Escherichia coli

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, which are classified into four groups, in the mouse.

Avian Pathol, 2002 Aug, 31(4), 371 - 6
Major histocompatibility complex effect on cellulitis among different chicken lines; Macklin KS et al.; The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance/susceptibility to several bacterial, parasitic, and viral diseases . Investigators have shown that the chicken MHC plays a major role in determining the outcome of a Marek's disease infection, in that standard B(13) is susceptible to the virus while B(21) confers resistance to the virus . Previous work with a broiler line has shown that B(21) is susceptible to an Escherichia coli-induced cellulitis infection and that B(13) conferred resistance to the infection . For this experiment, a broiler and a Leghorn chicken line shown to contain standard B(13) and B(21) were examined in a challenge model for cellulitis . The birds were challenged with a cellulitis-causing E . coli isolate . Homozygous B(21) had the highest incidence of cellulitis development compared with either homozygous B(13) or the heterozygous B(13)/B(21) for both the broiler and Leghorn lines . Additionally, cellulitis lesion severity was measured in both lines and shown to be independent of MHC type.

Xenobiotica, 2002 Sep, 32(9), 749 - 59
Characterization of mixtures of recombinant human cytochrome p450s as a screening model for metabolic stability in drug discovery; Hagen N et al.; 1 . Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue . Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery . 2 . In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)) . Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters . Results were compared with data obtained in human liver microsomes (HLM) . 3 . Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max) . K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold . V(max) differed up to 3-fold between matrices for the CYP forms investigated . However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM . The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged . 4 . In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters . Furthermore, addition of antioxidant can in certain cases increase catalytic activity.

J Membr Biol, 2002 Oct 1, 189(3), 191 - 9
Altered sugar selection and transport conferred by spontaneous point and deletion mutations in the lactose carrier of Escherichia coli; Shinnick SG et al.; Spontaneous mutants harboring the lacY gene on an F'-factor were isolated . Those mutants that failed to grow on 5 mM lactose minimal media plates were chosen for further study . The mutants showed striking mutations in the lactose carrier as well as in sugar selection properties during transport assays . DNA sequencing of the lacY gene of the mutants revealed the following mutations: M-1-I, R-144-W, G-370-C and a deletion of residues 387-392, located in helix 12 of the carrier . Transport studies indicated that ONPG transport ranged between 8 and 25% of normal for the M-1-I, G-370-C and D387-392 mutants and 51% of normal for the R-144-W mutant . The downhill transport of lactose was 2-fold greater than for melibiose in cells harboring the M-1-I mutation and 3-fold higher for cells with the G-370-C mutation . On the other hand, cells with the D387-392-deletion mutation showed no lactose downhill transport, but 47% melibiose transport . Accumulation of TMG, a lactose analog, was 3-fold higher than the accumulation of melibiose in cells with the G-370-C mutation . On the other hand, in cells with the D387-392 mutation, TMG accumulation was completely defective, whereas melibiose accumulation was 50-fold higher than that of TMG, indicating that one or more of these residues in helix 12 of the carrier play a role in the active transport of b-galactoside, but not a-galactoside sugars . Initial lactose downhill transport rates were too unreliable to obtain trustworthy kinetic data . TMG and melibiose accumulation activities were present, but severely reduced in the mutant containing the R144W mutation, confirming that Arg-144 is important for active transport . All transport data were normalized for expression levels . The results indicate that the affected residues play a role in dictating sugar specificity and transport in the lactose carrier . The results here are novel in that they represent mutations in unique locations along the lactose carrier protein . For example, the M-1-I mutation was located at the N-terminal cytoplasmic tail of the carrier . Furthermore, G-370-C was located in the periplasmic loop between helices 11 and 12, suggesting a role for residues in this loop in mediating sugar selection.

Mol Genet Genomics, 2002 Oct, 268(2), 206 - 13 Epub 2002 Aug 27.
Transient repressor effect of Fis on the growth phase-regulated osmE promoter of Escherichia coli K12; Bordes P et al.; Transcription of the gene osmE of Escherichia coli is osmotically inducible and regulated by the growth phase . Expression of osmE is directed by a single promoter, osmE (p), which is recognized by Esigma(70) and Esigma(s), two forms of RNA polymerase using, respectively, the sigma factors sigma(70) and sigma(s) . Esigma(s) transcribes osmE (p) during entry into stationary phase . Esigma(70) is responsible for osmotic induction of osmE (p) during the exponential growth phase . In a search for proteins that can modulate osmE (p) expression in trans, we performed electrophoretic mobility shift experiments using a DNA fragment carrying osmE (p) and crude extracts from E . coli . One major retarded band was observed in these experiments . The Fis protein is responsible for this retarded band, and binds to several sites upstream and downstream of, and overlapping, the promoter region of osmE . In a fis mutant background, the kinetics of in vivo transcription of osmE (p) during growth demonstrated that Fis is not responsible for the repression of the promoter seen during early exponential phase . In contrast, expression of osmE (p) at elevated osmolarity during the mid-exponential growth phase is increased in the absence of Fis, demonstrating that Fis is able to act as a repressor in vivo at a particular stage of growth.

Crit Care Med, 2002 Oct, 30(10), 2306 - 12
Distinct pathways of lipopolysaccharide priming of human neutrophil respiratory burst: role of lipid mediator synthesis and sensitivity to interleukin-10; Sibelius U et al.; OBJECTIVE: Exposure of neutrophils to low doses of bacterial lipopolysaccharides enhances their readiness to respond with inflammatory mediator generation including oxygen radical formation to a subsequently applied inflammatory stimulus ("priming") . In the present study, we investigated the role of lipid mediator synthesis and the impact of the anti-inflammatory cytokine interleukin-10 on the lipopolysaccharide-dependent priming of human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine . DESIGN: Prospective, experimental study . SETTING: Research laboratory at a university hospital . SUBJECTS: Isolated neutrophils from healthy volunteers . INTERVENTIONS: Incubation of isolated neutrophils with endotoxin . MEASUREMENTS AND MAIN RESULTS: Evidence for two distinct priming mechanisms was obtained . The first was strictly serum component dependent, proceeded via CD14, and was not inhibited by even high concentrations of interleukin-10 . The second priming mechanism was serum component independent but nevertheless proceeded via CD14 . It was linked with neutrophil synthesis of the platelet activating factor and resulted in the appearance of leukotrienes, in particular leukotriene B4, as far as exogenous arachidonic acid was provided . The employment of a platelet-activating factor receptor antagonist (WEB 2086) blocked leukotriene synthesis, and both WEB 2086 and a 5-lipoxygenase inhibitor (MK-886) suppressed the respiratory burst linked with this second priming pathway . This sequence of priming events was inhibited by interleukin-10, when this cytokine was coadministered with the priming agent lipopolysaccharide, whereas late interleukin-10 admixture was ineffective . CONCLUSIONS: We conclude that two mechanisms of lipopolysaccharide priming of human neutrophil respiratory burst can be differentiated . One displays serum component dependence, is independent of neutrophil lipid mediator generation, and is not affected by interleukin-10 . The other is serum independent although being operated via CD14, employs autocrine loops of platelet-activating factor and leukotriene B4 synthesis, and is sensitive to the inhibitory capacity of interleukin-10 . These features may be relevant when the goal is to pharmacologically modify neutrophil functions in septic events.

Coron Artery Dis, 2002 Aug, 13(5), 269 - 74
Elevated levels of oxidative DNA damage in patients with coronary artery disease; Botto N et al.; BACKGROUND: Somatic DNA damage has been suggested to contribute to the pathogenesis of atherosclerosis . However, little is known about the role of oxidative DNA damage in patients with coronary artery disease (CAD) . METHODS: In this study, we used the comet assay to measure oxidative DNA damage (DNA strand breaks and enzyme-sensitive sites) in peripheral blood lymphocytes from 13 patients with angiographically documented CAD and 11 age- and sex-matched control participants . RESULTS: Mean values of DNA strand breaks, oxidized pyrimidines and altered purines were significantly higher in CAD patients than in the control group (11.9 +/- 1.4, 18.0 +/- 2.7 and 18.1 +/- 3.1 compared with 3.3 +/- 0.2, 2.7 +/- 0.5 and 4.5 +/- 1.1; P < 0.0001, P < 0.0001 and P = 0.0009, respectively) . Moreover, oxidized purines (for example, 8-oxo-guanine) increased with the number of affected vessels and positively correlated with the extent of CAD measured by means of the number of the coronary lesions (P = 0.76, P = 0.003) and the Duke scoring system (P = 0.66, P = 0.01) . Diabetic patients showed higher levels of oxidized pyrimidines (31.3 +/- 5.5 compared with 14.1 +/- 2.7; P = 0.013), while patients with dyslipidemia had elevated altered purines compared with normal patients (20.4 +/- 2.6 compared with 4.9 +/- 3.1; P = 0.03) . CONCLUSIONS: These data indicate an overall elevation of oxidative DNA damage in CAD patients correlated with the severity of the disease and some atherogenic risk factors, suggesting a possible role of oxidative genetic damage in the pathogenesis of atherosclerosis .

Acta Crystallogr D Biol Crystallogr, 2002 Nov, 58(Pt 11), 1897 - 900 Epub 2002 Oct 21.
The SUPERFAMILY database in structural genomics; Gough J; The SUPERFAMILY hidden Markov model library representing all proteins of known structure predicts the domain architecture of protein sequences and classifies them at the SCOP superfamily level . This analysis has been carried out on all completely sequenced genomes . The ways in which the database can be useful to crystallographers is discussed, in particular with a view to high-throughput structure determination . The application of the SUPERFAMILY database to different target-selection strategies is suggested: novel folds, novel domain combinations and targeted attacks on genomes . Use of the database for more general inquiry in the context of structural studies is also explained . The database provides evolutionary relationships between target proteins and other proteins of known structure through the SCOP database, genome assignments and multiple sequence alignments.

J Biol Chem, 2002 Dec 27, 277(52), 50365 - 72 Epub 2002 Oct 21.
Ca2+-myristoyl switch in the neuronal calcium sensor recoverin requires different functions of Ca2+-binding sites; Senin II et al.; Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner . It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to EF-hand 2 and 3 . We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca(2+) binding . EF-hand 2 and 3 display different properties and serve different functions . Binding of Ca(2+) to recoverin is a sequential process, wherein EF-hand 3 is occupied first followed by the filling of EF-hand 2 . After EF-hand 3 bound Ca(2+), the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes . In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold . We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.

J Biol Chem, 2002 Dec 20, 277(51), 49841 - 9 Epub 2002 Oct 21.
Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly; Pittman MS et al.; Assembly of Escherichia coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown . Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm . Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner . New pleiotropic cydD phenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation . Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d . Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective in orf299, encoding a transporter of the major facilitator superfamily . A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations . We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

J Biol Chem, 2002 Dec 27, 277(52), 50396 - 402 Epub 2002 Oct 21.
Processing of Escherichia coli alkaline phosphatase . Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide; Kajava AV et al.; Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2 . This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions . Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation . Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing . In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing . To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor . The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation . These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.

J Biol Chem, 2002 Dec 27, 277(52), 50899 - 906 Epub 2002 Oct 18.
An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate; Rodrigues CO et al.; We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX) . The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa . LmPPX differs from other exopolyphosphatases previously investigated . Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg(2+) requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity . LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, or p-nitrophenylphosphate . Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization . High levels of short chain (21.4 +/- 3.0 mm) and long chain polyP (55.9 +/- 5.6 mm) were detected in L . major promastigotes . The unique characteristics of LmPPX and L . major polyP metabolism may facilitate the development of novel antileishmanial agents.

J Virol Methods, 2002 Dec, 106(2), 225 - 33
The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease; Hust M et al.; A single chain variable fragment antibody (scFv; anti-NIa scFv102) was selected from a synthetic human antibody library by using a NIa protease of Plum pox virus (PPV) as an antigen, which was expressed in bacteria . The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins . The NIa protein was detected with anti-NIa scFv102 after expression in Escherichia coli cells as well as from PPV-infected Nicotiana benthamiana plants . Furthermore, the scFv102 has the ability to identify not only PPV from infected plants but also can detect other infections with members of the potyviruses . Nineteen different potyviruses were recognized by the scFv102 in various infected plants tested through dot blot assays . Therefore, the antibody scFv102 has the potential of becoming a general tool to detect potyvirus infections in different plant species .

Biosens Bioelectron, 2002 Dec, 17(11-12), 993 - 8
Escherichia coli and its application in a mediated amperometric glucose sensor; Ito Y et al.; Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor . The E . coli modified glucose sensor, which was prepared by immobilizing E . coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme . This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution . The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM . Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current . The E . coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA) . When E . coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.

Biosens Bioelectron, 2002 Dec, 17(11-12), 953 - 63
Effect of cysteine mutations on direct electron transfer of horseradish peroxidase on gold; Ferapontova E et al.; Surface exposed cysteines were genetically engineered in the structure of recombinant horseradish peroxidase (rHRP) . Recombinant forms of HRP with either a His-tag or a Strep-tag at the C-terminus were produced, which additionally had cysteines at positions 57, 189 or 309 (C-terminus) of the polypeptide chain . An E . coli expression system was exploited . The effect of these mutations on the direct electron transfer (ET) between Au and the enzyme was studied in the reaction of the bioelectrocatalytic reduction of H(2)O(2), at -50 mV versus Ag/AgCl, on rHRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell . Adsorptive immobilisation of rHRPs on pre-oxidised Au from the protein solution at pH 6.0 provided a high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct (mediatorless) ET between Au and the active site of the rHRPs . Comparative analysis of the direct ET rate constants, estimated from the amperometric data on direct and mediated ET in the presence of catechol at pH 7.4 and 6.0, gave evidence that the introduction of the His-tag or cysteine in the C-terminal area of the enzyme resulted in an increased efficiency of direct ET due to a favourable coupled electron and proton transfer pathway . Due to the high efficiency of direct ET, the sensitivity was independent on the addition of the mediator or change of pH indicating that the response to H(2)O(2) is determined solely by the mass transfer of the analyte to the active site of HRP . The sensitivities obtained for the Au electrodes modified with rHRPs (2.0+/-0.1 A M(-1) cm(-2)) and the low detection limit for H(2)O(2) (10 nM) paves the way to develop the P-chip (peroxidase chip)--a biosensors system of a microscopic size for a mediatorless detection of H(2)O(2) based on direct ET between Au and the recombinant forms of HRP.

Int J Parasitol, 2002 Nov, 32(12), 1477 - 85
Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum; Millership JJ et al.; Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism . Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3) . We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain . Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C . parvum genome-sequencing project . The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C . parvum . The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein . However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum . Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites . However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites . Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays . The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties . Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT) . This study indicates that C . parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.

Biochem Pharmacol, 2002 Nov 1, 64(9), 1317 - 24
Expression and purification of a recombinant form of human aromatase from Escherichia coli; Zhang F et al.; Aromatase converts androgen to estrogen, a hormone that plays an important role in the development of breast cancer . Aromatase inhibitors have been shown to be a useful endocrine regimen for estrogen-dependent breast cancer . Structure-function studies of aromatase can generate critical structural information for designing highly potent and specific inhibitors . However, aromatase structure-function studies have been hampered by a lack of purified protein . In this report, we describe the construction and expression of a recombinant derivative of human aromatase in Escherichia coli using the pET vector system, and the purification of the enzyme by means of nickel-agarose affinity chromatography . We examined the expression of the full-length, Del-38, C-6xHis-tagged Del-38, and NC-6xHis-tagged Del-38 forms of aromatase . The recombinant aromatase without the first 38 amino acids from the amino-terminus (i.e . Del-38) was found to have a higher activity than the full-length enzyme . Moreover, the addition of two separate hexameric histidine tags at both the amino and the carboxyl-termini (i.e . NC-6xHis-tagged Del-38) increased the binding affinity of the recombinant enzyme to the nickel-agarose . The expressed aromatase (i.e . NC-6xHis-tagged Del-38 aromatase) was eluted from the nickel-agarose with 80 mM EDTA . The total aromatase activity of the 80 mM EDTA-eluted fractions was significantly higher than the detergent-solubilized protein extract, indicating a renaturation process during the nickel-agarose affinity chromatography . Purified aromatase exhibited a single band when analyzed by SDS-PAGE, and activity up to 5.8 nmol/mg/min was obtained using the tritiated water release assay . The K(m) value for androstenedione was determined to be 62+/-24 nM by enzyme kinetic analysis . The recombinant aromatase preparation was also characterized by reduced CO-difference spectral analysis, reaction product extraction assay, and inhibition studies using two aromatase inhibitors (letrozole and anastrozole) . The results indicate that the recombinant aromatase from E . coli has catalytic properties identical to those of the enzyme expressed in human tissue and will be very useful for further structure-function studies of aromatase.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 45 - 8
Induction of the soxRS regulon of Escherichia coli by glycolaldehyde; Benov L et al.; The ability of short-chain sugars to cause oxidative stress has been examined using glycolaldehyde as the simplest sugar . Short-chain sugars autoxidize in air, producing superoxide and alpha,beta-dicarbonyls . In Escherichia coli the soxRS regulon mediates an oxidative stress response, which protects the cell against both superoxide-generating agents and nitric oxide . In superoxide dismutase-deficient E . coli mutants, glycolaldehyde induces fumarase C and nitroreductase A, which are regulated as members of the soxRS regulon . A mutational defect in soxRS eliminates that induction . This establishes that glycolaldehyde can cause induction of this defensive regulon . This effect of glycolaldehyde was oxygen-dependent, was not shown by glyoxal, and was not seen in the superoxide dismutase-replete parental strain, and it was abolished by a cell-permeable SOD mimetic . All of these suggest that superoxide radicals produced by the oxidation of glycolaldehyde played a key role in the induction.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 32 - 8
The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p; Luo YS et al.; One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein . The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate . Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed . Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies . Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay . These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates . Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar . Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p . The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 25 - 31
Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin; Ishigami A et al.; Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner . Rodents have four isoforms of PAD (types I, II, III, and IV), each of which is distinct in substrate and tissue specificity . In fact, the only tissue in which all four PAD mRNAs have been detected is the epidermis . In this study, we found PAD activity in HSC-1 human cutaneous squamous carcinoma cells in vitro, and this activity increased during cultivation . Using a homology-based strategy, we cloned a full-length cDNA encoding human PAD type II . The cDNA was 2348 bp long and encoded a 665-amino-acid sequence with a predicted molecular mass of 75 kDa . The predicted protein shared 93% identity with the rat and mouse PAD type II sequence . Alignment of the amino acid sequences from both species revealed notable conservation in the C-terminal region, suggesting the presence of a functional region such as an enzyme catalytic site and/or a calcium-binding domain . Gene organization analysis established that human PAD type II on chromosome 1p35.2-p35.21 spanned more than 50 kb and contained 16 exons and 15 introns . A recombinant PAD protein subsequently produced in Escherichia coli proved to be enzymatically active, with substrate specificities similar to those of the rat PAD type II . In an immunohistochemical study of human skin, the type II enzyme was expressed by all the living epidermal layers, suggesting that PAD type II is functionally important during terminal differentiation of epidermal keratinocytes.

