obg/Gtp1 subfamily are essential proteins in most bacterial species and are evolutionarily conservative from bacteria to humans . However, their specific functions in the regulation of cellular processes are largely unknown . Here we demonstrate that overproduction of a member of the Obg/Gtp1 subfamily, cgtA ( yhbZ, obgE) gene product, in Escherichia coli is deleterious for bacterial growth . However, syntheses of DNA, RNA, and proteins were not significantly affected under these conditions as measured by efficiency of incorporation of radioactive precursors . On the other hand, flow cytometry studies revealed that cgtA-overexpressing bacteria form enlarged cells with significantly changed distribution of chromosomal DNA . These results strongly suggest that overproduction of a GTP-binding protein from the Obg/Gtp1 subfamily impairs regulation of some chromosomal functions in E . coli, especially synchronization of DNA replication initiation and possibly also partitioning of daughter chromosomes after a replication round. Nephrol Dial Transplant, 2002 Nov, 17(11), 1964 - 70 IL-1beta, TNF-alpha and IL-6 release from monocytes in haemodialysis patients in relation to dialytic age; Malaponte G et al.; BACKGROUND: It has been suggested that changes in immune response to infectious agents in patients on haemodialysis might be due to impaired monocyte function; uraemic and haemodialysed patients overproduce proinflammatory cytokines, such as interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) . METHODS: We quantitated the cytokines released into the plasma and into the supernatants of 24-h cultured purified monocytes, under basal conditions and after stimulation by lipopolysaccharide from Escherichia coli, in 15 healthy subjects (CON), 20 uraemic patients who had not yet started dialysis (CRF) and 60 haemodialysed patients (HD), who were divided into three groups of 20 patients corresponding to short-, medium- and long-term dialysis . RESULTS: Monocytes from HD patients spontaneously secreted significantly higher levels of cytokines than those from controls and uraemic patients who had not yet started dialysis . After stimulation with lipopolysaccharide (LPS), cytokine levels in culture supernatants of cells from HD patients were significantly lower than those from controls and uraemic patients . Moreover, levels of cytokines in monocyte supernatants and plasma from short-, medium- and long-term haemodialysed patients decreased progressively with dialytic age . Monocytes from haemodialysed patients tended to be constitutively active, but their ability to secrete proinflammatory cytokines was inversely correlated with dialytic age . CONCLUSIONS: These results indicate that prolonged treatment with dialysis can be considered a form of chronic stress that causes the progressive activation of monocytes, which ultimately leads to monocyte exhaustion and dysfunction. J Biol Chem, 2002 Dec 27, 277(52), 51068 - 76 Epub 2002 Oct 24. The link module from human TSG-6 inhibits neutrophil migration in a hyaluronan- and inter-alpha -inhibitor-independent manner; Getting SJ et al.; TSG-6 protein (the secreted product of the tumor necrosis factor-stimulated gene-6), a hyaluronan-binding protein comprised mainly of a Link and CUB module arranged in a contiguous fashion, has been shown previously to be a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (Wisniewski, H . G., Hua, J . C., Poppers, D . M., Naime, D., Vilcek, J., and Cronstein, B . N . (1996) J . Immunol . 156, 1609-1615) . It was hypothesized that this activity of TSG-6 was likely to be mediated by its potentiation of inter-alpha-inhibitor anti-plasmin activity (causing a down-regulation of the protease network), which was reliant on these proteins forming a stable, probably covalent approximately 120-kDa complex . Here we have shown that the recombinant Link module from human TSG-6 (Link_TSG6; expressed in Escherichia coli) has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein (which we produced in a Drosophila expression system) . The active dose of 1 microg of Link_TSG6 per mouse (administered intravenously) also resulted in a significant reduction in the concentrations of various inflammatory mediators (i.e . tumor necrosis factor-alpha, KC, and prostaglandin E(2)) in air pouch exudates . Link_TSG6, although unable to form a stable complex with inter-alpha-inhibitor (under conditions that promote maximum complex formation with the full-length protein), could potentiate its anti-plasmin activity . This demonstrates that formation of an approximately 120-kDa TSG-6.inter-alpha-inhibitor complex is not required for TSG-6 to enhance the serine protease inhibitory activity of inter-alpha-inhibitor . Six single-site Link_TSG6 mutants (with wild-type folds) were compared for their abilities to inhibit neutrophil migration in vivo, bind hyaluronan, and potentiate inter-alpha-inhibitor . These experiments indicate that all of the inhibitory activity of TSG-6 resides within the Link module domain, and that this anti-inflammatory property is not related to either its hyaluronan binding function or its potentiation of the anti-plasmin activity of inter-alpha-inhibitor. J Biol Chem, 2002 Dec 27, 277(52), 50487 - 90 Epub 2002 Oct 24. Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts; Elder RH et al.; In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase . However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations . In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice . We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts . Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines. Chem Biol, 2002 Oct, 9(10), 1141 - 8 NikR repressor: high-affinity nickel binding to the C-terminal domain regulates binding to operator DNA; Chivers PT et al.; E . coli NikR repressor binds operator DNA in a nickel-dependent fashion . The pM affinity of NikR for nickel is mediated by its C-terminal 86 residues . Nickel binding induced additional secondary structure, decreased the compactness, and increased the stability of NikR . Tetramer formation by the C-terminal domain and intact NikR did not require nickel . High-affinity nickel binding decreased the NikR concentration needed to half maximally protect operator DNA from undetectable levels to 30 nM . The intracellular concentration of NikR in E . coli is high enough that saturation of the high-affinity nickel sites should lead to substantial occupancy of the nik operator . Nickel binding to a set of low-affinity NikR sites resulted in an additional large increase in operator affinity and substantially increased the size of the NikR footprint on the operator. Chem Biol, 2002 Oct, 9(10), 1055 - 7 Sensing nickel . NikRs with two pockets; Helmann JD; NikR represses expression of a nickel transporter in response to elevated levels of Ni(II) . Recent results suggest that repression is elicited by binding of nickel to a high-affinity site, but a low-affinity binding pocket may also play a role. J Microbiol Methods, 2003 Jan, 52(1), 29 - 38 Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction; Rose P et al.; Competitive approaches have shown promise for overcoming some of the difficulties in the use of PCR for assessment of specific bacterial species in soil . A competitive touchdown PCR (cTD-PCR) protocol specific for the rrsB gene of Escherichia coli was developed for tracking the organism in environments impacted by human wastes . Regression of product ratios from co-amplification of varying amounts of analyte and competitor DNA templates was linear . To test the robustness of the method, reactions were titrated with an extract of sterilized soil; no significant effect was detected . The cTD-PCR was used to assay recovery of E . coli DNA from soil . Stock DNA was spiked onto two sterilized soils during extraction, and the purified extracts were analyzed by cTD-PCR . Recovery of DNA spiked at a rate of 180 ng g(-1) was 34+/-7% (mean+/-S.D.) for an agricultural silt loam . DNA spiked at 1.8 pg g(-1) was recovered at a mean rate of 6.1+/-1.3% . DNA in these extracts was not directly quantifiable by image analysis . The cTD-PCR method provides a useful means of quantifying small amounts of E . coli DNA, and could be modified for other specific targets in a mixture of DNA from a variety of organisms. Ann Thorac Surg, 2002 Oct, 74(4), 1161 - 6; discussion 1166 Gene transfer to coronary artery bypass conduits; Chiu-Pinheiro CK et al.; BACKGROUND: Gene therapy is a rational approach to prevention of stenosis in saphenous vein grafts used as conduits for coronary artery bypass grafting . To explore this possibility we developed methods for adenoviral-mediated gene transfer to canine saphenous veins . METHODS: During a single procedure, autogenous canine saphenous vein segments were transduced ex vivo and used as coronary artery bypass grafts . The proximal end of each vein was ligated, adenovirus containing the Escherichia coli beta-galactosidase gene (Ad.CMVLacZ) was delivered at titers of 2.5 x 10(9) or 5 x 10(9) plaque-forming units (pfu)/mL to the lumen through a distal heparin lock, and the segment was immersed in the viral solution for 1 hour at 37 degrees C . Control segments were exposed to diluent alone in an identical manner . Aortocoronary anastomoses were made using cardiopulmonary bypass . Transgene expression was assessed qualitatively and quantitatively after 3 days . RESULTS: Beta-galactosidase levels showed a dose-dependent increase: 0.00 +/- 0.00 ng/mg total protein for controls; 5.60 +/- 2.27 ng/mg total protein for a viral titer of 2.5 x 10(9) pfu/mL and 11.97 +/- 6.14 ng/mg for 5 x 10(9) pfu/mL . The two dosage groups differed significantly from each other (p = 0.035) and from controls (p = 0.003) . X-gal staining demonstrated mostly endothelial and scattered adventitial transgene expression . CONCLUSIONS: Transgene expression after ex vivo gene transfer into saphenous vein grafts in a canine coronary artery bypass model indicates that this method may be useful for delivery of therapeutic genes to prevent or retard vein graft arteriosclerosis. Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1981 - 4 Increase in the rate of L-pipecolic acid production using lat-expressing Escherichia coli by lysP and yeiE amplification; Fujii T et al.; Biotransformation of L-lysine (L-Lys) to L-pipecolic acid (L-PA) using lat-expressing Escherichia coli has been reported (Fujii et al., Biosci . Biotechnol . Biochem., 66, 622-627 (2002)) . The rate-limiting step of this biotransformation seemes to be the transport of L-Lys into cells . To improve the L-PA production rate, we attempted to increase the rate of L-Lys uptake . E . coli BL21 carrying a plasmid with lat and lysP (pRH125) caused a 5-fold increase in the rate of L-PA production above the level of cells carrying a plasmid with lat (pRH124) . Moreover, E . coli BL21 carrying a plasmid with lat, lysP, and yeiE (pRH127) caused a 6.4-fold increase in the rate of L-PA production above the level of cells carrying pRH124 . Our results from RT-PCR experiments and the sequence similarity of YeiE to LysR transcriptional regulators suggest the possibility that yeiE expression induces lysP expression . The amplification of lysP, or rather both lysP and yeiE, increases the rate of L-PA production using lat-expressing E . coli. Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1967 - 71 Kinetics of hyperprocessing reaction of human tyrosine tRNA by ribonuclease P ribozyme from Escherichia coli; Ando T et al.; Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme . The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hair-pin-like structure . The hyperprocessing reaction of these tRNAs requires magnesium ions . We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme . The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed . With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates . When mature tRNA was the substrate, the apparent K(M) was almost the same as in the usual reaction, but k(cat) was smaller . These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes. Science, 2002 Oct 25, 298(5594), 824 - 7 Network motifs: simple building blocks of complex networks; Milo R et al.; Complex networks are studied across many fields of science . To uncover their structural design principles, we defined "network motifs," patterns of interconnections occurring in complex networks at numbers that are significantly higher than those in randomized networks . We found such motifs in networks from biochemistry, neurobiology, ecology, and engineering . The motifs shared by ecological food webs were distinct from the motifs shared by the genetic networks of Escherichia coli and Saccharomyces cerevisiae or from those found in the World Wide Web . Similar motifs were found in networks that perform information processing, even though they describe elements as different as biomolecules within a cell and synaptic connections between neurons in Caenorhabditis elegans . Motifs may thus define universal classes of networks . This approach may uncover the basic building blocks of most networks. J Bacteriol, 2002 Nov, 184(22), 6198 - 206 Effects of environmental changes on expression of the oligopeptide permease (opp) genes of Borrelia burgdorferi; Wang XG et al.; We analyzed expression of a putative oligopeptide permease (Opp) of Borrelia burgdorferi . Unlike the opp operons of other bacteria for which there is a single substrate binding protein, B . burgdorferi codes for three substrate binding proteins (OppA-I to -III) in its opp operon and an additional two homologs on plasmids (OppA-IV and -V) . Instead of a single promoter region regulating transcription of the entire operon, as seen in other bacterial opp operons, it appears that among oppA-I, -II, and -III, as well as oppA-IV and -V, each has a potential upstream promoter region . We tested the function of these putative promoter sequences by fusion to a promoterless beta-galactosidase reporter gene in pCB182 . Each of the promoter regions was found to be active . The level of activity in the reporter constructs closely paralleled the level of expression of each gene in in vitro-grown B . burgdorferi . Changes in carbon and nitrogen availability differentially affected individual promoters, but no changes in promoter activity were seen when Escherichia coli bacteria (with the promoter constructs) were grown in various concentrations of phosphate and leucine and changes in pH . Expression of specific oppA genes with B . burgdorferi varied significantly between its mouse and fed and unfed tick hosts . Differences in regulation of opp gene expression suggest a potential role in environmental response by the organism. J Bacteriol, 2002 Nov, 184(22), 6115 - 22 Expression, purification, and characterization of AknX anthrone oxygenase, which is involved in aklavinone biosynthesis in Streptomyces galilaeus; Chung JY et al.; In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes . aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus . In order to identify its function, the aknX gene was expressed in Escherichia coli . The cell extract prepared from E . coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin . This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6 . The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography . The native molecular mass of AknX was estimated to be 42 kDa by gel filtration . Thus, native AknX is considered to have a homotrimeric subunit structure . AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation . Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction . Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction . A possible mechanism for the reaction via peroxy anion intermediate is proposed. J Biol Chem, 2002 Dec 27, 277(52), 50482 - 6 Epub 2002 Oct 23. Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana; Dobrzanska M et al.; The sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives . Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH . The deduced amino acid sequence of AtNUDT1 contains 147 amino acids . The recombinant AtNUDT1 was expressed in Escherichia coli and purified . In the presence of Mn(2+) and the optimal pH of 7 . 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0 . 36 mm . A V(max) of 12 . 7 units mg (-1) for NADH was determined . The recombinant AtNUDT1 migrated as a dimer on a gel filtration column . Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A . thaliana is a NADH pyrophosphatase. J Biol Chem, 2002 Dec 20, 277(51), 49755 - 60 Epub 2002 Oct 23. The role of intersubunit interactions for the stabilization of the T state of Escherichia coli aspartate transcarbamoylase; Chan RS et al.; Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state . These two alternate states are stabilized by a series of interdomain and intersubunit interactions . The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state . When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions . These characteristics are consistent with an enzyme locked in the functional R state . Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized . The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states . The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions . A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated . The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states . Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme . These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition. Genetics, 2002 Oct, 162(2), 557 - 66 Fitness evolution and the rise of mutator alleles in experimental Escherichia coli populations; Shaver AC et al.; We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment . We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift . In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci . In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation . The spread of the mutator allele from rarity to fixation took >1000 generations in each population . We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations . We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations. FEMS Microbiol Lett, 2002 Oct 8, 215(2), 273 - 8 Functional analysis of the Escherichia coli zinc transporter ZitB; Lee SM et al.; The membrane transporter ZitB responsible for Zn(II) efflux in Escherichia coli was studied by site-directed mutagenesis to elucidate the function of individual amino acid residues . Substitutions of several charged or polar residues, H53, H159, D163 and D186, located in predicted transmembrane domains resulted in loss of ZitB function . In contrast, neither the amino-terminal nor the carboxy-terminal regions, both histidine-rich, were required for function. Phys Rev Lett . 2002 Oct 21;89(17):178101 . Epub 2002 Oct 08. Time-delayed spread of viruses in growing plaques; Fort J et al.; The spread of viruses in growing plaques predicted by classical models is greater than that measured experimentally . There is a widespread belief that this discrepancy is due to biological factors . Here we show that the observed speeds can be satisfactorily predicted by a purely physical model that takes into account the delay time due to virus reproduction inside infected cells . No free or adjustable parameters are used. J Biomol NMR, 2002 Aug, 23(4), 289 - 301 Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles; Hilty C et al.; Sequence-specific assignments have been obtained for side chain methyl resonances of Val, Leu and Ile in the outer membrane protein X (OmpX) from Escherichia coli reconstituted in 60 kDa micelles in aqueous solution . Using previously established techniques, OmpX was uniformly 2H,13C,15N-labeled with selectively protonated Val-gamma(1,2), Leu-delta(1,2) and Ile-delta1 methyl groups . The thus labeled protein was studied with the novel experiments 3D (H)C(CC)-TOCSY-(CO)-{15N,1H}-TROSY and 3D H(C)(CC)-TOCSY-(CO)-{15N,1H}-TROSY . Compared to the corresponding conventional experimental schemes, the TROSY-type experiments yielded a sensitivity gain of about 2 at 500 MHz . The overall sensitivity of the experiments was further enhanced more than two-fold by the use of a cryoprobe . Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the delta1-methyls of Ile . The present approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to about 100 kDa, and adds to the potentialities of solution NMR for de novo structure determination as well as for functional studies, such as ligand screening with proteins in large structures. Microbiol Res, 2002, 157(3), 191 - 5 Characteristics of capsules in enterotoxemic Escherichia coli O139:K12 strains causing swine edema disease; Meno Y et al.; The characteristics of the capsule of the enterotoxemic Escherichia coli (ETEEC) O139:K12 strains that strongly adhere to Hep-2 cells were examined . Electron microscopic studies using the freeze-substitution technique revealed that ETEEC strains had a capsule of approximately 25 nm . These strains show hydrophobic surface properties and strong adherence to human polymorphonuclear leukocytes (PMNs) . In contrast, ETEEC strains RK-O139 and ED-1 show weak adherence to HEp-2 cells and fail to express the capsule layer on the cell surface . These ETEEC strains possess hydrophilic surface properties and also adhere to PMNs . The lipopolysaccharide (LPS) analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that ETEEC strains had the same LPS profile and long O-side chains of LPS . Furthermore, all strains were resistant to serum killing activity . These results suggest that the capsule of ETEEC strains does not contribute as an antiphagocytic factor, but as an adherence factor to host cells. Int J Med Microbiol, 2002 Sep, 292(3-4), 169 - 83 Consequences of EHEC colonisation in humans and cattle; Smith DG et al.; While many factors have been associated with human EHEC infection, the full role these play in both human and ruminant hosts are not yet clear despite much investigation . It is hoped that the continued intense international research effort into EHEC will provide further insights into the commensal versus pathogenic lifestyles of E . coli and lead to approaches to reduce EHEC carriage in ruminants as well as prevent or treat human disease. J Endod, 2002 Oct, 28(10), 694 - 6 Radiographic evaluation of the effect of endotoxin (LPS) plus calcium hydroxide on apical and periapical tissues of dogs; Nelson-Filho P et al.; The aim of this study was the radiographic evaluation of the apical and periapical region of dog teeth submitted to intracanal bacterial endotoxin (lipopolysaccharide, LPS), associated or not with calcium hydroxide . After removal of the pulp, 60 premolars were divided into four groups and were filled with bacterial endotoxin (group 1), bacterial endotoxin plus calcium hydroxide (group 2), saline solution (group 3), or periapical lesions were induced with no treatment (group 4), for a period of 30 days . Similar periapical lesions were observed in groups 1 and 4 . The lamina dura was intact in groups 2 and 3 . Bacterial endotoxin (LPS) caused radiographically visible periapical lesions, but when associated with calcium hydroxide, this endotoxin was detoxified. Biofizika, 2002 Sep-Oct, 47(5), 847 - 51 {Electrochemical study of proton translocation function of hydrogenase 3}; Bagramian KA; The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied . The extent of acidification was dependent on the amount of formate added . An average H+/formate ratio of 1.3 was obtained . The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone . Inverted vesicles of E . coli were found to translocate protons upon oxidation of formate at pH 6.5 . The extent of alkalization was also dependent on the amount of formate added . The maximum H+/formate ratio for this reaction was close to 0.6 . Formate oxidation by inverted vesicles from E . coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone . It was supposed that the hydrogenase 3 (hyc) component of E . coli formate hydrogen lyase is responsible for the translocation of protons at low pH. Biofizika, 2002 Sep-Oct, 47(5), 809 - 19 {Levels in the structural organization of Escherichia coli promoter DNA}; Ozolin' ON et al.; A number of additional structural elements were identified by statistic analysis of nucleotide sequences in promoters recognized by Escherichia coli RNA polymerase . Together with canonical hexanucleotides, these elements characterize different levels in the structural organization of promoter DNA . Sequence motifs exhibiting the highest statistical significance, which dominate in the contact regions with RNA polymerase alpha and sigma subunits, are considered as targets for specific interaction with RNA polymerase . A typical feature of these elements is the presence of easily deformable dinucleotides (TG, CA and TA) or tracts containing only A/T base pairs . Thus, we noticed that the frequency of occurrence of TA in the promoter DNA is essentially higher than the average value for the genome . Besides the regions of specific interaction with RNA polymerase, these dinucleotides are often located in the number of other sites periodically distributed along the promoter DNA . This preferred disposition suggests that deformable elements participate in the adaptive conformational transitions of the promoter DNA favoring optimal configuration of the transcription complex . Probably, the most important feature of promoter DNA revealed by statistic analysis is the presence of A/T-tracts regularly distributed in the wide range from -160 up to +75 relative to the transcription start point . Both of these spatially distributed elements (TA dinucleotides and A/T-tracts) are linked with canonical regions and, therefore, may contribute to the conformational or dynamic features of the transcription machinery . Having high statistic significance, these elements might be considered as additional factors discriminating the promoter DNA on the background of other nucleotide sequences in the genome. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14964 - 9 Epub 2002 Oct 23. The phage lambda CII transcriptional activator carries a C-terminal domain signaling for rapid proteolysis; Kobiler O et al.; ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates . One of these is the lambda CII protein, which plays a key role in the phage lysis-lysogeny decision . Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis . Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII . However, the mutations abrogate CII degradation by FtsH . We have established an in vitro assay for the lambda CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation . Phage lambda carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development . In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP . We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity . These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein. Development, 2002 Nov, 129(21), 4931 - 40 Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms; Andrioli LP et al.; The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo . The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb) . As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position . The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border . We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion . This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain . Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors . Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip . Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position . Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer . These results suggest a common mechanism for preventing anterior activation of three different eve enhancers. J Biol Chem, 2002 Dec 20, 277(51), 49621 - 30 Epub 2002 Oct 22. Degradation of cellulose substrates by cellulosome chimeras . Substrate targeting versus proximity of enzyme components; Fierobe HP et al.; A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Belaich, A., Lamed, R., Shoham, Y., Belaich, J.-P., and Bayer, E . A . (2001) J . Biol . Chem . 276, 21257-21261), based on the high affinity species-specific cohesin-dockerin interaction . Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins . The activities of the resultant ternary complexes were assayed using different types of cellulose substrates . Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates . On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate . For certain enzyme pairs, however, one effect appeared to predominate over the other . The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites. J Biol Chem, 2002 Dec 27, 277(52), 50985 - 90 Epub 2002 Oct 22. Functionally significant mobile regions of Escherichia coli SecA ATPase identified by NMR; Chou YT et al.; SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane . SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein . SecA is proposed to undergo major conformational changes during translocation . These conformational changes are accompanied by major rearrangements of SecA structural domains . To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility . The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564-579) and to the extreme C-terminal segment of SecA (residues 864-901), both of which are essential for preprotein translocation activity . Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA. J Biol Chem, 2002 Dec 20, 277(51), 49685 - 90 Epub 2002 Oct 22. Kinetic analysis of Arabidopsis phospholipase Ddelta . Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate; Qin C et al.; Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation . Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes . To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis . PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles . V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate . PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)) . Maximal activation was observed at a PIP(2) molar ratio around 0.01 . Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface . Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B) . This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme. J Biol Chem, 2003 Jan 17, 278(3), 2030 - 5 Epub 2002 Oct 22. Comparative enzymology of 11 beta -hydroxysteroid dehydrogenase type 1 from glucocorticoid resistant (Guinea pig) versus sensitive (human) species; Shafqat N et al.; Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo . We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity . Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli . Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels . Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme . Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells . Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme . The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs . Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species. Am J Respir Cell Mol Biol, 2002 Nov, 27(5), 575 - 82 Functions of IkappaB proteins in inflammatory responses to Escherichia coli LPS in mouse lungs; Mizgerd JP et al.; Acute inflammation induced by intrapulmonary LPS requires nuclear factor (NF)-kappaB RelA . This study elucidates the effects of intrapulmonary LPS on IkappaB proteins, endogenous inhibitors of RelA, and the effects of deficiency of IkappaB-beta . IkappaB-alpha, IkappaB-beta, and IkappaB-epsilon each complexed with RelA in uninfected murine lungs . Intratracheal instillation of LPS induced the degradation of IkappaB-alpha and IkappaB-beta, as measured by the loss of immunoreactive proteins in non-nuclear fractions . Degradation was apparent by 2 h and sustained through 6 h . In contrast, net IkappaB-epsilon content increased over this period . The small amounts of IkappaB-alpha and IkappaB-beta that were detected in nuclear fractions from the lungs also decreased over this time frame, whereas intranuclear NF-kappaB content (including both RelA and p50) increased . The hypophosphorylated form of IkappaB-beta, which facilitates transcription induced by NF-kappaB, was not detected . Neutrophil recruitment and edema accumulation did not differ between wild type mice and gene-targeted mice deficient in IkappaB-beta, suggesting that IkappaB-beta is not specifically required for these responses . Altogether, these data suggest that RelA is liberated during LPS-induced pulmonary inflammation by the regulated degradation of both IkappaB-alpha and IkappaB-beta . In the absence of IkappaB-beta, IkappaB-alpha or other inhibitory proteins can regulate NF-kappaB functions essential to acute neutrophil emigration in the lungs. J Interferon Cytokine Res, 2002 Aug, 22(8), 883 - 9 Expression of interleukin-18 by porcine airway and intestinal epithelium; Muneta Y et al.; In this study, we investigated the expression of interleukin-18 (IL-18) in porcine airway and intestinal epithelium . We found constitutive protein expression of precursor IL-18 in primary culture of porcine airway epithelium . Immunohistochemical staining revealed that porcine IL-18 was localized in the porcine airway epithelium and that it was significantly upregulated with experimental endotoxemia induced by Escherichia coli lipopolysaccharide (LPS) inoculation . We also confirmed by immunohistochemical staining that IL-18 was expressed in porcine intestinal epithelial cells . Moreover, the concentration of IL-18 in intestinal cell lysates of 1-day-old piglets was about 3-fold and 6-fold less than that in those of 1-month-old and 6-month-old piglets, respectively . Exogenous IL-18 was able to induce interferon-gamma (IFN-gamma) in the peripheral blood of 1-day-old piglets, whereas concanavalin A (ConA) was not able to induce IFN-gamma in the same condition . These results suggest that mucosal epithelial cells are among the major sources of IL-18 in pig and that IL-18 may be useful as a therapeutic agent for the enhancement of immune responses and as a vaccine adjuvant, especially in neonatal piglets. J Interferon Cytokine Res, 2002 Sep, 22(9), 981 - 93 Genomic structure of the mouse 2',5'-oligoadenylate synthetase gene family; Kakuta S et al.; 2',5'-Oligoadenylate synthetase (2-5OAS) is one of the interferon (IFN)-induced proteins and mediates the antiviral action of IFN . In human, three classes of 2-5OAS genes (OAS1, OAS2, and OAS3) and one OAS-like gene (OASL) are reported . In mice, however, OAS genes corresponding to human OAS2 and OAS3 have not been identified . In this report, we identified six novel OAS family genes in mice by screening mouse genomic library and expressed sequence tag (EST) database . These genes include three homologs of the human OAS1 and each homologous gene of the human OAS2, OAS3, and OASL, respectively . Each gene displays 52%-65% amino acid identity to the corresponding human homologs . Nine 2-5OAS genes, except for two OASL genes, locate within the 210-kb genomic region and form a cluster . Each novel 2-5OAS gene showed a characteristic expression pattern among different tissues, and all of them were induced by polyinosinic-polycytidylic acid . Biochemical analyses using recombinant proteins produced in Escherichia coli showed that all the novel mouse 2-5OAS molecules have double-stranded RNA (dsRNA) binding ability, but they do not have 2-5OAS activity except for the OAS2 and OAS3 mouse homologs . These results show that there are at least 11 OAS genes,