Eur J Biochem, 2002 Nov, 269(21), 5323 - 9
Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase; Sillero MA et al.; Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented . The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN) . In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector . The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm . Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values . In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively . With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme . These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.

Eur J Biochem, 2002 Nov, 269(21), 5271 - 9
tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase; Gruic-Sovulj I et al.; The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code . Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine {Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., Soll, D . & Weygand-Durasevic, I . (1997) J . Biol . Chem.272, 1136-1141} . Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone . Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine . This may proceed via a tRNA-induced conformational change in the enzyme's active site . The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site . Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner . The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.

Eur J Biochem, 2002 Nov, 269(21), 5264 - 70
Biosynthesis of vitamin B2; Kaiser J et al.; GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, the first committed intermediate in the biosynthesis of riboflavin . The enzyme was shown to contain one zinc ion per subunit . Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate . However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide-type intermediate analog . The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring . Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon-nitrogen bonds.

Eur J Biochem, 2002 Nov, 269(21), 5259 - 63
Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli; Sati SP et al.; The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins . Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum . It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein . Six different constructs were made and each of the fusion proteins were expressed and purified . Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields . Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher . Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein . Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner . The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion . These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E . coli . Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal 'solubilization tag' . This system may allow large-scale production of those proteins that have a tendency to misfold during expression.

Eur J Biochem, 2002 Nov, 269(21), 5182 - 91
Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold; Ye K et al.; Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress . pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site . Such a translational arrest may mediate overall adaptation of cells to environmental conditions . We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy . The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet . The 23 C-terminal residues of the protein are disordered . Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1 . Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits . Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site . The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity . It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.

Eur J Biochem, 2002 Nov, 269(21), 5137 - 48
Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue; Hunter T et al.; The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network . The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli) . Although site-directed mutant enzymes lacking this glutamine residue (FeSOD{Q69G} and MnSOD{Q146A}) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types . FeSOD double mutants (FeSOD{Q69G/A141Q}), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD{G77Q/Q146A}) increased to 150% (relative to wild-type MnSOD) . Both double mutants showed reduced selectivity toward their metal . Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD{A141Q} and MnSOD{G77Q}), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile . Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD . We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.

J Appl Microbiol, 2002, 93(5), 835 - 9
Construction of green fluorescent protein (GFP)-marked strains of Bradyrhizobium for ecological studies; Bhatia R et al.; AIM: To introduce the gfp gene encoding green fluorescent protein (GFP) into bradyrhizobia for their identification in nodules, soil and carrier-based inoculants . METHODS AND RESULTS: Bradyrhizobium sp . strains M29 and GN7, which nodulate mungbean (Vigna radiata), were conjugated with Escherichia coli S17-1 carrying plasmid EDS 15 (a suicide plasmid carrying a promoterless gfp gene fused with Tn5) . The GFP-marked strain expressed the gfp gene from a Bradyrhizobium promoter and gave green fluorescence when observed under an epifluorescent microscope or u.v . transilluminater . All the GFP-marked strains were able to nodulate mungbean and fix nitrogen . The GFP-marked bradyrhizobia were recovered at a frequency of 90-100% and 16-63% from nodules formed under sterilized and unsterilized conditions, respectively . The GFP-marked bradyrhizobia were identified from soil and from charcoal-based inoculants on the basis of green fluorescence . CONCLUSIONS: The GFP-marked Bradyrhizobium was successfully identified on the basis of green fluorescence to study its competition and survival in the soil and in charcoal-based inoculants . SIGNIFICANCE AND IMPACT OF THE STUDY: Introduction of the gfp gene into Bradyrhizobium provides a simple, specific and cost-effective method of strain identification for ecological studies.

J Appl Microbiol, 2002, 93(5), 758 - 64
Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test; Perelle S et al.; AIMS: The aims of the study were to characterize the O91 O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O91 and to provide the basis for a specific PCR test for rapid detection of E . coli O91 . METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E . coli and related species were used to amplify the 10-kbp O91 O-antigen biosynthesis locus of STEC O91 . A DNA library representative of this cluster allowed two O91 specific probes to be identified, and two specific PCR O91 serotyping tests to be successfully developed . CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E . coli allow rapidly a specific O-antigen PCR assay to be designed . SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of PCR-assays available to replace the classical O-serotyping among E . coli O-antigen.

Genetika, 2002 Sep, 38(9), 1223 - 34
{Functional interrelationship between elements of the Escherichia coli udp gene promotor responsible for binding regulatory proteins CytR, CRP, and RNA polymerase}; Zolotukhina MA et al.; Site-directed mutagenesis was conducted in the regulatory region of the Escherichia coli udp gene at promoter sites responsible for binding regulatory proteins CRP and CytR as well as RNA polymerase (the core-promoter containing the--10 sequence) . In mutants with an "improved"--10 region, a partial relief from the control of the cAMP-CRP transcription activation complex occurred, and the negative CytR repressor regulation was reduced . In contrast, mutant promoters with a weak Pribnow block or with a deletion that completely eliminates the core-promoter exhibited an increased ability to titrate the CytR protein in vivo . On the other hand, the affinity of CytR for DNA in mutants with an altered--10 region was the same as in the wild-type udp promoter . After introduction of mutations affecting binding sites for CRP (CRP1 and CRP2), the negative effect of the CytR protein on promoter transcription was fully abolished . The CRP1 binding site was shown to play the main role in the activation of the promoter by the cAMP-CRP complex, whereas the CRP2 site participates in the formation of the repressor complex . Mutations in the main and additional CytR binding sites were isolated and characterized . On the basis of these data, it is concluded that the modification of each structural element of the udp regulatory region (binding sites for CytR, CRP, or RNA polymerase) caused changes in the overall pattern of the promoter regulation.

Genetika, 2002 Sep, 38(9), 1215 - 22
{Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication}; Sukhodolets VV; The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes . Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium . In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude . These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication . Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants . The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.

Genetika, 2002 Sep, 38(9), 1196 - 202
{Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selective marker}; Ladygin VG et al.; To transform Chlamydomonas reinhardtii Dang . Cells, plasmid pCTVHyg was constructed with the use of the Escherichia coli hygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter . Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10(3) hygromycin-resistant (HygR) clones per 10(6) recipient cells . The exogenous DNA integrated in the Ch . reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character . Codon usage was compared for the hpt gene and Ch . reinhardtii nuclear genes . The results testified that codon usage bias, which is characteristic of Ch . reinhardtii, is not the major factor affecting foreign gene expression . The advantages of the selective system for studying Ch . reinhardtii transformation with heterologous genes are discussed.

DNA Seq, 2002 Jun, 13(3), 167 - 72
Molecular characterization of ribonucleotide reductase from Cryptosporidium parvum; Akiyoshi DE et al.; The Apicomplexan enteric parasite, Cryptosporidium, infects a broad range of mammals, birds, fish and reptiles . Cryptosporidium parvum, the principal causative agent of human cryptosporidiosis, has emerged as a major contributor to waterborne outbreaks of this disease . The absence of effective drugs against C . parvum has necessitated the search for new drug targets . One attractive class of target enzymes is ribonucleotide reductase, an essential enzyme required for the de novo synthesis of deoxyribonucleotides . We report the cloning and sequencing of the small and large subunits of ribonucleotide reductase from a C . parvum genotype 2 isolate, GCH1 . Southern blot analysis showed that these subunits are encoded by single copy genes . Both subunits have been expressed as recombinant proteins in Escherichia coli.

Kansenshogaku Zasshi, 2002 Sep, 76(9), 730 - 7
{Four serotypes highly positive for pathogenic factor-related genes found among Escherichia coli isolates from sporadic diarrheal cases in Fukui Prefecture}; Ishiguro F et al.; To evaluate the prevalence of pathogenic factor-related genes in 964 O-serotype Escherichia coli isolated from sporadic diarrhea cases at 2 hospitals in Fukui during 1997 to 2001, we checked all of these strains for H-serotype and examined them for 4 pathogenic factor-related genes (LT, ST, stx and invE) by PCR . Of these strains, 409 except for most of 9 serotypes which are usually low prevalence of pathogenic factor-related genes, and additional 16 strains isolated from other hospitals in Fukui Prefecture were also examined for 3 pathogenic factor-related genes (eaeA, astA and aggR) by PCR . It was found that 4 serotypes, O6:H16, O25:HNM, O111:H21 and O126:H27, were highly positive for pathogenic factor-related genes; O6:H16 (11/12 strains) positive for LT, ST, astA, O 25:HNM (10/14 strains) positive for ST and astA, O111:H21 (22/22 strains) positive for astA or aggR, and O126:H27 (8/9) positive for astA and aggR genes . According to the drug susceptibility test, these serotypes as O6:H16, O25:HNM, O111:H21 and O126:H27 showed a significant resistance to some drugs in 7/12, 4/14, 21/22 and 9/9 strains, respectively . Such results indicate that both O111 and O126-serotypes are highly positive for pathogenic factor-related genes and also drug-resistant, namely this fact suggests that the O-serotyping as laboratory screening is one of the useful measures of clinical management . Additionally, the pulsedfied gel electrophoresis patterns found in each of the 4 serotypes mentioned revealed that a part of E . coli in Fukui might be derived from the same source of infection.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14037 - 40 Epub 2002 Oct 21.
An approach to membrane protein structure without crystals; Sorgen PL et al.; The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H(+) across the cell membrane . It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes . Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family . However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts . Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better.

J Appl Physiol, 2003 Jan, 94(1), 43 - 52 Epub 2002 Sep 13.
Role of extracellular HSP72 in acute stress-induced potentiation of innate immunity in active rats; Campisi J et al.; Acute stress can compromise acquired, and potentiate innate, immunity . Recent evidence suggests that the impact of stress on measures of immunity can be modulated by the physical activity status of the organism and that extracellular heat shock protein 72 (eHSP72) contributes to the activation of innate immunity produced by stress . Therefore, this study investigated whether physical activity status would impact the immunologically enhancing effects of stressor exposure {inescapable tail-shock stress (IS)} on innate immunity and whether changes in eHSP72 responses could play a role . Adult, male Fischer 344 rats lived with mobile (physically active) or immobile (sedentary) running wheels . After 6 wk, rats were exposed to IS or to no stress . Immediately after IS, all rats were injected subcutaneously with live Escherichia coli . Inflammation was assessed daily, and plasma eHSP72 was measured at various time points . Rats exposed to IS resolved their inflammation faster than nonstressed rats, but the beneficial impact of stress on recovery was greater in physically active rats . All rats had equal increases in circulating eHSP72 after IS . Splenocytes harvested from a separate cohort of nonstressed rats were cultured with eHSP72, and nitric oxide and cytokines were measured . Physically active rats responded to eHSP72 stimulation in vitro with a greater nitric oxide and cytokine response than sedentary rats . Thus physically active rats both recover faster than sedentary rats after bacterial challenge + IS exposure and demonstrate potentiated cellular responses to eHSP72 activation that could be important for bacterial recovery.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jan, 22(1), 22 - 4
Cloning and identification of human tissue-type plasminogen activator gene; Li ZL et al.; OBJECTIVE: To acquire the gene fragments coding for human tissue-type plasminogen activator (ht-PA) . METHOD: Total RNA was extracted from Bowes Melanoma cell line and reverse transcriptase-"touchdown" PCR was employed to amplify ht-PA cDNA that underwent digestion with the restriction enzymes and was subsequently cloned into vector pSP72 for transforming E.coli TG1 . The cloned fragment was subjected to restriction mapping and sequence analysis for confirmation . RESULT: The restriction maps of the selected clones were consistent with those generated by computer-based analysis and was identical with that of reported ht-PA cDNA . CONCLUSION: The gene fragment encoding ht-PA was successfully cloned, which lays a foundation for further study and artificial modification of this gene.

Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 427 - 9
Establishment of rat model for Pneumocystis carinii infection and cloning of the surface glycoprotein A gene of Pneumocystis carinii; Ouyang LS et al.; OBJECTIVE: To establish a rat model for Pneumocystis carinii infection and to clone surface glycoprotein A (GpA) gene of Pneumocystis carini . METHODS: Immunosuppression was induced in 5 rats with an immunosuppresion regimen consisting mainly of dexamethasone in a course of 2 weeks, after which pulmonary homogenates of rats infected with Pneumocystis carinii were fed to the immunosuppressed rats . Immunosuppression was maintained for approximately 4 weeks after the feeding to induce Pneumocystis pneumonia in the rats . GpA gene was subsequently amplified from the rats with pneumonia as confirmed by microscope and PCR detection, and was subcloned into T-vector for the transformation of Escherichia coli JM109 strain . RESULTS: Pneumocystis carinii was detected by microscope and PCR detection in rats with immunosuppression . The length of PCR product was 319 bp as shown by agarose electrophoresis . CONCLUSION: The rat model of Pneumocystis carinii infection can be established by immunosuppression with dexamethasone and a single oral administration of the pathogen.

BMC Struct Biol . 2002 Oct 11;2(1):6.
Crystal structure of LIR-2 (ILT4) at 1.8 A: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18; Willcox BE et al.; BACKGROUND: Leukocyte Immunoglobulin-like Receptor-1 (LIR-1) and LIR-2 (also known as ILT2 and ILT4 respectively) are highly related cell surface receptors that bind a broad range of class I MHC molecules with low (microM) affinities . Expressed on monocytic cells and macrophages, both molecules transmit inhibitory signals after binding ligands . In addition to binding host class I MHC, the LIR-1 molecule, which is also expressed on lymphoid tissues, binds with a high (nM) affinity to UL18, a class I MHC homolog encoded by Human Cytomegalovirus (HCMV) . In comparison, LIR-2 binds UL18 only weakly (microM KD) . To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1 . RESULTS: Recombinant LIR-2 (domains 1 and 2) was produced in E . coli and crystallized using streak seeding to optimize the crystal morphology . A data set complete to 1.8 A was collected at 100 K from a single crystal in the P4(1)2(1)2 spacegroup . The structure was solved by molecular replacement, using a search model based on the LIR-1 structure . CONCLUSIONS: The overall structure of LIR-2 D1D2 resembles both LIR-1, and Killer Inhibitory Receptors, in that the A strand in each domain forms hydrogen bonds to both beta sheets, and there is a sharp angle between the two immunoglobulin-like domains . However, differences from LIR-1 are observed in each domain, with two key changes apparent in the ligand-binding domain, D1 . The region corresponding to the residue 44-57 helix of LIR-1 adopts a topology distinct from that of both LIR-1 and the KIR structures, involving a shortened 310 helix . Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76-84 loop mainchain is displaced 11 A with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation . In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.

Liver, 2002 Oct, 22(5), 394 - 403
Involvement of platelet-activating factor in hepatic apoptosis and necrosis in chronic ethanol-fed rats given endotoxin; Murohisa G et al.; BACKGROUND/AIMS: Platelet-activating factor (PAF)-a potent activator of neutrophils-plays an important role in the pathogenesis of endotoxin-induced tissue injury . However, the role of PAF in hepatic damage during alcoholic hepatitis remains unclear . The aims of the present study were to test whether PAF contributes to hepatic injury in an animal model of alcoholic hepatitis and to investigate the involvement of the Fas-receptor/Fas-ligand system in this process . METHODS: Male Sprague-Dawley rats were pair-fed with Lieber-DeCarli ethanol liquid diet or isocaloric control diet for 6 weeks . Liver injury was induced by the intravenous (i.v.) injection of lipopolysaccharide (LPS) (1 mg/kg) . Rats were pretreated with a specific PAF receptor antagonist (TCV-309; 100 mg/kg i.v.) or vehicle 1 h before LPS treatment . RESULTS: Chronic ethanol administration remarkably sensitized the rats to the effects of LPS, with resultant severe hepatocellular injury, accompanied by significant increases in serum levels of alanine aminotransferase (ALT), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 (CINC/gro) . Histological examination of the damaged livers showed hepatocyte apoptosis and necrosis with extensive infiltration by neutrophils, whereas immunohistochemical studies revealed enhanced Fas-receptor expression on hepatocytes and hepatic accumulation of neutrophils expressing Fas-ligand . Pretreatment with the PAF receptor antagonist protected against hepatic injury, suppressing hepatocyte apoptosis and necrosis, infiltration of neutrophils, expression of Fas-receptor and Fas-ligand, and serum TNF-alpha levels . CONCLUSIONS: Our study suggests that PAF is an important mediator of hepatic injury in the ethanol/endotoxin model of alcoholic hepatitis.

Genes Cells, 2002 Nov, 7(11), 1125 - 34
ADP stabilizes the human Rad51-single stranded DNA complex and promotes its DNA annealing activity; Kim HK et al.; BACKGROUND: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination . These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step . Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins . RESULTS: Measurements using the fluorescence ssDNA analogue, poly(1,N6-etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state . While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex . ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs . CONCLUSIONS: The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state . The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step . However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.

Biochemistry, 2002 Oct 29, 41(43), 13096 - 105
Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases; D'souza VM et al.; Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated . Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively . These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively . The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II . Both specific activity and K(m) values increased with increasing temperature . An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C . The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).

Biochemistry, 2002 Oct 29, 41(43), 13039 - 45
Binding of hydrophobic D-galactopyranosides to the lactose permease of Escherichia coli; Sahin-Toth M et al.; Binding of alpha- and beta-D-galactopyranosides with different hydrophobic aglycons was compared using substrate protection against N-ethylmaleimide alkylation of single-Cys148 lactose permease . As demonstrated previously, methyl- or allyl-substituted alpha-D-galactopyranosides exhibit a 60-fold increase in binding affinity (K(D) = 0.5 mM), relative to galactose (K(D) = 30 mM), while methyl beta-D-galactopyranoside binds only 3-fold better . In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in alpha and beta anomeric configurations, were synthesized . Surprisingly, relative to methyl alpha-D-galactopyranoside, binding of cyclohexyl alpha-D-galactopyranoside to lactose permease is essentially unchanged (K(D) = 0.4 mM), and phenyl alpha-D-galactopyranoside exhibits only a modest increase in binding affinity (K(D) = 0.15 mM) . Nitro- or methyl-substituted phenyl alpha-D-galactopyranosides bind with significantly higher affinities (K(D) = 0.014-0.067 mM), and the strongest binding is observed with analogues containing para substituents . In contrast, D-galactopyranosides with a variety of large hydrophobic substituents (isopropyl, cyclohexyl, phenyl, o- or p-nitrophenyl) in beta anomeric configuration exhibit uniformly weak binding (K(D) = 1.0-2.3 mM) . The results confirm and extend previous observations that hydrophobic aglycons of D-galactopyranosides increase binding affinity, with a clear predilection toward alpha-substituted sugars . In addition, the data suggest that the primary interaction between the permease and hydrophobic aglycons is directed toward the carbon atom bonded to the anomeric oxygen . The different positioning of this carbon atom in alpha- or beta-D-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for alpha anomers.

Biochemistry, 2002 Oct 29, 41(43), 13012 - 20
Dimerization of human XPA and formation of XPA2-RPA protein complex; Yang ZG et al.; XPA plays an important role in the DNA damage recognition during human nucleotide excision repair . Here we report that the XPA is a homodimer either in the free state or as a complex with human RPA in solution under normal conditions . The human XPA protein purified from baculovirus-infected sf21 insect cells has a molecular mass of 36 317 Da, as determined by mass spectroscopy . However, the apparent molecular mass of XPA determined by the native gel filtration chromatography was about 71 kDa, suggesting that XPA is a dimer . This observation was supported by a native PFO-PAGE and fluorescence spectroscopy analysis . XPA formed a dimer (XPA2) in a broad range of XPA and NaCl concentrations, and the dimerization was not due to the disulfide bond formation . Furthermore, a titration analysis of the binding of XPA to the human RPA indicated that it was the XPA2 that formed the complex with RPA . Finally, the difference between the mass spectrometric and the calculated masses of XPA implies that the protein contains posttranslational modifications . Taken together, our data suggest that the dimerization of XPA may play an important role in the DNA damage recognition of nucleotide excision repair.

Biochemistry, 2002 Oct 29, 41(43), 13003 - 11
Structure and function of the C-terminal domain of methionyl-tRNA synthetase; Crepin T et al.; The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain . The minimal MetRS behaves as a monomer . In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide . Upon truncation of this C-terminal domain, subunits dissociate irreversibly . Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied . It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea . The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS . Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity . However, dimer formation is required to evidence the gain in affinity.

Biochemistry, 2002 Oct 29, 41(43), 12975 - 85
Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC; Ionescu CN et al.; Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA . The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits . To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein . Recombinant wild-type and mutant PCNAs were purified from E . coli, and they were tested for their properties in vitro . Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations . In contrast to what has been reported for a monomeric form of the beta clamp in E . coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC) . Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC . The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA . They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy . These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

Am J Reprod Immunol, 2002 Aug, 48(2), 117 - 25
Effect of active and passive immunization of male and female rats with a recombinantly expressed bonnet monkey pituitary GnRH receptor fragment; Santra S et al.; Gonadotropin releasing hormone (GnRH) exerts its action by binding to the specific receptor which belongs to the family of G-protein coupled receptors that are characterized by the presence of seven transmembrane domains linked together by extracellular and intracellular loops . A fragment of the pituitary receptor of the bonnet monkey (Macaca radiata) corresponding to amino acids 164-266 was cloned and expressed in Escherichia coli . This was used to raise antibodies to the receptor in rabbits . Active and passive immunization studies in both male and female rats were carried out using, both the 'overexpressed' fragment, as well as antibodies raised to the receptor fragment . Both active, as well as passive immunization in the male rats brought about an agonistic effect in terms of increase in serum LH level, as well as increase in serum and testicular testosterone levels . However, in the female rats, active immunization with the receptor fragment did not have any effect on the gonadal steroid levels but had a selective effect on the uterine morphology.

Tijdschr Diergeneeskd, 2002 Oct 1, 127(19), 582 - 8
{Avian pathogenic Escherichia coli (APEC)}; Vandemaele F et al.; Escherichia coli infections are being increasingly detected among poultry flocks, indicating the growing importance of this pathogen to the industry . The infection begins as a respiratory infection of the trachea, followed by colonization of the air sacs and lungs, from where it invades the blood-stream, leading to infection of the deeper organs (liver, heart, oviduct, and peritoneum) . A number of factors play a crucial role in the virulence and pathogenesis of infection . The F1 and P pili are particularly important in establishing the infection at the level of the tracheal epithelium cell . Other important factors are aerobactin, capsule, and serum resistance . Treatment is with antibiotics, but the growing bacterial resistance of avian E . coli and stricter regulations mean that attention is turning to prophylactic, preventative, measures, such as vaccination . Current vaccines provide limited homologous protection against the pathogen . Research is needed to develop a good, broad-spectrum vaccine.

Am J Obstet Gynecol, 2002 Oct, 187(4), 1059 - 65
Intra-amniotic endotoxin induces lung maturation by direct effects on the developing respiratory tract in preterm sheep; Moss TJ et al.; OBJECTIVE: Our purpose was to determine whether improved preterm lung function caused by intraamniotic endotoxin treatment requires endotoxin entry into the respiratory tract . STUDY DESIGN: We assessed lung inflammation 2 days after intra-amniotic endotoxin (10 mg, Escherichia coli 055:B5) or saline solution in preterm lambs (123 days' gestation) that had undergone surgery to isolate the gastrointestinal or respiratory systems from the amniotic sac . In other sheep longer-term effects were assessed 7 days after we isolated the fetal respiratory tract and gave endotoxin or saline solution directly to the lungs or into the amniotic sac . We measured pulmonary inflammation, lung function, and surfactant 1 week after treatment (approximately 125 days) . RESULTS: Pulmonary inflammation was present after intra-amniotic endotoxin only if there was communication between the respiratory tract and the amniotic sac . Lung function was improved and surfactant was increased only in preterm lambs that received direct pulmonary endotoxin . CONCLUSION: Endotoxin causes functional improvement of the preterm lung by direct effects on the developing respiratory system.

J Gen Virol, 2002 Nov, 83(Pt 11), 2857 - 67
Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129; IJkel WF et al.; Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus . One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV) . Se17/18 was transcribed in cultured S . exigua 301 cells, as a polyadenylated transcript of 1.1 kb . 5'-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon . These sites overlap with a baculovirus consensus early promoter motif . Se17/18 transcripts were detected by Northern blot analysis and RT-PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i . A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells . A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E . coli . However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S . exigua larvae, neither in concentrated BV and ODV preparations . These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts . Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed.

J Virol, 2002 Nov, 76(22), 11748 - 52
Mutation of capsid protein phosphorylation sites abolishes cauliflower mosaic virus infectivity; Chapdelaine Y et al.; The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56) . We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells . Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets . A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity . The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing . We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.

J Virol, 2002 Nov, 76(22), 11343 - 9
A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction; Nakai H et al.; Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo . However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established . To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 x 10(8) and 1.1 x 10(13) vector genomes (vg)/mouse, in three- to sixfold increments . A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 x 10(9) to 3.0 x 10(11) vg/mouse . Vector doses above 3.0 x 10(11) vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 x 10(12) vg/mouse . In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 x 10(12) vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose . Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes . Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

J Pharmacol Exp Ther, 2002 Nov, 303(2), 476 - 86
Protection of NRK-52E cells, a rat renal proximal tubular cell line, from chemical-induced apoptosis by overexpression of a mitochondrial glutathione transporter; Lash LH et al.; The dicarboxylate carrier (DCC) is one of two carriers responsible for glutathione (GSH) transport into rat kidney mitochondria . The central hypothesis of the present study was that overexpression of this carrier in renal proximal tubular cells increases content of mitochondrial GSH, which in turn can protect these cells from chemical-induced injury . We first cloned the carrier protein and verified its properties . This was accomplished by reverse transcribing total rat kidney RNA and polymerase chain reaction amplification with primers based on the complete cDNA sequence for the mitochondrial DCC protein . DCC was expressed as a His(6)-tagged protein, purified from Escherichia coli inclusion bodies, and reconstituted into proteoliposomes for transport assays . Time- and concentration-dependent uptake of both L-{(3)H-glycyl}GSH and {2-(14)C}malonate was observed with kinetics, substrate specificity, and inhibitor sensitivities similar to those observed in rat kidney proximal tubules . We next transiently transfected NRK-52E cells with the cDNA for rat kidney DCC to overexpress the protein . The presence of the recombinant DCC-His(6) protein was confirmed by immunoblots . Transport of both GSH and malonate into the mitochondrial fraction of transfected cells was enhanced 2.45- to 11.3-fold, compared with that in wild-type cells . Transfected cells exhibited markedly less apoptosis from tert-butyl hydroperoxide or S-(1,2-dichlorovinyl)-L-cysteine than did wild-type cells, validating the central hypothesis and providing us with a valuable and novel tool with which to further study GSH and thiol redox status in renal mitochondria, and the function of GSH transport in regulation of processes such as apoptosis and oxidative phosphorylation.

J Biol Chem, 2002 Dec 20, 277(51), 50046 - 53 Epub 2002 Oct 17.
A role for DNA polymerase beta in mutagenic UV lesion bypass; Servant L et al.; We report here that DNA polymerase beta (pol beta), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure . To investigate the potential role of pol beta in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta . We firstly demonstrated that mammalian cells overexpressing pol beta are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD) . By using in vitro primer extension reactions with purified pol beta, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed . pol beta mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively . In the case of CPD, efficient and error-prone extension of the correct dATP was found . These data support a biological role of pol beta in UV lesion bypass and suggest that deregulated pol beta may enhance UV-induced genetic instability.

Am J Physiol Renal Physiol, 2003 Feb, 284(2), F399 - 410 Epub 2002 Oct 08.
Carbonic anhydrase XII mRNA encodes a hydratase that is differentially expressed along the rabbit nephron; Schwartz GJ et al.; Membrane-bound carbonic anhydrase (CA) facilitates acidification in the kidney . Although most hydratase activity is considered due to CA IV, some in the basolateral membranes could be attributed to CA XII . Indeed, CA IV is glycosylphosphatidylinositol anchored, connoting apical polarization, but CA IV immunoreactivity has been detected on basolateral membranes of proximal tubules . Herein, we determined whether CA XII mRNA was expressed in acidifying segments of the rabbit nephron . The open reading frame of CA XII was sequenced from a rabbit kidney cortex cDNA library; it was 83% identical to human CA XII and coded for a 355-amino acid single-pass transmembrane protein . Northern blot analysis revealed an abundant 4.5-kb message in kidney cortex, medulla, and colon . By in situ hybridization, CA XII mRNA was expressed by proximal convoluted and straight tubules, cortical and medullary collecting ducts, and papillary epithelium . By RT-PCR, CA XII mRNA was abundantly expressed in cortical and medullary collecting ducts and thick ascending limb of Henle's loop; it was also expressed in proximal convoluted and straight tubules but not in glomeruli or S3 segments . FLAG-CA XII of approximately 40 kDa expressed in Escherichia coli showed hydratase activity that was inhibited by 0.1 mM acetazolamide . Unlike CA IV, expressed CA XII activity was inhibited by 1% SDS, suggesting insufficient disulfide linkages to stabilize the molecule . Western blotting of expressed CA XII with two anti-rabbit CA IV peptide antibodies showed no cross-reactivity . Our findings indicate that CA XII may contribute to the membrane CA activity of proximal tubules and collecting ducts.

Am J Physiol Lung Cell Mol Physiol, 2002 Dec, 283(6), L1247 - 54 Epub 2002 Aug 09.
Modulation of endotoxin-induced NF-kappa B activation in lung and liver through TNF type 1 and IL-1 receptors; Koay MA et al.; We investigated the requirement for tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 receptors in the pathogenesis of the pulmonary and hepatic responses to Escherichia coli lipopolysaccharide (LPS) by studying wild-type mice and mice deficient in TNF type 1 receptor {TNFR1 knockout (KO)} or both TNF type 1 and IL-1 receptors (TNFR1/IL-1R KO) . In lung tissue, NF-kappaB activation was similar among the groups after exposure to aerosolized LPS . After intraperitoneal injection of LPS, NF-kappaB activation in liver was attenuated in TNFR1 KO mice and further diminished in TNFR1/IL-1R KO mice; however, in lung tissue, no impairment in NF-kappaB activation was found in TNFR1 KO mice and only a modest decrease was found in TNFR1/IL-1R KO mice . Lung concentrations of KC and macrophage-inflammatory peptide 2 were lower in TNFR1 KO and TNFR1/IL-1R KO mice after aerosolized and intraperitoneal LPS . We conclude that LPS-induced NF-kappaB activation in liver is mediated through TNF-alpha- and IL-1 receptor-dependent pathways, but, in the lung, LPS-induced NF-kappaB activation is largely independent of these receptors.

Am J Physiol Gastrointest Liver Physiol, 2003 Feb, 284(2), G328 - 39 Epub 2002 Oct 16.
Progastrin1-80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites; Singh P et al.; Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide . It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly . Full-length recombinant human PG (rhPG(1-80)) was generated to examine possible direct effects of PG on IEC cells . Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor . Even though IEC cells did not express CCK(1) and CCK(2) receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK(1)-R and CCK(2)-R on IEC cells . High-affinity (K(d) = 0.5-1.0 nM) binding sites for (125)I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG >or= COOH-terminally extended G17 >or= G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05) . In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Am J Physiol Endocrinol Metab, 2003 Jan, 284(1), E228 - 36 Epub 2002 Sep 03.
Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver; Beigneux AP et al.; Infection is associated with low serum thyroid hormones and thyrotropin levels . Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression . In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration . Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha(1), TR-alpha(2), and TR-beta levels were reduced by 55, 87, and 41%, respectively . We previously reported that LPS rapidly decreases RXR protein levels in liver . Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls . A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding . We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.

Math Biosci, 2002 Nov-Dec, 180, 237 - 53
Alternative designs for a genetic switch: analysis of switching times using the piecewise power-law representation; Savageau MA; Some genes are thought to be switched discontinuously ON or OFF in response to environmental or developmental stimuli, whereas other genes are thought to be switched in a continuously variable fashion . We have previously identified criteria that distinguish between discontinuous and continuous genetic switches for an inducible catabolic pathway . These two types of switches exhibit several additional characteristics, beyond their qualitatively distinct behaviors, that influence their natural selection . These characteristics include threshold value, magnitude of the input signal required for switching ('switching effort'), magnitude of the corresponding output signal, duty cycle, switching time, and robustness . In order to characterize the biological design principles governing such switches, we have developed mathematical models of generic gene circuits and analyzed their behavior . Here we report the results of a comparative study designed to identify essential differences in switching time . This study has been greatly facilitated by use of the piecewise power-law representation, which was first developed by systems engineers in the 1940s and adapted for biochemical systems in the early 1970s . With this approach, we have been able to derive analytical expressions for switching time . When the alternative designs are made as nearly equivalent as possible, by the method of mathematically controlled comparison, we find that the switching times for the continuous case are less than that for the corresponding discontinuous case.We also find that ON times are faster than OFF times in all cases . These results are discussed in the specific context of the inducible lactose operon of Escherichia coli.

FEBS Lett, 2002 Oct 23, 530(1-3), 233 - 8
A novel GTP-binding protein hGBP3 interacts with NIK/HGK; Luan Z et al.; A novel human guanylate-binding protein (GBP) hGBP3 was identified and characterized . Similar as the two human guanylate-binding proteins hGBP1 and hGBP2, hGBP3 has the first two motifs of the three classical guanylate-binding motifs, GXXXXGKS (T) and DXXG, but lacks the N (T) KXD motif . Escherichia coli-expressed hGBP3 protein specifically binds to guanosine triphosphate (GTP) . Using a yeast two-hybrid system, it was revealed that the N-terminal region of hGBP3 binds to the C-terminal regulatory domain of NIK/HGK, a member of the group I GCK (germinal center kinase) family . This interaction was confirmed by in vitro glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays.

Mol Biochem Parasitol, 2002 Sep-Oct, 124(1-2), 37 - 45
Schistosoma mansoni ferredoxin NADP(H) oxidoreductase and its role in detoxification; Girardini JE et al.; Ferredoxin NADP(H) oxidoreductases (FNR) are flavoenzymes that catalyze the electron transfer between NADP(H) and a wide range of compounds including ferredoxins and bacterial flavodoxins . FNRs are classified into two major groups: plant- and vertebrate-type . Plant-type FNRs are implicated in photosynthesis and nitrogen fixation in plastids and photosynthetic bacteria, and were recently implicated in cell protection against reactive oxygen species (ROS) . Vertebrate-type FNRs are mitochondrial enzymes implicated in steroid hormone biosynthesis in mammals and in Fe(+) uptake and metabolism in yeasts . We have cloned and sequenced a cDNA coding for the vertebrate-type Schistosoma mansoni FNR . Gel diaphorase activity and western blot assays demonstrated that SmFNR represented the major diaphorase activity of adult worms . An active recombinant SmFNR was expressed in Escherichia coli that made the bacteria tolerant to oxygen peroxide, cumene hydroperoxide and the superoxide-generating herbicide, methyl viologen (MV).

Mol Biochem Parasitol, 2002 Sep-Oct, 124(1-2), 23 - 36
Two casein kinase 1 isoforms are differentially expressed in Trypanosoma cruzi; Spadafora C et al.; The cDNAs for two casein kinase 1 (CK1) homologues, TcCK1.1 and TcCK1.2, have been isolated from Trypanosoma cruzi . Both isoforms showed strong identity with other known CK1s . Their corresponding genes encode proteins of 312- and 330-amino acid residues with apparent molecular weights of 16 and 37 kDa, respectively . TcCK1.1 is a two-copy gene while TcCK1.2 is tandemly repeated, an arrangement not yet found in any other CK1 . TcCK1.1 has been overexpressed in Escherichia coli and the recombinant protein exhibited properties characteristic of the CK1 family . Northern blot indicated that both TcCK1s are expressed differentially during the life stages of the parasite: the isoform TcCK1.1 shows low levels of mRNA expression in epimastigotes and increased expression in trypomastigotes while TcCK1.2 presents an augmented expression in amastigotes as compared with the other two life stages of the parasite . The CK1-like activity of amastigotes and trypomastigotes is significantly higher than that of epimastigotes and, independent of the life stage of the parasite, a constitutive activity is observed which, in the epimastigote forms, is found predominantly in the microsomal fraction . Also in the epimastigote forms, the CK1-like activity increases in the log phase of growth of the parasites, and, through synchronization studies, this activity has been most conspicuously circumscribed to the S and M phases of the cell cycle.

Mol Biochem Parasitol, 2002 Sep-Oct, 124(1-2), 11 - 21
Secretion of a novel developmentally regulated chitinase (family 19 glycosyl hydrolase) into the perivitelline fluid of the parasitic nematode, Ascaris suum; Geng J et al.; The early development of the parasitic nematode, Ascaris suum, occurs within a chitinous eggshell and an abundant chitinase (As-p50) has been identified in the perivitelline fluid (PVF) surrounding the infective larva prior to hatching . A cDNA encoding As-p50 was cloned, sequenced and the protein expressed in Escherichia coli . As-p50 is a member of glycosyl hydrolase family 19, previously identified only in plants, making the characterization of As-p50 the first family 19 glycosyl hydrolase from any animal species . As expected, the chitinase activity of recombinant As-p50 or isolated PVF was insensitive to allosamidin . As-p50 expression was developmentally regulated . As-p50 mRNA appeared between days 5 and 8 of development prior to the formation of the first-stage larva (L1) . The As-p50 protein and chitinase activity appeared later between days 8 and 15 and remained at constant levels until hatching . GFP-promoter constructs of C08B6.4, the most closely related Caenorhabditis elegans As-p50 homologue, were expressed in hypodermal cells of 3-fold stage larvae and L1s with a timing similar to that of As-p50 and the fusion protein was secreted into the space between the hypodermis and the cuticle . Taken together, these results suggest that As-p50 is involved in the formation of the L1 cuticle and/or the initial molt; however, As-p50 may be multifunctional and also responsible for the digestion of the eggshell during hatching.

Biochem Biophys Res Commun, 2002 Oct 25, 298(2), 216 - 24
Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli; Lee KH et al.; An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL . On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library . A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned . Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E . coli), recombinant ALDH2 and ALDH2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E . coli chaperonin proteins . Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively . Co-expression of ALDH2 with ALDH2(2) in the presence of E . coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer . Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation . Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system . The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly . Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.

Biochemistry (Mosc), 2002 Sep, 67(9), 978 - 85
Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion; Golovastov VV et al.; The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids . It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region . The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14 . In contrast, secretion of the protein withamino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine . The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.

Chem Res Toxicol, 2002 Oct, 15(10), 1254 - 8
Endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K additively enhance arsenic-induced DNA strand breaks in human cells; Wang TS et al.; We report here that sequential digestion with endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K in Tris buffer markedly increased the sensitivity for detecting DNA damage in arsenic-treated cells . These three enzymes increased DNA strand breaks in an additive manner . By using this sequential-enzyme-digestion comet assay, we demonstrated that trivalent inorganic arsenic induced more DNA damage than monomethylarsonous acid, monomethylarsonic acid, and dimethylarsinic acid in human blood cell lines . However, trivalent inorganic arsenic was far less potent than monomethylarsonous acid in inhibiting pyruvate dehydrogenase activity . Therefore, different mechanisms are involved in inhibiting pyruvate dehydrogenase activity and inducing DNA damage . Our results also indicate while trivalent inorganic arsenic induced more endonuclease III-digestible adducts, monomethylarsonous acid and monomethylarsonic acid induced more proteinase K-digestible adducts . These results suggest there is a difference in the mechanism for inducing DNA damage between inorganic and organic methylated arsenic compounds.

Acta Virol, 2002, 46(2), 85 - 90
Selection of phage-display peptides that bind specifically to the outer coat protein of Rice black streaked dwarf virus; Bai FW et al.; Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library . The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein . The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system . The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein . The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein . Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus . Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.

Arch Insect Biochem Physiol, 2002 Nov, 51(3), 136 - 50
Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis; Silver GM et al.; The degradation of cat immunoglobulin G (IgG) in blood-fed adult C . felis midguts was examined . SDS-PAGE analysis of dissected midgut extracts obtained from C . felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested . A 31-kDa serine protease with IgG degrading activity was purified from fed C . felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography . Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG . N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG . The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea . A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated . When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein . Arch . Insect Biochem .

Arch Insect Biochem Physiol, 2002 Nov, 51(3), 111 - 20
A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori; Makka T et al.; It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development . In order to know the function of egg ecdysteroids in embryonic development of B . mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B . mori eggs . First, using the ecdysteroid receptor of B . mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B . mori was analyzed . The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate . Next, several egg ecdysteroids of B . mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage) . Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg) . In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development . These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B . mori embryos . Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development .

Cancer Gene Ther, 2002 Nov, 9(11), 935 - 45
Comparison of safety, delivery, and efficacy of two oncolytic herpes viruses (G207 and NV1020) for peritoneal cancer; Bennett JJ et al.; G207 and NV1020 are two replication-competent, multimutant oncolytic herpes simplex viruses evaluated in the current studies for their anticancer effects in the treatment of gastric cancer . Deletion of both gamma(1)34.5 genes and inactivation of ICP6 (ribonucleotide reductase) allows G207 to selectively replicate within tumor cells . NV1020 is another attenuated recombinant herpes virus with deletions of the HSV joint region, with deletion of only one copy of the gamma(1)34.5 gene, and with the ICP6 gene intact . In vitro, both G207 and NV1020 effectively infected, replicated, and killed human gastric cancer cells, with NV1020 being more effective at lower concentrations of virus . In a murine xenograft model of peritoneally disseminated gastric cancer, both NV1020 and G207 reduced tumor burden when given intraperitoneally (i.p.) at higher doses . When viral doses were lowered or when advanced tumor was treated, i.p . NV1020 was superior to i.p . G207 . In vitro viral replication and cytotoxicity predicted the in vivo antitumor response . Intravenous delivery of either G207 or NV1020 failed to reduce tumor burden, demonstrating the importance of regional therapy as treatment for compartmentalized malignancy . Both agents were safe for use in animals, and immunohistochemistry performed on mouse tissue revealed selective viral targeting of tumor . Oncolytic therapy using genetically engineered HSVs represents a promising strategy for peritoneal malignancies.

J Biol Chem, 2002 Dec 20, 277(51), 49408 - 16 Epub 2002 Oct 16.
Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation; Chen Y et al.; Hec1 (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase . Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation . The mechanisms by which Hec1 is regulated, however, are not known . Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M . Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo . Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1 . When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it . Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 mutant changing Ser(201) to Ala does not . Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation . These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

J Biol Chem, 2002 Dec 27, 277(52), 50716 - 24 Epub 2002 Oct 16.
Escherichia coli K1 internalization via caveolae requires caveolin-1 and protein kinase Calpha interaction in human brain microvascular endothelial cells; Sukumaran SK et al.; The morbidity and mortality associated with Escherichia coli K1 meningitis during the neonatal period have remained significant over the last decade and are once again on the rise . Transcytosis of brain microvascular endothelial cells (BMEC) by E . coli within an endosome to avoid lysosomal fusion is crucial for dissemination into the central nervous system . Central to E . coli internalization of BMEC is the expression of OmpA (outer membrane protein A), which interacts with its receptor for the actin reorganization that leads to invasion . However, nothing is known about the nature of the signaling events for the formation of endosomes containing E . coli K1 . We show here that E . coli K1 infection of human BMEC (HBMEC) results in activation of caveolin-1 for bacterial uptake via caveolae . The interaction of caveolin-1 with phosphorylated protein kinase Calpha (PKCalpha) at the E . coli attachment site is critical for the invasion of HBMEC . Optical sectioning of confocal images of infected HBMEC indicates continuing association of caveolin-1 with E . coli during transcytosis . Overexpression of a dominant-negative form of caveolin-1 containing mutations in the scaffolding domain blocked the interaction of phospho-PKCalpha with caveolin-1 and the E . coli invasion of HBMEC, but not actin cytoskeleton rearrangement or the phosphorylation of PKCalpha . The interaction of caveolin-1 with phospho-PKCalpha was completely abrogated in HBMEC overexpressing dominant-negative forms of either focal adhesion kinase or PKCalpha . Treatment of HBMEC with a cell-permeable peptide that represents the scaffolding domain, which was coupled to an antennapedia motif of a Drosophila transcription factor significantly blocked the interaction of caveolin-1 with phospho-PKCalpha and E . coli invasion . These results show that E . coli K1 internalizes HBMEC via caveolae and that the scaffolding domain of caveolin-1 plays a significant role in the formation of endosomes.

J Biol Chem, 2002 Dec 27, 277(52), 50693 - 702 Epub 2002 Oct 16.
Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo . Evidence for a role in antioxidant biology; Bennaars-Eiden A et al.; 4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging . Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function . Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo . 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates . Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A . The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids . 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence . Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP . Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells . These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.

Circ Res, 2002 Oct 18, 91(8), 704 - 11
Identification of novel interactions between domains of Myosin binding protein-C that are modulated by hypertrophic cardiomyopathy missense mutations; Moolman-Smook J et al.; Cardiac myosin binding protein-C (cMyBPC) is a modular protein consisting of 11 domains whose precise function and sarcomeric arrangement are incompletely understood . Identification of hypertrophic cardiomyopathy (HCM)--causing missense mutations in cMyBPC has highlighted the significance of certain domains . Of particular interest is domain C5, an immunoglobulin-like domain with a cardiac-specific insert, which is of unknown function yet is the site of two HCM-causing missense mutations . To identify interactors with this region, a human cardiac cDNA library was screened in a yeast two-hybrid (Y2H) assay using the C5 sequence as bait . Screening >7x10(6) clones surprisingly revealed that domain C5 preferentially bound to clones encoding C-terminal fragments of cMyBPC; the interacting region was narrowed to domain C8 by deletion mapping . A surface plasmon resonance assay using purified recombinant cMyBPC domains was used to measure the affinity of C5 and C8 in vitro (K(a)=1x10(5) mol/L(-1)) . This affinity was decreased about 2-fold by the HCM mutation R654H, and by at least 10-fold by the mutation N755K . Further Y2H assays also demonstrated specific binding between domains C7 and C10 of cMyBPC . Based on these novel interactions, and previous biochemical and structural data, we propose that cMyBPC molecules trimerize into a collar around the thick filament, with overlaps of domains C5-C7 of one cMyBPC with C8-C10 of another . We speculate that this interaction may be dynamically formed and released, thereby restricting or favoring cross-bridge formation, respectively . We suggest that the HCM mutations act by altering the cMyBPC collar, indicating its importance in thick filament structure and regulation.

J Biochem Mol Biol Biophys, 2002 Oct, 6(5), 357 - 64
Transient DNA binding by a proteolytic peptide from m5C-DNA methyltransferase MspI; Bhattacharya SK et al.; A peptide fragment (p26) generated as a result of limited tryptic proteolysis of methyltransferase MspI retains transient but non-specific DNA binding capability . The transient DNA binding by p26 was characterized with respect to physicochemical factors . Limited proteolysis was performed to probe gross structural deviation from the reported two-domain organization for m5C-MTases, in light of topoisomerase activity shown by MspI, resulted in two peptide fragments; a large fragment p26 and a small fragment p18, consistent with the other reported m5C-MTase structures . The purified large peptide fragment p26, spans between 6 and 251 in the amino acid sequence of M.MspI . The peptide p26 does not bind S-adenosylmethionine, although in the intact protein the AdoMet binding region can be mapped to a region in the protein that is present in this peptide . Such transient DNA binding has not been reported for other protolytic product of any other m5C-DNA methyltransferase.

J Biochem Mol Biol Biophys, 2002 Oct, 6(5), 347 - 50
Cloning and expression of the nucleocapsid protein gene of nipah virus in different strains of Escherichia coli; Ong ST et al.; The coding region of the nucleocapsid (N) gene was amplified from the viral RNA and inserted into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935 . The N protein was expressed as a fusion protein containing the myc epitope and His-tag at its C-terminal end . The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum . Hence, the N(fus) protein produced in E . coli could serve as an alternative antigen for the detection of anti-NiV in swine.

Biochim Biophys Acta, 2002 Nov 20, 1588(2), 106 - 12
Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin, reveals its resistance to proteinaceous trypsin inhibitors; Fukuoka S et al.; Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells . Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK) . However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown . Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated . P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen . Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied . Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen . Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100% . P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin . P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI . Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.

Mech Dev, 2002 Nov, 119(1), 109 - 14
Expression of COUP-TFII in metabolic tissues during development; Zhang P et al.; In mammals, the COUP-TF-family consisting of two structurally related proteins, COUP-TFI and COUP-TFII belongs to the orphan member of the steroid/thyroid hormone receptor superfamily . In an attempt to gain insights into the role of COUP-TFII, we examined developmental expression pattern of the mouse COUP-TFII focusing our studies on endoderm-derived tissues, pancreas and liver in particular . Independent lines of transgenic mice expressing Escherichia coli beta-galactosidase driven by the COUP-TFII promoter were generated . Embryonic expression of the beta-gal protein at day 9 of gestation was detected in the notochord, the ventral neural tube and, interestingly, in the gut endoderm, a site where COUP-TFII has not been detected previously . Between 9.5 and 11.5 dpc, beta-gal expression pattern that was established earlier persisted and sections revealed a staining of the common atrial chamber of the heart . At 15.5 dpc, beta-gal activity was found in all endoderm-derived tissues . We found that COUP-TFII mRNA and protein were present in fetal and adult hepatocytes . Finally, COUP-TFII expression was detected in pancreas, as judged by co-expression of the beta-gal in some of the glucagon and PDX1 positive-cells at 12.5 dpc and co-expression with insulin positive-cells at 15.5 dpc . In adult pancreas, COUP-TFII protein was present in the endocrine islet cells .

Am J Gastroenterol, 2002 Oct, 97(10), 2585 - 8
A double blind, randomized, placebo-controlled study of SP-303 (Provir) in the symptomatic treatment of acute diarrhea among travelers to Jamaica and Mexico; DiCesare D et al.; OBJECTIVE: The study was designed to evaluate the effectiveness of SP-303 (Provir), a plant-derived product with novel antisecretory properties, in the treatment of travelers' diarrhea . METHODS: A total of 184 persons from the United States who acquired diarrhea in Jamaica or Mexico were enrolled in a double-blind, placebo-controlled study examining the effectiveness of three doses of SP-303 in reducing illness . Subjects were treated with 125 mg, 250 mg, or 500 mg SP-303 or a matching placebo four times a day for 2 days . Subjects kept daily diaries of symptoms and were seen each day for 3 days . Of the subjects, 169 (92%) were included in the efficacy analysis . RESULTS: The most common etiological agent identified was enterotoxigenic Escherichia coli, found in 19% of subjects . The mean time interval from taking the first dose of medication until passage of the last unformed stool during 48 h therapy (TLUS48) was 38.7 h for the placebo group . TLUS48 was shortened by SP-303: 30.6 h for the 125-mg dose group (p = 0.005); 30.3 h for the 250-mg group; and 32.6 h for the 500-mg group (p = 0.01) . Treatment failures were seen in 29.3% in the placebo group compared with 7.3% (p = 0.01), 4.3 (p = 0.002), and 9.8 (p = 0.026) in the three treatment groups . SP-303 was well tolerated at all doses . CONCLUSIONS: SP-303 was effective in shortening the duration of travelers' diarrhea by 21% . This antisecretory approach works directly against the pathophysiology of travelers' diarrhea and is not likely to potentiate invasive forms of diarrhea or to produce posttreatment constipation.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 509 - 11
{The functional expression of humanized ScFv-urokinase fusion protein in Escherichia coli}; Liu ZG et al.; The fusion protein of Humanized mouse anti-human fibrin ScFv and the low molecular weight urokinase (IIn-UK) contained seven disulfide bonds and formed inclusion body while expressing in normal E . coli strain . By coexpressing DsbC and using the special E . coli strain Origami(DE3) which was trxB/gor double mutant, the fusion protein IIn-UK was functionally expressed in the cytoplasm of E . coli . The expressed fusion protein in the soluble fraction was purified by using affinity chromatography specific against urokinase . The purified fusion protein could combine the thrombus in vitro, and the specific activity of urokinase reached 80,000 IU/mg fusion protein . The result showed that the fusion protein retained the activity of two moieties, and this study laid a foundation for further research of targeting thrombolytic agent.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 505 - 8
{Enhancing hGH expression level in insect cells by shortening the 5'-UTR of hGH cDNA}; Geng ZH et al.; The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex . In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined . A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR . The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH . After transforming into E . coli . DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired . By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed . Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 463 - 7
{The study of aggregate of the ORF2 peptide of hepatitis E virus expressed in Escherichia coli}; Li SW et al.; A fragment of hepatitis E virus open reading frame-2(ORF2), located from amino acid residues 394 to 604, was expressed in E . coli . The recombinant protein NE2 was found to form homodimer mostly in SDS-PAGE, which can be dissociated to monomers when treated with urea, and it was recognized more strongly in its dimeric form than the monomer by HEV reactive human serum in Western blotting . Besides, many aggregated form of NE2 from dimer to at least hexamer can be seen in MALDI-TOF-MS . And when the hydrated dynamic semidiameter of NE2 moleculars in PBS was measured as about 4 nm by Dynamic Light Scattering (DLS), being equal to tetramer, but with high polydispersity, which suggested that the NE2 moleculars were existed in PBS in many different sizes . These results suggested that the recombinant NE2 can aggregate into several oligomer forms, the association in the dimer is most strong, and dimers can assemble further to form some super-structure.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 431 - 7
{Establishment of gene replacement/disruption system through homologous recombination in Amycolatopsis mediterranei U32}; Ding XM et al.; A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers . Through two-step selection, ahbas gene in U32 was replaced by a promoterless alpha-amylase gene constructed on the plasmid pDK110 of E . coli . The first single-crossover and the second double-crossover frequencies were approximately 0.5%-0.7% and 2%, respectively . Denaturation of the plasmid pDK110 increased the integration frequency about 7-10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds . Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30-50 transformants per microgram of DNA.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 424 - 30
{Expression of TPO mimetic peptide chimeric proteins with human IgG1 Fc fragments and their biological characters}; Li YX et al.; Many antineoplastic agents can cause myelosuppression and thrombocytopenia . Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels . We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo . For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments . The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids . By transforming the four recombinant plasmids into E . coli . BL21 (DE3) respectively, we got 3 kinds of engineered E . coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column . The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts . Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc . These studies have laid basis for production of TPO mimetic peptide by genetic engineering.

Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 420 - 3
{Construction of a temperature inducible shuttle expression vector and its application in Streptomyces}; Tao MF et al.; pHZ1080, an E . coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces . A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp . FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067 . The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E . coli . The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot . The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E . coli and Streptomyces.

Eur J Immunol, 2002 Nov, 32(11), 3066 - 70
Leukemia inhibitory factor is a mediator of Escherichia coli lipopolysaccharide-induced acute thymic atrophy; Sempowski GD et al.; Septic shock in animals and humans is associated with thymic atrophy and generalized lymphocyte apoptosis that impairs T cell responses . Injection of animals with E . coli lipopolysaccharide (LPS) mimics bacterial sepsis-induced thymic atrophy . Leukemia inhibitory factor (LIF) induces pituitary ACTH release in vivo, and LIF administration to mice induces acute thymic atrophy . We have investigated the role of LIF in LPS-induced thymic atrophy . LPS significantly elevated serum LIF within 1 h and induced thymic LIF mRNA within 6 h . Moreover, pretreatment of mice with anti-LIF antibody significantly reduced LPS-induced thymic atrophy . Using adrenalectomized mice and 17-day fetal thymic organ cultures treated with the steroidogenic enzyme inhibitor metyrapone, we demonstrated that LIF induced both systemic and intrathymic corticosteroid production . These data demonstrate systemic and intrathymic pathways of E . coli LPS-induced acute thymic atrophy mediated by LIF and corticosteroids.

Nucleic Acids Res, 2002 Oct 15, 30(20), 4548 - 55
Use and misuse of correspondence analysis in codon usage studies; Perriere G et al.; Correspondence analysis has frequently been used for codon usage studies but this method is often misused . Because amino acid composition exerts constraints on codon usage, it is common to use tables containing relative codon frequencies (or ratios of frequencies) instead of simple codon counts to get rid of these amino acid biases . The problem is that some important properties of correspondence analysis, such as rows weighting, are lost in the process . Moreover, the use of relative measures sometimes introduces other biases and often diminishes the quantity of information to analyse, occasionally resulting in interpretation errors . For instance, in the case of an organism such as Borrelia burgdorferi, the use of relative measures led to the conclusion that there was no translational selection, while analyses based on codon counts show that there is a possibility of a selective effect at that level . In this paper, we expose these problems and we propose alternative strategies to correspondence analysis for studying codon usage biases when amino acid composition effects must be removed.

Nucleic Acids Res, 2002 Oct 15, 30(20), 4406 - 13
The Drosophila transcription factor tramtrack (TTK) interacts with Trithorax-like (GAGA) and represses GAGA-mediated activation; Pagans S et al.; In this study, we report the interaction of the Drosophila transcription factors Trithorax-like (GAGA) and tramtrack (TTK) . This interaction is documented both in vitro, through GST pull-down assays, as well as in vivo, in yeast and Schneider S2 cells . GAGA and TTK share in common the presence of an N-terminal POZ/BTB domain that was found to be necessary and sufficient for GAGA-TTK interaction . Structural models that could account for this interaction are discussed . GAGA is known to activate the expression of many genes in Drosophila . On the other hand, TTK was proposed to act as a maternally provided repressor of several pair-rule genes, such as even-skipped (eve) . As with many Drosophila genes, eve contains at its promoter region binding sites for GAGA and TTK . Here, in transient expression experiments, we showed that GAGA activates transcription from the eve stripe 2 promoter element and that TTK inhibits this GAGA-dependent activation . Repression by TTK of the eve promoter requires its activation by GAGA and depends on the presence of the POZ/BTB domains of TTK and GAGA . These results indicate that GAGA-TTK interaction contributes to the regulation of gene expression in Drosophila.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14083 - 8 Epub 2002 Oct 16.
Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases; Schwarzer D et al.; Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids . Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme . A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation . However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure . Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)) . Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs . For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis . Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis . However, this reaction is unlikely to be of physiological relevance . Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf) . These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.

Cancer Res, 2002 Oct 15, 62(20), 5641 - 4
Hypermutable bases in the p53 cancer gene are at vulnerable positions in DNA secondary structures; Wright BE et al.; A DNA folding analysis indicates that the most hypermutable bases in exons 5, 7, and 8 of the p53 tumor suppressor gene are located immediately next to stems in stable DNA stem-loop structures . On the basis of the highest negative energy (-DeltaG) value of the structures containing each mutable bases and on the extent to which each base is unpaired during transcription, their relative mutabilities are calculated using a new computer algorithm . These predicted mutation frequencies correlate well with those observed in 14,000 human cancers (R(2) = 0.76), whereas there is no such correlation (R(2) = 0.0005) for nearby control bases . The correlation of hypermutable base frequencies with -DeltaG values is poor (R(2) = 0.19), indicating that the extent to which a base is unpaired during transcription is a significant contribution to predicting mutation frequencies.

J Biol Chem, 2002 Dec 27, 277(52), 50326 - 32 Epub 2002 Oct 15.
The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist; Roeb E et al.; Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane . Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit . In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis . To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli . After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography . In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity . The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance . From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin . In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner . These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion . Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.

J Biol Chem, 2002 Dec 27, 277(52), 50923 - 33 Epub 2002 Oct 15.
The 2.2-A crystal structure of human pro-granzyme K reveals a rigid zymogen with unusual features; Hink-Schauer C et al.; Granzyme K (GzmK) belongs to a family of trypsin-like serine proteases localized in electron dense cytoplasmic granules of activated natural killer and cytotoxic T-cells . Like the related granzymes A and B, GzmK can trigger DNA fragmentation and is involved in apoptosis . We expressed the Ser(195) --> Ala variant of human pro-GzmK in Escherichia coli, crystallized it, and determined its 2.2-A x-ray crystal structure . Pro-GzmK possesses a surprisingly rigid structure, which is most similar to activated serine proteases, in particular complement factor D, and not their proforms . The N-terminal peptide Met(14)-Ile(17) projects freely into solution and can be readily approached by cathepsin C, the natural convertase of pro-granzymes . The pre-shaped S1 pocket is occupied by the ion paired residues Lys(188B)-Asp(194) and is hence not available for proper substrate binding . The Ser(214)-Cys(220) segment, which normally provides a template for substrate binding, bulges out of the active site and is distorted . With analogy to complement factor D, we suggest that this strand will maintain its non-productive conformation in mature GzmK, mainly due to the unusual residues Gly(215), Glu(219), and Val(94) . We hypothesize that GzmK is proteolytically active only toward specific, as yet unidentified substrates, which upon approach transiently induce a functional active-site conformation.

Blood, 2002 Nov 1, 100(9), 3383 - 91
Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis; Coakley RJ et al.; Following phagocytosis in vivo, acidification of extracellular pH (pH(o)) and intracellular metabolic acid generation contribute to cytosolic proton loading in neutrophils . Cytosolic pH (pH(i)) affects neutrophil function, although its regulation is incompletely understood . Its effect on mechanisms of neutrophil death is also uncertain . Thus, we investigated pH(i) regulation in Escherichia coli-exposed neutrophils, at various pathogen-to-phagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu in vivo and correlated pH(i) changes with mechanisms of neutrophil death . Following phagocytosis, proton extrusion was dominated early by passive proton conductance channels, and later by Na(+)/H(+) exchange (NHE) . H(+)-translocating adenosine triphosphatase (V-ATPase) pH(i) regulation was evident primarily at lower bacterial densities . At physiologic pH(o), lower pathogen-to-phagocyte ratios alkalinized pH(i) and inhibited apoptosis, whereas higher ratios acidified pH(i) (despite proton extrusive mechanisms) and promoted apoptosis . Necrosis was induced by high-density bacterial exposure at reduced pH(o) . Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl(2), or bafilomycin, respectively) moderately hyperacidified pH(i) and accelerated apoptosis . However, in combination they profoundly acidified pH(i) and induced necrosis . Proinflammatory mediators in vivo might affect both pH(i) regulation and cell death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy subjects . Only CF BAL fluid alkalinized pH(i) and suppressed apoptosis at physiologic pH(o), but failed to prevent necrosis following phagocytosis at low pH(o) . Thus, a precarious balance between cytosolic proton loading and extrusion after phagocytosis dictates the mode of neutrophil cell death . pH(i)/pH(o) might be therapeutically targeted to limit neutrophil necrosis and protect host tissues during necrotizing infections.

Blood, 2002 Nov 1, 100(9), 3233 - 9
LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system; Zhao L et al.; Intravenous injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in the lung and liver . When degradation of the accumulated platelets occurs, anaphylactoid shock follows rapidly, the severity of the shock paralleling the quantity of platelets accumulated in the lung . Here we examined the contributions made by LPS structure and the complement system to the platelet response to LPS . BALB/c mice were injected with an LPS from Escherichia coli O8, O9, O111, or K-12, or from recombinant mutants of K-12 . The O-regions of the O8 and O9 LPSs consist of a mannose homopolysaccharide (MHP), while that of O111 consists of a heteropolysaccharide (not including mannose), and K-12 LPS lacks an O-region . O111 LPS was devoid of the ability to induce the platelet response or shock, while the ability of K-12 LPS was weak . The 2 recombinant LPSs-each having an O-region (from O8 or O9) linked to K-12 LPS-exhibited activities similar to or stronger than those of their original LPSs . Mannose-binding lectin (MBL) complexed with MBL-associated serine proteases (MASPs) bound strongly to LPSs containing MHP and caused C4 activation . Moreover, the abilities of these LPSs to activate the complement system corresponded well with their abilities to induce the platelet response and rapid shock . These results suggest that the structure of the O-antigen region is important for the platelet response to LPS, and that activation of the lectin pathway of the complement system is involved in this response.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3386 - 93
Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF; Sanchez P et al.; We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF . SmeT belongs to the TetR and AcrR family of transcriptional regulators . The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes . Experiments with S . maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor . S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5' end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PSMET: The level of expression of smeT is higher in smeDEF-overproducing S . maltophilia strain D457R, which suggests that SmeT represses its own expression . Band-shifting assays have shown that wild-type strain S . maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region . That cellular factor(s) was absent from smeDEF-overproducing S . maltophilia strain D457R . The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein . This change allowed overexpression of both smeDEF and smeT in D457R . It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R . This suggests that the wild-type protein is not dominant over the mutant SmeT.

Gene, 2002 Sep 4, 297(1-2), 221 - 8
Nucleotide variations in the lxd region of Drosophila melanogaster: characterization of a candidate modifier of lifespan; Tahoe NM et al.; We have investigated the structure and function of several proteins that might influence adult lifespans in Drosophila melanogaster . The present report focuses on the gene lxd ('low xanthine dehydrogenase'), which lies in a region of chromosome III identified by QTL-mapping as potentially important for lifespan . DNA sequence of a 3780 bp genomic fragment containing the lxd locus reveals differences between long-lived and control inbred lines . In order to determine the importance of nucleotide replacements, the intron/exon boundaries have been determined, based on peptide alignment and conserved amino acids . We identified four exons in the lxd coding region . The deduced amino acid sequence of exon 4 shows 46.5% identity with Escherichia coli MoaC sequences . There are eight nucleotide substitutions in exons differentiating the inbred lines, three in exon 3 and five in exon 4 . One of the exon 4 substitutions has resulted in a Thr-Ile replacement at the protein surface, but not entirely solvent exposed . This substitution is potentially a modifier of lifespan via oxygen defense, but since the activities of three molybdoenzymes are unaffected in inbred lines, this possibility seems remote.

Neuropharmacology, 2002 Oct, 43(5), 889 - 98
Contribution of peroxynitrite to fatal cardiovascular depression induced by overproduction of nitric oxide in rostral ventrolateral medulla of the rat; Chan SH et al.; We evaluated the contribution of peroxynitrite to the fatal cardiovascular depression induced by overproduction of nitric oxide (NO) after activation of inducible NO synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone . In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of E . coli lipopolysaccharide (LPS) bilaterally into the RVLM elicited significant hypotension, bradycardia, reduction in sympathetic vasomotor tone and mortality . There was also a discernible elevation of iNOS expression in the ventrolateral medulla, followed by a massive production of nitrotyrosine, an experimental index for peroxynitrite . Co-administration bilaterally into the RVLM of the selective iNOS inhibitor, S-methylisothiourea (50, 100 or 250 pmol), an active peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis- (N-methyl-4'-pyridyl)-porphyrinato iron (III) (10 or 50 pmol), a peroxynitrite scavenger, L-cysteine (5, 50 or 100 pmol), or a superoxide dismutase mimetic, Mn(III)-tetrakis-(4-benzoic acid) porphyrin (1 or 10 pmol), significantly prevented mortality, reduced nitrotyrosine production and reversed the NO-induced cardiovascular suppression after application of LPS into the RVLM . We conclude that the formation of peroxynitrite by a reaction between superoxide anion and NO is primarily responsible for the fatal cardiovascular depression induced by overproduction of NO after activation of iNOS at the RVLM.

J Theor Biol, 2002 Oct 21, 218(4), 507 - 20
Modular response analysis of cellular regulatory networks; Bruggeman FJ et al.; The sheer complexity of intracellular regulatory networks, which involve signal transducing, metabolic, and genetic circuits, hampers our ability to carry out a quantitative analysis of their functions . Here, we describe an approach that greatly simplifies this type of analysis by capitalizing on the modular organization of such networks . Steady-state responses of the network as a whole are accounted for in terms of intermodular interactions between the modules alone; processes operating solely within modules need not be considered when analysing signal transfer through the entire network . The intermodular interactions are quantified through (local) response coefficients which populate an interaction map (matrix) . This matrix can be derived from a biochemical or molecular biological analysis of (macro) molecular interactions that constitute the regulatory network . The approach is illustrated by two examples: (i) mitogenic signalling through the mitogen-activated protein kinase cascade in the epidermal growth factor receptor network and (ii) regulation of ammonium assimilation in Escherichia coli.

Peptides, 2002 Oct, 23(10), 1719 - 25
Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation; Balass M et al.; Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10 . Using a 15-mer phage-peptide library, we identified two 2A10-epitopes on human Mdm2 (hdm2): at positions 255-266 (LDSEDYSLSEEG) and 389-400 (QESDDYSQPSTS) . Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A10 to Mdm2 with high (4.5 x 10(-9)M) and moderate affinity (1.1 x 10(-7)M), respectively . Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10 . These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation.

Vet Immunol Immunopathol, 2002 Oct 28, 89(3-4), 115 - 25
Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide; Yagi Y et al.; A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion . TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected . A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period . The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells . TUNEL-positive PMN observed in normal animals showed a reduced migration capacity . The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity . Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis . The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging .

Vet Microbiol, 2002 Nov 6, 89(4), 277 - 89
Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp . mycoides SC; Alonso JM et al.; Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp . mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster . In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals . Using this strategy, a genome fragment has been isolated and characterised . The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E . coli . The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp . capricolum (Mcc) . Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster . The full recombinant EF-Tu was produced in E . coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.

Gene, 2002 Aug 21, 296(1-2), 179 - 85
Construction of a mariner-based transposon for epitope-tagging and genomic targeting; Chiang SL et al.; Mobile genetic elements are often employed for constructing gene fusions or to perform mutagenesis . mariner transposons are well-suited to such applications because of their low site specificity, in vitro activity, and exceptionally broad host range . This report describes a mariner-based method for rapidly creating a large number of insertion mutants that can be converted to in-frame epitope fusions in a single step . First, a mariner-based vector is used to deliver a FLP recombinase substrate randomly into a target molecule . Expression of the FLP recombinase is then induced to catalyse the excision of sequences flanked by FLP recombinase target recognition sites, leaving behind a triple-FLAG epitope . The reversibility of the excision event provides opportunities for using genomic targeting methods easily to create transcriptional or translational fusions to genes of interest.

Gene, 2002 Aug 21, 296(1-2), 129 - 37
The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA; Mlynarova L et al.; Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP . We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA . In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre . The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account . It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event.

Eur J Biochem, 2002 Oct, 269(20), 4969 - 80
The glucose-specific carrier of the Escherichia coli phosphotransferase system; Garcia-Alles LF et al.; Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS) . 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate . Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates . They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter . 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter . The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake . Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site . EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d) . Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421 . Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter . Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells . This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.

Eur J Biochem, 2002 Oct, 269(20), 4960 - 8
Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy; Encinas MV et al.; Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 . The interaction of the enzyme with the substrates originates important domain movements in the protein . In this work, the interaction of several substrates and ligands with E . coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct . The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding . The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal.mol-1, which is 3.2 kcal.mol-1 more favorable than in the complex with Mg2+ . For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal.mol-1) were found for either metal ion . The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns . These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates . Molecular models of the P-pyridoxyl-E . coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.

Eur J Biochem, 2002 Oct, 269(20), 4948 - 59
Diffusion through channel derivatives of the Escherichia coli FhuA transport protein; Braun M et al.; FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports {Fe3+}ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, phi80 and UC-1, and to colicin M and microcin J25 . The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5 . The FhuA crystal structure reveals 22 antiparallel transmembrane beta-strands that form a beta-barrel which is closed by a globular N-terminal domain . FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5-160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane . Here it is shown that isolated FhuADelta5-160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels . The double mutant FhuADelta5-160 Delta322-336 lacking in addition to the globular domain most of the large surface loop 4 which partially constricts the channel entrance, displayed an increased single-channel conductance but formed no stable channels . It transported in vivo{Fe3+}ferrichrome with 45% of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics . In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335-355 comprising one-third of surface loop 4 and half of the transmembrane beta-strand 8 formed stable channels in lipid bilayers with a large single-channel conductance of 2.5 nS in 1 m KCl . Cells that synthesized FhuADelta5-160 Delta335-355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane . FhuADelta5-160 Delta335-355 showed no FhuA specific functions such as active transport of {Fe3+}ferrichrome or sensitivity to the other FhuA ligands . It is concluded that FhuADelta5-160 Delta335-355 assumes a conformation that is incompatible with any of the FhuA functions.

Eur J Biochem, 2002 Oct, 269(20), 4913 - 20
Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli; Tramonti A et al.; Glutamate decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the irreversible alpha-decarboxylation of glutamate to yield 4-aminobutyrate . In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5) . The contribution of the active-site lysine residue (Lys276) to the catalytic mechanism of E . coli glutamate decarboxylase has been determined . Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable . The purified mutants contain very little (K276A) or no (K276H) cofactor at all . However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly . The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form . Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400-fold less than that of the reaction between glutamate and pyridoxal 5'-phosphate in solution . Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism . Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.

Immunology, 2002 Oct, 107(2), 183 - 9
Cross-presentation of cell-associated antigens by CD8alpha+ dendritic cells is attributable to their ability to internalize dead cells; Schulz O et al.; In the mouse, cross-presentation is an exclusive property of the CD8alpha+ subset of dendritic cells (DC) but the basis for this selectivity remains unclear . Here we report that splenic CD8alpha+ DC are much superior to other DC subsets in internalizing dying cells in vitro . In contrast, CD8alpha+, CD8alpha- CD4+ and CD8alpha- CD4- DC subsets phagocytose bacteria or latex beads to a similar extent . Although CD8alpha+ DC are better than CD4+ DC at presenting ovalbumin (OVA)-loaded splenocytes to naive OT-I T lymphocytes, CD4+ DC are better at presenting OVA-expressing Escherichia coli to the same T cells . In both cases, presentation is abrogated by lactacystin . These results show that both splenic CD8alpha+ and CD8alpha- DC can present exogenous antigens on major histocompatibility complex (MHC) class I via a proteasome-dependent pathway and suggest that the specialized cross-presenting function of CD8alpha+ DC is a result of their ability to endocytose dying cells rather than a unique pathway for handling endosomal contents.

Biochem J, 2003 Feb 1, 369(Pt 3), 497 - 507
Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase; Iyengar A et al.; CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH(3) or L-glutamine as the nitrogen source . GTP is required as an allosteric effector to promote glutamine hydrolysis . In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102-118) within the synthase domain of Escherichia coli CTP synthase . Mutant K102A CTP synthase exhibited wild-type activity with respect to NH(3) and glutamine; however, the R105A, D107A, L109A and G110A enzymes exhibited wild-type NH(3)-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation . The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH(3) . Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2-4-fold, suggesting that these residues do not play a significant role in GTP binding . In the presence of GTP, the k (cat) values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase . Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH(3) tunnel has its normal structure altered or fails to form . L109F CTP synthase was prepared to block totally the putative NH(3) tunnel; however, this enzyme's rate of glutamine-dependent CTP formation and glutaminase activity were both impaired . In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzyme's glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.

Biochem J, 2003 Feb 1, 369(Pt 3), 627 - 34
Interdomain communication in the molecular chaperone DnaK; Han W et al.; DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain . Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP.DnaK and GrpE-liganded ADP.DnaK . Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions . Binding of peptide increased the intrinsic fluorescence of both ATP.DnaK and ADP.DnaK.GrpE and rendered their spectra similar to the spectrum of ADP.DnaK with closed conformation of the ATPase domain . These results, together with the differential kinetics of peptide binding to ADP.DnaK on the one hand, and to ATP.DnaK or ADP.DnaK.GrpE on the other, suggest that ligands for either domain, i.e . ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.

Cell Transplant, 2002, 11(5), 481 - 8
Lentiviral transfer of the LacZ gene into human endothelial cells and human bone marrow mesenchymal stem cells; Totsugawa T et al.; Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells . The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro . For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E . coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system . The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs . Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity . There was no significant difference in transduction efficacy between early and late-passage phases in both cells . LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay . These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

Extremophiles, 2002 Oct, 6(5), 359 - 67 Epub 2002 Apr 18.
Holliday junction-resolving enzymes from eight hyperthermophilic archaea differ in reactions with cruciform DNA; Neef K et al.; Holliday junction-resolving enzymes have been identified in a broad variety of organisms and tissues . In this study, six new Holliday junction-cleaving enzymes (Hjcs) were obtained from hyperthermophilic crenarchaeal and euryarchaeal species, including Pyrococcus horikoshii, Pyrococcus abyssi, Methanococcus jannaschii, Methanobacterium thermautotrophicum, Archaeoglobus fulgidus, and Aeropyrum pernix . The genes were cloned and overexpressed in Escherichia coli, and the respective proteins were purified from crude extracts to homogeneity . For an initial characterization of the enzymatic activities, synthetic heat-stable fixed and mobile cruciform DNA substrates were used at 75 degrees C . The Hjcs from Pyrococcus furiosus, Sulfolobus solfataricus, and the archaeal virus SIRV2 were included in the study for comparison . Despite their sequence homology, the enzymes showed marked differences in their reactions with individual cruciform DNAs . While the fixed cruciform structure was cleaved by all enzymes at only one major position, the mobile cruciform structure displayed different cleavage patterns for individual Hjcs, each with several cleavage positions . Furthermore, a strong bias for cleavage of one direction across the junction was observed with the fixed cruciform DNA for all enzymes . In contrast, the mobile cruciform DNA displayed different preferences, depending on the enzyme used.

Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 134 - 8 Epub 2002 Sep 06.
The effect of a disrupted yhjQ gene on cellular morphology and cell growth in Escherichia coli; Kim MK et al.; The 5' upstream region of the cellulose synthase operon ( bcs operon) has been isolated by cloning from Escherichia coli . A gene encoding YhjQ is located 1.0 kb upstream of the bcs operon in E . coli . The function of YhjQ remains unknown . Insertional inactivation of the yhjQ gene causes abnormal cell division, resulting in incomplete partition of the chromosome and filamentous cells of various sizes . These results suggest that the product of yhjQ may affect normal doubling and cellular morphology.

J Biosci, 2002 Sep, 27(5), 515 - 20
Comparative analysis of naturally occurring L-amino acid osmolytes and their D-isomers on protection of Escherichia coli against environmental stresses; Shahjee HM et al.; Adaptation to high salinity and low or high temperature is essential for bacteria to survive . Accumulation of exogenous osmolytes is one of the ways that helps bacteria to survive under such extracellular stress . We have analysed the capability of various L-amino acids and their D-isomers to act as osmolytes and thus enable Escherichia coli cells to survive under various stress conditions . E . coli cells were grown in the presence or absence of L- and D-proline, alanine, serine and lysine under salt, heat and cold stresses . Of the various amino acids tested, L-proline, closely followed by L-serine turned out to be highly protective against environmental stresses . L-proline provided excellent protection (95%) against salt stress, followed by cold (60%) and heat (40%) stresses . D-amino acids on the other hand, proved to be highly inhibitory under stress conditions . Thus L-amino acids were found to be growth protectants under stress while their D-isomers were inhibitory during stress as well as normal conditions.

J Biol Chem, 2002 Dec 20, 277(51), 49360 - 5 Epub 2002 Oct 14.
Bax-type apoptotic proteins porate pure lipid bilayers through a mechanism sensitive to intrinsic monolayer curvature; Basanez G et al.; During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism . We have proposed that Bax and DeltaN76Bcl-x(L) (the Bax-like cleavage fragment of Bcl-x(L)) function by forming pores that are at least partially composed of lipids (lipidic pore formation) . Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation . Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization . The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or DeltaN76Bcl-x(L) . Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.

J Biol Chem, 2002 Dec 20, 277(51), 49383 - 8 Epub 2002 Oct 14.
Association of the histone methyltransferase Set2 with RNA polymerase II plays a role in transcription elongation; Li J et al.; The Saccharomyces cerevisiae protein, Set2, has recently been shown to be a histone methyltransferase . To elucidate the function of Set2, its associated proteins were identified using tandem affinity purification and mass spectrometry . We found that Set2 associates with RNA polymerase II . The interaction between the Set2 protein and RNA polymerase II requires the WW domain in Set2 and phosphorylation of the carboxyl-terminal domain of the largest subunit of RNA polymerase II . Set2 directly binds to the carboxyl-terminal domain with phosphorylated Ser(2) in the heptapeptide repeats . set2 deletion mutant is sensitive to 6-azauracil, a property often associated with impaired transcription elongation . Together, our results suggest that Set2 through association with the elongating form of RNA polymerase II plays an important role in transcription elongation.

J Natl Cancer Inst, 2002 Oct 16, 94(20), 1527 - 36
Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer; Feng Z et al.; BACKGROUND: Mutations in ras genes are commonly found in human cancers and in animal models . Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers . To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells . METHODS: We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo{a}pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras . We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method . RESULTS: Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively . The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12 . There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot . Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes . CONCLUSION: These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.

Anal Biochem, 2002 Oct 1, 309(1), 137 - 42
Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase; Takamune N et al.; Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins . To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis . Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus . The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody . Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate . This was followed by absorbance measurement (lambda(450)-lambda(630)) . In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies . Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity . This is the first report of the measurement of NMT activity by the ELISA system.

Anal Biochem, 2002 Oct 1, 309(1), 102 - 8
Screening for recombinant glutathione transferases active with monochlorobimane; Eklund BI et al.; A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli . The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB) . This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants . Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light . The fluorescence is visible instantly . One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies . The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries . Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.

Anal Biochem, 2002 Oct 1, 309(1), 27 - 34
Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein; Oelschlaeger P et al.; A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described . The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP) . The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain . The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required . Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h . This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes . In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv . The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R(2)>0.99 . The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B.

J Mol Biol, 2002 Oct 18, 323(2), 309 - 25
Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase, a TIM barrel protein; Wu Y et al.; A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed . The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase . Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28 . The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases . A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases . Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface . Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively . The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state . The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms . The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.

J Mol Biol, 2002 Oct 18, 323(2), 253 - 62
Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography; Chu R et al.; To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein . Four residues expected to form a hydrophobic core were mutated . One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme . The other three positions were randomly mutated . In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c . This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins . The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two . Nevertheless, the selected proteins were thermodynamically very stable . The structure of a selected protein was characterized using multi-dimensional NMR . All four helices were formed in the structure . Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection . This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues . These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.

J Food Prot, 2002 Oct, 65(10), 1646 - 50
Evaluation of household sanitizers for reducing levels of Escherichia coli on iceberg lettuce; Vijayakumar C et al.; Diluted solutions of various household sanitizers (apple cider vinegar, white vinegar, bleach, and a reconstituted lemon juice product) were tested for their effectiveness in reducing counts of inoculated Escherichia coli and naturally present aerobic, mesophilic bacteria on lettuce . Sanitization treatments were carried out at 4 degrees C and at room temperature (ca . 21 degrees C) with and without agitation and at different exposure times (0, 1, 5, and 10 min) . Of the sanitizers tested, 35% white vinegar (1.9% acetic acid) was the most effective in reducing E . coli levels (with a 5-log10 reduction after 5 min with agitation and after 10 min without agitation) and in reducing aerobic plate counts (with a >2-log10 reduction after 10 min with agitation) . Lettuce samples treated with diluted household sanitizers were analyzed for consumer acceptability by sensory evaluation using a 9-point hedonic scale . The sanitized samples did not differ in acceptability (P > 0.05), except for samples treated with white vinegar . Samples treated with the white vinegar for 10 min were noticeably sour and slightly wilted in appearance . Consumer acceptability was maintained with all sanitization treatments, including those involving 35% white vinegar.

Nat Struct Biol, 2002 Nov, 9(11), 849 - 54
Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex; Stark H et al.; The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP . Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex . The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center . The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket . These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.

Infect Immun, 2002 Nov, 70(11), 5913 - 23
The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system; Burton CL et al.; The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of Escherichia coli in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an "autoinducer" (AI) which, in turn, promotes E . coli growth in the absence of NE . Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations . Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate E . coli growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine . Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe(3+) to saturate transferrin, growth inhibition was relieved . Other metal cations, including Mg(2+), Ca(2+), and Zn(2+), had no effect . These data suggested that the stimulation of E . coli growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine . Consistent with this hypothesis, E . coli strains with mutations in ferrienterobactin transport (fepA or tonB) or enterobactin biosynthesis (entA) did not respond to NE . Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum . The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of E . coli and mutations in fepA or tonB abolished the DBS-dependent growth stimulation . In contrast to NE, however, DBS stimulated the growth of the entA mutant . Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the E . coli entA(+) parent but not of the entA mutant contained AI, i.e., stimulated the growth of E . coli in SAPI+serum . Taken together, these data show that when low numbers of E . coli are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system . This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.

J Biol Chem, 2002 Dec 13, 277(50), 48386 - 94 Epub 2002 Oct 11.
Deletion mutagenesis of human cystathionine beta-synthase . Impact on activity, oligomeric status, and S-adenosylmethionine regulation; Oliveriusova J et al.; Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine . We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme . Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation . Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity . Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme . Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner . The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme . The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability . Removal of both of these domains resulted in stable constitutively activated enzyme . Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.

J Biol Chem, 2002 Dec 27, 277(52), 50676 - 82 Epub 2002 Oct 11.
Role of hydration in the binding of lac repressor to DNA; Fried MG et al.; The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA . Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules . If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A . This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator . The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules . Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules . The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.

J Endocrinol, 2002 Oct, 175(1), 261 - 7
Recombinant interleukin-1 beta activates the hypothalamic-pituitary-interrenal axis in rainbow trout, Oncorhynchus mykiss; Holland JW et al.; The present study provides the first direct evidence that implicates fish cytokines as the effector molecules by which the immune system signals the neuroendocrine system and activates the hypothalamic-pituitary-interrenal stress axis . I.p . injections of trout recombinant interleukin-1 beta (rIL-1 beta) or E . coli lipopolysaccharide (LPS), at concentrations known to induce immune/inflammatory responses in vivo (0.1-0.6 nmol/kg and 1.3 mg/kg respectively), significantly elevated plasma cortisol levels in a dose- and/or time-dependent manner . However, in contrast to general stress responses in fish, under the conditions employed in this study, no specific treatment effects on plasma glucose levels could be demonstrated . The trout IL-1 beta peptides (P1 and P3), which are homologous to receptor-binding sequences of human IL-1 beta, failed to influence the prevailing cortisol concentration even though an equivalent dose has been found to have immunostimulatory properties in vivo . Blockade of endogenous ACTH release by administration of the synthetic glucocorticoid dexamethasone prevented the rIL-1 beta/LPS-mediated elevation of plasma cortisol, suggesting that IL-1 beta and LPS modulate cortisol secretion via effects at the level of the hypothalamic-pituitary axis . These data indicate that, with respect to IL-1 beta, cytokine signalling between the immune and neuroendocrine systems in mammals appears to be conserved in lower vertebrates.

J Endocrinol, 2002 Oct, 175(1), 61 - 73
The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis; Schreiber G; In larger mammals, thyroid hormone-binding plasma proteins are albumin, transthyretin (TTR) and thyroxine (T4)-binding globulin . They differ characteristically in affinities and release rates for T4 and triiodothyronine (T3) . Together, they form a 'buffering' system counteracting thyroid hormone permeation from aqueous to lipid phases . Evolution led to important differences in the expression pattern of these three proteins in tissues . In adult liver, TTR is only made in eutherians and herbivorous marsupials . During development, it is also made in tadpole and fish liver . More intense TTR synthesis than in liver is found in the choroid plexus of reptilians, birds and mammals, but none in the choroid plexus of amphibians and fish, i.e . species without a neocortex . All brain-made TTR is secreted into the cerebrospinal fluid, where it becomes the major thyroid hormone-binding protein . During ontogeny, the maximum TTR synthesis in the choroid plexus precedes that of the growth rate of the brain and occurs during the period of maximum neuroblast replication . TTR is only one component in a network of factors determining thyroid hormone distribution . This explains why, under laboratory conditions, TTR-knockout mice show no major abnormalities . The ratio of TTR affinity for T4 over affinity for T3 is higher in eutherians than in reptiles and birds . This favors T4 transport from blood to brain providing more substrate for conversion of the biologically less active T4 into the biologically more active T3 by the tissue-specific brain deiodinases . The change in affinity of TTR during evolution involves a shortening and an increase in the hydrophilicity of the N-terminal regions of the TTR subunits . The molecular mechanism for this change is a stepwise shift of the splice site at the intron 1/exon 2 border of the TTR gene . The shift probably results from a sequence of single base mutations . Thus, TTR evolution provides an example for a molecular mechanism of positive Darwinian evolution . The amino acid sequences of fish and amphibian TTRs are very similar to those in mammals, suggesting that substantial TTR evolution occurred before the vertebrate stage . Open reading frames for TTR-like sequences already exist in Caenorhabditis elegans, yeast and Escherichia coli genomes.

Mutat Res, 2002 Oct 31, 508(1-2), 107 - 19
Chemotherapeutically induced deletion of expanded triplet repeats; Hashem VI et al.; The number of neurodegenerative disorders associated with the expansion of DNA repeats, currently about 18, continues to increase as additional diseases caused by this novel type of mutation are identified . Typically, expanded repeats are biased toward further expansion upon intergenerational transmission, and disease symptoms show an earlier age of onset and greater severity as the length of the triplet repeat tract increases . Most diseases exhibit progressive neurological and/or muscular degeneration that can lead to total disability and death . As yet, no treatment exists for the genetic basis of any repeat disease . Given that the severity of these diseases is related to repeat tract length, reducing repeat lengths might delay the onset and reduce disease severity . Here, we test the hypothesis that the introduction of damage into DNA, which results in subsequent repair events, can lead to an increased rate of repeat deletion . Applying a sensitive genetic assay in Escherichia coli {Mut . Res . 502 (2002) 25}, we demonstrate that certain DNA damaging agents, including EMS, ENU, UV light, and anticancer agents mitomycin C, cisplatin, and X-rays increase the rate of deletion of (CTG).(CAG) repeats in a length and orientation dependent fashion . In addition, oxidative damage to DNA also increases the deletion rate of repeats . These results suggest that a chemotherapeutic approach to the reduction in triplet repeat length may provide one possible rationale to slow, stop, or reverse the progression of these diseases.

Mutat Res, 2002 Oct 31, 508(1-2), 71 - 81
Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli; Cai Z et al.; This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU) . A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU . Base substitution mutations were investigated by direct sequencing methods . These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites . These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.

J Immunol Methods, 2002 Dec 15, 270(2), 247 - 57
Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display; Tuckey CD et al.; It has recently been reported that single amino acid residues can strongly influence the expression of recombinant antibody fragments in Escherichia coli . Prediction of these critical positions can be difficult even with prior knowledge of the primary sequence and the three-dimensional folded structure of the antibody . To circumvent this, a Fab phage display library containing random point mutations was generated from a hybridoma specific for activated p44/p42 mitogen-activated protein (MAP) kinases . Clones that express Fab were selected by panning against the target antigen . It was found that a cysteine-to-serine substitution at position 91 in the CDR3 of the light chain was responsible for allowing expression of Fab . Site-directed mutagenesis was performed to effect this substitution and others at cysteine 91 on a nonexpressing clone . Mutants containing serine, glycine or alanine at position 91 expressed Fab and bound to target antigen . In contrast, tyrosine mutants had moderate Fab expression but no detectable binding to antigen . These results demonstrate that by using phage display, one can select for the expression of antibody fragments while retaining biological activity.

Biochem Biophys Res Commun, 2002 Oct 18, 298(1), 37 - 40
Limitations on the recombinant plasmid selection by Lac(+)/Lac(-) colony phenotype detection; Gutkina N et al.; Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases . Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker . All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift . The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG . The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids.

Biochemistry, 2002 Oct 22, 41(42), 12739 - 44
Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine; Hayakawa H et al.; 8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells . 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors . To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation . We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine . On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex . Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified . Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not . YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth . We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.

Biochemistry, 2002 Oct 22, 41(42), 12697 - 705
Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus; Birkeland NK et al.; Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function . Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea . We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C . An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA . The purified AfalkA protein differs from E . coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) >> 3-methylguanine approximately 7-methyladenine >> 7-methylguanine . Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A . fulgidus . At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates . The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures . This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E . coli tag alkA double mutant . The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.

Biochemistry, 2002 Oct 22, 41(42), 12590 - 7
Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase; Lu H et al.; The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined . A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates . PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1 . The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5 . The roles of several active site residues in PICS TE were examined by site-directed mutagenesis . Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates . Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5 . Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.

Biochemistry, 2002 Oct 22, 41(42), 12582 - 9
The dual-specific active site of 7,8-diaminopelargonic acid synthase and the effect of the R391A mutation; Eliot AC et al.; 7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesis of biotin . The wild-type enzyme has a steady-state kcat value of 0.013 s(-1), and the K(m) values for SAM and KAPA are 150 and <2 microM, respectively . The k(max) and apparent K(m) values for the half-reaction of the PLP form of the enzyme with SAM are 0.016 s(-1) and 300 microM, respectively, while those for the reaction with DAPA are 0.79 s(-1) and 1 microM . The R391A mutant enzyme exhibits near wild-type kinetic parameters in the reaction with SAM, while the apparent K(m) for DAPA is increased 180-fold . The 2.1 A crystal structure of the R391A mutant enzyme shows that the mutation does not significantly alter the structure . These results indicate that the conserved arginine residue is not required for binding the alpha-amino acid SAM, but it is important for recognition of DAPA.

Biochemistry, 2002 Oct 22, 41(42), 12575 - 81
Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase; Kim J et al.; The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme . These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites . The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification . The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel . Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel . The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a substantially reduced rate of carbamoyl phosphate synthesis, but the rate of ATP turnover and glutamine hydrolysis was not significantly altered . These data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain . The partial reactions of CPS have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active sites has not been functionally perturbed.

World J Gastroenterol, 2002 Oct, 8(5), 923 - 7
Intestinal damage mediated by Kupffer cells in rats with endotoxemia; Gong JP et al.; AIM: To determine the in vivo effects of phagocytic blockade of Kupffer cell (KC) on the release of proinflammatory cytokines in small intestinal lesion and on the integrity of intestinal tract by using gadolinium chloride (GdCl(3)) during early endotoxemia . METHODS: Wistar rats were divided into three groups: Group A, rats were injected with endotoxin (E . coli O111:B(4), a dose of 12 mg x kg(-1)) only; Group B, rats were pretreated intravenously with 25 mg of GdCl(3) per kg 24 h are given endotoxin; and Group C, sham operation only . All animals were sacrificed 4 h after endotoxin injection . In portion of the rats of three groups, bile duct was cannulated, which the bile was collected externally . Morphological changes of ileum were observed under light microscopy and electronic microscopy . The KC were isolated from rats by collagenase perfusion and in KC, expression of TNF-alpha and IL-6 mRNA were determined by RT-PCR analysis . Plasma and bile TNF-alpha and IL-6 Levels were determined by enzyme-linked immunosorbent assay (ELISA) . RESULTS: In group A, there were neutrophil infiltration and superficial epithelial necrosis of the ileal villi, sloughing of mucosal epithelium, and disappearance of some villi . In group B, the ileal mucosal damage was much reduced . which in group C, no significant morphological changes were seen . GdCl(3) pretreatment decreased significantly the expression of TNF-alpha and IL-6 mRNA in group B (4.32+/-0.47 and 4.05+/-0.43) when compared to group A (9.46+/-1.21 and 9.04+/-1.09) (P<0.05) . There was no significant expression of TNF-alpha and IL-6 mRNA in group C (1.03+/-0.14 and 10.4+/-0.13) . In rats of group A, the levels of TNF-alpha and IL-6 in bile and plasma were 207+/-29 ng x L(-1), 1032+/-107 ng x L(-1), 213+/-33 ng x L(-1), and 1185+/-127 ng x L(-1), respectively . In group B, they were 113+/-18 ng x L(-1), 521+/-76 ng x L(-1), 147+/-22 ng x L(-1), and 572+/-54 ng x L(-1), respectively . In group C, they were 67+/-10 ng x L(-1), 72+/-13 ng x L(-1), 109+/-18 ng x L(-1), and 118+/-22 ng x L(-1) respectively . There were significant difference between the three group (P<0.05) . CONCLUSION: KC release cytokines TNF-alpha and IL-6 causing damage to the integrity of intestinal epithelium and play a crucial role in the initiation and progression of intestinal mucosal damage during early endotoxemia.

World J Gastroenterol, 2002 Oct, 8(5), 863 - 7
Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry; Zhong YW et al.; AIM: To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry . METHODS: The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate . After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen . The selected scFv clones were digested by SfiI/NotI and DNA was sequenced . Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv . The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody . RESULTS: The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp . ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B . CONCLUSION: We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.

Cell Motil Cytoskeleton, 2002 Dec, 53(4), 273 - 80
Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain; Hayashi M et al.; We have recently shown that rabbit actin can be introduced by electroporation into the Chlamydomonas ida5 mutant lacking conventional actin and rescue its mutant phenotype {Hayashi et al., 2001: Cell Motil . Cytoskeleton 49:146-153} . In this study, we explored the possibility of using electroporation for functional assay of a recombinant protein . The p28 light chain of inner-arm dyneins was expressed in Escherichia coli, purified to homogeneity, and introduced by electroporation into a non-motile mutant ida4oda6 that lacks it . Because this protein was insoluble in the low ionic strength solution used in the previous study, electroporation was performed at physiological ionic strength in the presence of Ca(2+) . Most cells shed their flagella after electroporation . Reflagellation took place within 3 h and up to 30% of the cells became motile, indicating that the introduced p28 retained its functional activity . Fluorescently-labeled p28 was equally effective; in this case fluorescence was observed along the flagella . The presence of Ca(2+) and deflagellation appeared to be important for efficient protein delivery, because a triple mutant with the fa1 mutation deficient in the flagellar shedding mechanism recovered motility only very poorly . Similar results were obtained with other combinations of recombinant proteins and mutants . This study thus demonstrates the feasibility of using electroporation for activity assays of recombinant proteins .

J Pediatr Surg, 2002 Oct, 37(10), 1435 - 40
A study of gut immunity to enteral endotoxin in rats of different ages: a possible cause for necrotizing enterocolitis; Chan KL et al.; PURPOSE: Immature gut immunity can be a predisposing factor for necrotizing enterocolitis (NEC) . The gut active immunity and innate defense to the Escherichia coli endotoxin lipopolysaccharide (LPS) in immature and mature rats were studied . METHODS: LPS, started at a dose of 10 mg/kg, was instilled into the stomachs of fetal, newborn, 1-month and 3-month-old rats . Boost doses and normal saline control instillations were given on day 14 . Rats that died after instillation had detailed postmortem examinations . For survivors, a group of 6 immunized and 6 controls were killed on day 7 for the collection of serum, spleens, mesenteric lymph nodes, and small intestines . Lymphocytes (10(6)) prepared from each tissue sample of individual group were cultured for 5 days . Serum and supernatant were analyzed for IgA and anti-E coli IgA levels . RESULTS: All control rats survived . The doses of LPS given were 10, 5, 2.5, and 1.25 mg/kg . All fetal rats died after LPS instillation . Half-lethal dose for newborns was 2.5 mg/kg . One-month and 3-month-old rats survived all doses of LPS . The cause of death was endotoxemia . The serum IgA and total supernatant anti- E coli IgA levels of rats of all ages studied showed no significant difference . CONCLUSION: The poor innate gut defense, not so much the active immunity, may provide an explanation for the susceptibility of the premature babies and newborn infants to the development of NEC .

Cell Mol Biol Lett, 2002, 7(3), 877 - 83
Methylation of the arginine-glycine-rich region in the fragile X mental retardation protein FMRP differentially affects RNA binding; Denman RB; The C-terminal end of the fragile X mental retardation protein contains a stretch of amino acid residues that are enriched in arginine and glycine . Recent studies using recombinant FMRPs have demonstrated that this region participates in RNA binding in vitro, with calculated Kds ranging from 1-10 nM depending on the RNA . It is known that other arginine-glycine-rich proteins are subject to site-specific methylation by protein arginine methyltransferases (PRMTs) that are particularly abundant in most cells . We have demonstrated that the interaction of homoribopolymer mimetic RNAs with human FMRP (hFMRP) made in PRMT-containing cell-free lysates is more sensitive to increasing salt concentrations than recombinant hFMRP expressed in bacteria . We have also shown that blocking methylation with adenosine-2', 3'-dialdehyde (AdOx) alters homoribopolymer binding and hFMRP target mRNA binding; both increases and decreases are observed as a function of methylation . These data suggest that changes in PRMT activity that occur during development, or arise via signal transduction may be a means of regulating the binding of hFMRP to mRNA in vivo.

Pancreatology, 2002, 2(5), 463 - 8
Gastric colonisation, intestinal permeability and septic morbidity in acute pancreatitis; McNaught CE et al.; INTRODUCTION: Bacterial translocation (BT) may represent an important cause of septic morbidity in patients with acute pancreatitis . We have previously demonstrated an association between BT, septic morbidity and colonisation of the proximal GI tract . Alterations in intestinal permeability (IP) may also predispose to BT . The aim of this study was to assess the extent of gastric colonisation, measure IP in patients with acute pancreatitis and relate these to both disease severity and septic complications . METHODS: Gastric colonisation was determined by culturing a sample of nasogastric aspirate, and IP was measured using a dual sugar probe technique (lactulose/rhamnose test) . Disease severity was assessed according to the modified Glasgow (Imrie) criteria . All septic complications were recorded prospectively . RESULTS: A total of 59 patients were studied (M:F ratio 32:27, median age 66 years, range 18-89), 24 (31%) of whom had severe disease . A nasogastric aspirate was obtained in 56 patients . There was a significantly higher incidence of colonisation with potentially pathogenic enteric bacteria in patients with severe disease compared to those with mild disease (57 vs . 6%, p < 0.001) . Septic morbidity occurred in 29% of severe patients and 11% mild patients (p = 0.17) . 33% of patients colonised with enteric organisms developed sepsis, compared to 16% with no enteric bacteria in the NG aspirate (p = 0.34) . Enteric bacteria caused 77% of the septic complications . Intestinal permeability was neither associated with disease severity nor was it predictive of septic morbidity . CONCLUSIONS: There is significantly higher incidence of gastric colonisation with enteric bacteria in patients with severe acute pancreatitis, but no difference in IP . Enteric bacteria were implicated in the majority of septic complications . These findings support the gut origin of sepsis hypothesis in acute pancreatitis .

Gut, 2002 Nov, 51(5), 634 - 40
Safety and efficacy of low dose Escherichia coli enterotoxin adjuvant for urease based oral immunisation against Helicobacter pylori in healthy volunteers; Banerjee S et al.; BACKGROUND AND AIMS: Escherichia coli heat labile enterotoxin (LT) at doses of 5 micro g or 10 micro g has adjuvant activity for oral immunisation in humans infected with Helicobacter pylori, but causes severe diarrhoea . This study was undertaken to establish a safe and effective dose of LT, to confirm the safety of recombinant urease, and to compare the immunogenicity of orally compared with enterically delivered urease . METHODS: 42 healthy adults without present or past H pylori infection were randomised to receive 60 mg recombinant H pylori urease in soluble or in encapsulated form, given with doses of LT ranging from 0 micro g to 2.5 micro g . Four oral doses were administered at day 1, 8, 29, and 57 . Specific IgG, IgA, and antibody secreting cells were measured as well as total alpha4beta7 integrin positive lymphocyte responses . RESULTS: Enterically delivered urease was well tolerated and no serious adverse events occurred . Mild diarrhoea (one to four loose stools) occurred after the first immunisation in 50% (6 of 12) of the volunteers exposed to 2.5 micro g LT (p=0.06; paired t test, compared with baseline) but not in volunteers exposed to lower LT doses . Immune responses occurred in five (p=0.048; Fisher's exact test), one, two, and one of six subjects exposed to 2.5 micro g, 0.5 micro g, 0.1 micro g, and no LT, respectively . Significant CD4(+), CD69(+), and CD45RO(hi) responses occurred over time among alpha4beta7(hi) lymphocytes in volunteers receiving 2.5 micro g LT . Enterically delivered urease induced higher lymphocyte responses than soluble urease . CONCLUSIONS: The safety of H pylori urease is confirmed . Oral LT may conserve its adjuvant activity at low doses with minimal side effects.

J Biol Chem, 2002 Dec 20, 277(51), 49945 - 51 Epub 2002 Oct 10.
HypF, a carbamoyl phosphate-converting enzyme involved in {NiFe} hydrogenase maturation; Paschos A et al.; HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active {NiFe} hydrogenases in Escherichia coli and other bacteria . To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity . The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors . As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate . In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate . This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate . Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate . The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.

J Biol Chem, 2002 Dec 13, 277(50), 48248 - 60 Epub 2002 Oct 10.
Carboxyl-terminal sequences critical for inositol 1,4,5-trisphosphate receptor subunit assembly; Galvan DL et al.; The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of conserved subunits that each contains six transmembrane regions (TMRs) localized near the carboxyl terminus . Receptor subunit assembly into a tetramer appears to be a multideterminant process involving an additive contribution of membrane spanning helices and the short cytosolic carboxyl terminus (residues 2590-2749) . Previous studies have shown that of the six membrane-spanning regions in each subunit, the 5th and 6th transmembrane regions, and the carboxyl terminus are strong determinants for assembly . The fifth and sixth TMRs contain numerous beta-branched amino acids that may participate in coiled/coil formation via putative leucine zipper motifs . InsP(3)R truncation mutants were expressed in COS-1 cells and analyzed by sucrose density gradient sedimentation and gel filtration for their ability to assemble . Chemical cross-linking with the homobifunctional reagents sDST or DMS of mammalian and bacterially expressed carboxyl-terminal containing receptor fragments reveals that sequences within the carboxyl terminus confer the formation of subunit dimers . A series of InsP(3) receptor carboxyl-terminal fragments and glutathione S-transferase (GST)/InsP(3)R chimeras were expressed in Escherichia coli and used in an in vitro assay to elucidate the minimal sequence responsible for association of the carboxyl termini into dimers . The results presented here indicate that this minimal sequence is approximately 30 residues in length and is localized between residues 2629 and 2654 . These residues are highly conserved between the three InsP(3)R isoforms ( approximately 80% identity) as well as the ryanodine receptor ( approximately 40% identity) and suggest that a conserved assembly motif may exist between the two intracellular receptor families . We propose that assembly of the InsP(3) receptor to a tetramer involves intersubunit interactions mediated through both the membrane-spanning regions and residues 2629-2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.

J Biol Chem, 2002 Dec 27, 277(52), 50621 - 8 Epub 2002 Oct 10.
GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle; Miyazaki T et al.; GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C . This state of arrest could be reversed with a simple increase in temperature . In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese . These complexes could be reactivated by a temperature shift to obtain active refolded protein . The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain . Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release . The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.

J Biol Chem, 2002 Dec 20, 277(51), 49422 - 7 Epub 2002 Oct 10.
Proteinaceous infectious behavior in non-pathogenic proteins is controlled by molecular chaperones; Ben-Zvi AP et al.; External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms . Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress . Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions . Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates . Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.

J Biol Chem, 2002 Dec 20, 277(51), 49282 - 6 Epub 2002 Oct 10.
Activation of the proton transfer pathway in catalysis by iron superoxide dismutase; Greenleaf WB et al.; Catalysis by Escherichia coli and Porphyromonas gingivalis iron superoxide dismutase was activated by addition of primary amines, as measured by pulse radiolysis and stopped-flow spectrophotometry . This activation was saturable for most amines investigated, and a free energy plot of the apparent second-order rate constant of activation was linear as a function of the pK(a) of the amine, indicating activation by proton transfer . Amines provide an alternate rather than the only pathway for proton transfer, and catalysis was appreciable in the absence of amines . Solvent hydrogen isotope effects were near unity for amine activation, which is consistent with rate-contributing proton transfer if the pK(a) of the proton acceptor on the enzyme is not in the region of the pK(a) values of the amines studied, from 7.8 to 10.6 . The activation of catalysis by these amines was uncompetitive with respect to superoxide, interpreted as proton transfer in a ternary complex of amine with the enzyme-bound peroxide dianion.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Oct, 22(10), 883 - 7
Expression and immunocompetence identification of Plasmodium falciparum glutamate dehydrogenase; Li LH et al.; OBJECTIVE: To express the fusion protein of glutamate dehydrogenase (GDH) with glutathione S-transferase (GST) of Plasmodium falciparum FCC1/HN in E . coli BL21 and assess the immunocompetence of the recombinant protein . METHODS: GDH gene of P . falciparum was specifically amplified with PCR, followed by double enzyme digestion and cloning the gene fragment into pGEX-4T-1 vector for the expression of the fusion protein GST . The recombinant plasmid was transformed into E . coli BL21 . Four mice (Kunming strain) were immunized with purified recombinant protein (antigen) and the polyclonal antibodies produced in response to the treatment were collected . Enzyme-linked immunosorbent assay and Western blotting were carried out to examine the immunocompetence of the recombinant protein . RESULTS: The fusion protein was successfully expressed, which exhibited specific reaction with the sera obtained from mice immunized with P . falciparum . Specific humoral responses were elicited after introducing the fusion protein in mice and the specific antibody titer was 1:16 in agar diffusion assay . CONCLUSION: GDH of P . falciparum may have successful expression in E . coli BL21 and the expressed protein possesses high antigenicity.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Oct, 22(10), 869 - 71
Conservative region of the genes encoding four adhesins of Helicobacter pylori: cloning, sequence analysis and biological information analysis; Bai Y et al.; OBJECTIVE: To clone the conserved regions of the genes encoding the 4 adhesins (BabA, AlpA, AlpB and HopZ) of Helicobacter pylori (H . pylori) and analyze their sequences and biological information, thus facilitating further research in the molecular mechanism and immunogenicity of H . pylori adhesins . METHODS: Common conserved region (designated as CB) was identified from the confirmed sequences (by ANTHEPROT V4.3c software package) of the 4 adhesin proteins . Their DNA sequences were deduced, according to which primers specific to CB were designed for subsequent PCR, and the products were inserted directionally into pET-22b(+) vector to construct recombinant clones of the conserved region . The DNA sequences were determined with the basic local alignment sequence tool (BLAST) and the biological properties analyzed with ANTHEPROT V4.3c software package . RESULTS: The recombinant plasmid containing the CB sequence was constructed . DNA sequencing showed an open reading frame of 588 bp in length, encoding 195 amino acids . The homogencity of conservative region of the 4 adhesion genes was above 50% . The corresponding protein possessed a relative molecular mass (Mr) of 22 500 as predicted by ANTHEPROT V4.3c software prediction, with excellent antigenicity and hydrophobicity . There were 836 767 sequences analyzed with BLAST, in which those with homogencity of 40% with the identified CB sequence were categorized into H . pylori sequences . CONCLUSION: There are conservative regions in the 4 adhesin genes with similar homogencity, suggesting similar molecular basis for adhesion of the adhesins . Biological information analysis indicates that CB has excellent immunogenicity and strict species specificity.

Dev Comp Immunol, 2002 Nov, 26(9), 797 - 804
Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement; Nikoskelainen S et al.; The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 degrees C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF . At 35 degrees C there was no difference between the total (TC) activity and the activity of the AP, but at 10 degrees C the TC was notably higher than the AP . Total activity peaked at 30 degrees C and gradually grew smaller towards 0 degrees C . The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 degrees C . When compared to human serum complement, the peak human TC activity at 37 degrees C was four times higher than the TC of rainbow trout at 30 degrees C . Human TC activity was 10.1-fold lower at 25 degrees C when compared to the activity at 37 degrees C . At 37 degrees C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 degrees C there was no difference between human TC and AP . In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 degrees C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.

Structure (Camb), 2002 Oct, 10(10), 1303 - 15
The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot; Michel G et al.; In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA . The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution . RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution . The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features . The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition . The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.

Pediatr Nephrol, 2002 Oct, 17(10), 852 - 5 Epub 2002 Sep 14.
Role of non-polio enterovirus infection in pediatric hemolytic uremic syndrome; De Petris L et al.; Verocytotoxin-producing Escherichia coli(VTEC) infections cause most cases of hemolytic uremic syndrome (HUS); 10-30% of patients, however, are negative for VTEC infection . The etiology of HUS in VTEC-negative cases remains poorly understood . Before the association between VTEC infection and HUS was recognized, sporadic cases of HUS with enterovirus infection were reported in the literature . Since May 1988, most cases of HUS in Italy have been reported to the Italian surveillance system, and in 73% of these, evidence of VTEC infection was demonstrated . The aim of this study was to determine whether the frequency of enteroviral infections was different in the acute phase of VTEC-positive and VTEC-negative HUS . Eighty-nine patients were investigated for enteroviral infection, of whom 58 were VTEC positive and 31 VTEC negative . Two serum samples from each patient were examined for seroconversion to enterovirus (coxsackie, echovirus, and picornavirus) by a complement fixation test . Serological evidence of acute infection with non-polio enterovirus was found in 33 patients (37%) {20/58 (34.5%) VTEC positive and 13/31 (41.9%) VTEC negative} . There was no statistically significant difference between the two groups . These results demonstrate that there are no significant differences for enteroviral infection in VTEC-positive and VTEC-negative patients and, therefore, enteroviral infections should not be considered a cause of HUS in VTEC-negative children.

Plant Physiol, 2002 Oct, 130(2), 857 - 64
Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis; Chassin Y et al.; The Arabidopsis genome contains 14 genes encoding the serine protease DegP . Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane . We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically . Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers . Proteolytic activity was characterized using beta-casein as a model substrate . DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures . DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen . The results of this work are discussed in the context of the recent elucidation of the structure of the E . coli homolog and the possible physiological role of the protease in the chloroplast lumen.

Plant Physiol, 2002 Oct, 130(2), 847 - 56
An essential role of s-adenosyl-L-methionine:L-methionine s-methyltransferase in selenium volatilization by plants . Methylation of selenomethionine to selenium-methyl-L-selenium- methionine, the precursor of volatile selenium; Tagmount A et al.; Selenium (Se) phytovolatilization, the process by which plants metabolize various inorganic or organic species of Se (e.g . selenate, selenite, and Se-methionine {Met}) into gaseous Se forms (e.g . dimethylselenide), is a potentially important means of removing Se from contaminated environments . Before attempting to genetically enhance the efficiency of Se phytovolatilization, it is essential to elucidate the enzymatic pathway involved and to identify its rate-limiting steps . The present research tested the hypothesis that S-adenosyl-L-Met:L-Met S-methyltransferase (MMT) is the enzyme responsible for the methylation of Se-Met to Se-methyl Se-Met (SeMM) . To this end, we identified and characterized an Arabidopsis T-DNA mutant knockout for MMT . The lack of MMT in the Arabidopsis T-DNA mutant plant resulted in an almost complete loss in its capacity for Se volatilization . Using chemical complementation with SeMM, the presumed enzymatic product of MMT, we restored the capacity of the MMT mutant to produce volatile Se . Overexpressing MMT from Arabidopsis in Escherichia coli, which is not known to have MMT activity, produced up to 10 times more volatile Se than the untransformed strain when both were supplied with Se-Met . Thus, our results provide in vivo evidence that MMT is the key enzyme catalyzing the methylation of Se-Met to SeMM.

Plant Physiol, 2002 Oct, 130(2), 784 - 95
Inventory and functional characterization of the HAK potassium transporters of rice; Banuelos MA et al.; Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions . Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters . To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare) . By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes . Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions . The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment . A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake . We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane . Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

J Biol Chem, 2002 Dec 20, 277(51), 49256 - 66 Epub 2002 Oct 09.
Modulation of induction properties of glucocorticoid receptor-agonist and -antagonist complexes by coactivators involves binding to receptors but is independent of ability of coactivators to augment transactivation; He Y et al.; Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes . These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors . We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity . Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities . DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities . The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved . The ability of a TIF2.4 fragment (i.e . TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2 . Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids . These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes . In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).

Carcinogenesis, 2002 Oct, 23(10), 1751 - 7
Mutations induced by alpha-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats; Chen T et al.; The antiestrogen tamoxifen is widely used for the treatment of breast cancer and more recently for the prevention of breast cancer . A concern over the use of tamoxifen as a chemopreventive agent is its carcinogenicity in rat liver, through a genotoxic mechanism involving alpha-hydroxylation, esterification, and DNA adduct formation, primarily by reaction with dG . In a recent study {Gamboa da Costa et al., Cancer Lett., 176, 37-45 (2002)}, we demonstrated a significant increase in the mutant frequency in the lacI gene of Big Blue rats treated with tamoxifen, and a further increase in rats administered alpha-hydroxytamoxifen . In the present study, we have assessed mutation induction by tamoxifen and alpha-hydroxytamoxifen in the liver cII gene of Big Blue rats and have characterized the types of mutations induced by alpha-hydroxytamoxifen in the liver lacI and cII genes . The mutant frequencies in the liver cII gene were 80 +/- 13 x 10(-6) in the control, 112 +/- 13 x 10(-6) in the tamoxifen-treated group (P < 0.01 vs . control), and 942 +/- 114 x 10(-6) in the alpha-hydroxytamoxifen-treated animals (P < 0.001 vs . control; P < 0.001 vs . tamoxifen) . Molecular analysis of the mutants indicated that the alpha-hydroxytamoxifen-induced mutational spectrum differed significantly from the control spectrum, but was very similar to the spectrum induced by tamoxifen for both the lacI and cII genes {Davies et al., ENVIRON: Mol . Mutagen., 28, 430-433 (1996); Davies et al., Carcinogenesis, 20, 1351-1356 (1999)} . G:C --> T:A transversion was the major type of mutation induced by alpha-hydroxytamoxifen and tamoxifen, while G:C --> A:T transition was the main type of mutation in the control . These results support the hypothesis that alpha-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats treated with tamoxifen.

Bioinformatics, 2002 Oct, 18(10), 1374 - 81
Finding motifs in the twilight zone; Keich U et al.; MOTIVATION: Gene activity is often affected by binding transcription factors to short fragments in DNA sequences called motifs . Identification of subtle regulatory motifs in a DNA sequence is a difficult pattern recognition problem . In this paper we design a new motif finding algorithm that can detect very subtle motifs . RESULTS: We introduce the notion of a multiprofile and use it for finding subtle motifs in DNA sequences . Multiprofiles generalize the notion of a profile and allow one to detect subtle patterns that escape detection by the standard profiles . Our MULTIPROFILER algorithm outperforms other leading motif finding algorithms in a number of synthetic models . Moreover, it can be shown that in some previously studied motif models, MULTIPROFILER is capable of pushing the performance envelope to its theoretical limits . AVAILABILITY: http://www-cse.ucsd.edu/groups/bioinformatics/software.html

Am J Physiol Lung Cell Mol Physiol, 2002 Nov, 283(5), L952 - 62
Neutrophils play a critical role in development of LPS-induced airway disease; Savov JD et al.; We investigated the role of neutrophils in the development of endotoxin-induced airway disease via systemic neutrophil depletion of C3H/HeBFeJ mice and coincident inhalation challenge with lipopolysaccharide (LPS) over a 4-wk period . Mice were made neutropenic with intraperitoneal injections of neutrophil antiserum before and throughout the exposure period . Experimental conditions included LPS-exposed, antiserum-treated; LPS-exposed, control serum-treated; air-exposed, antiserum-treated; and air-exposed, control serum-treated groups . Physiological, biological, and morphological assessments were performed after a 4-wk exposure and again after a 4-wk recovery period . After the 4-wk exposure, LPS-induced inflammation of the lower airways was significantly attenuated in the neutropenic mice, although airway responsiveness (AR) to methacholine (MCh) remained unchanged . After the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals . Morphometric data indicate that the 4-wk exposure to LPS leads to a substantial expansion of the subepithelial area of the medium-sized airways (90-129 microm diameter) in nonneutropenic mice but not neutropenic mice, and this difference persisted even after the recovery period . Expression of bronchial epithelial and subepithelial transforming growth factor-beta1 (TGF-beta1) was diminished in the challenged neutropenic mice compared with the neutrophil-sufficient mice . These studies demonstrate that neutrophils play a critical role in the development of chronic LPS-induced airway disease.

Prep Biochem Biotechnol, 2002 Aug, 32(3), 239 - 51
Construction and expression of a reshaped VH domain against human CD28 molecules; Cheng J et al.; Compared with the amino acid sequence of a mouse anti-human CD28 VH domain antibody, the two most homologous sequences of human antibodies were pulled out from Genbank . One of them was used as the main template for the framework regions of the reshaped VH domain . While the original mouse antibody CDRs were inserted into the human acceptor FRs, some residues in human acceptor FRs, which were different from those of the original mouse FRs in corresponding positions, were then determined or, alternatively, mutagenized to their conservative properties in kappa classification . Based on the amino acid sequences of the designed VH domain, the nucleotide sequence was deduced by using E . coli bias codons . The sequence was split into ten 30 to 60 nucleotide fragments for synthesizing, then annealed and amplified by overlap PCR . Taq DNA polymerase was used in a buffer with high Mg2+ concentration to induce more random mutations, both in FRs and CDRs . A phage display library was constructed by cloning these PCR products . After three rounds of panning, several reshaped VH with high antigen binding activity were obtained . One of them had the same CDR amino acid sequences as that of the original mouse VH domain . Further study showed that it retained a high antigen binding affinity after being expressed in E . coli BL21 (DE3).

Arch Microbiol, 2002 Nov, 178(5), 358 - 69 Epub 2002 Aug 22.
A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly; Cook GM et al.; cydDC genes encode a heterodimeric ABC transporter required for assembly of the membrane-bound cytochrome bd quinol oxidase and periplasmic cytochromes . Here, we demonstrate that overexpression of functional cydDC genes on a multicopy plasmid results in elevated levels of cytochromes b and d, but most notably formation in anaerobically grown cells of a novel haem-containing component P-574 . The pigment has a distinctive absorbance at 574-579 nm and 448 nm in reduced minus oxidised spectra and renders over-producing cells reddish in colour . The highest levels of P-574 were observed in mutants (cydAB) in the structural genes for the polypeptides of cytochrome bd . P-574 is labile; its spectral signal is reduced in cells that are frozen-thawed or subjected to mechanical disruption . P-574 was not detected in cytoplasmic or periplasmic fractions and was predominantly associated with the cell membrane . P-574 did not bind CO or cyanide . Production of P-574 was dependent on haem biosynthesis indicating that it is a haem-containing molecule or derived from haem biosynthesis . These findings suggest that P-574 may result from association of a haem compound with overexpressed transporter subunits, but not with oxidase subunits, and are consistent with an intimate link between the transporter and haem processing during oxidase assembly.

Arch Microbiol, 2002 Nov, 178(5), 331 - 7 Epub 2002 Aug 15.
Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes; Koledin T et al.; Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria . The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis . In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins) . Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant . Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M . smegmatis genome, as well as the orthologous gene Rv0819 in the M . tuberculosis genome . Cloning and expression of mshD from M . tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.

Microb Ecol, 2002 Nov, 44(4), 354 - 64 Epub 2002 Oct 14.
Participation of oxygen and role of exogenous and endogenous sensitizers in the photoinactivation of Escherichia coli by photosynthetically active radiation, UV-A and UV-B; Muela A et al.; We studied the mechanisms by which photosynthetically active radiation (PAR) and ultraviolet (UV-A and UV-B) radiation damage Escherichia coli suspended in water . The roles played by oxygen and exogenous and endogenous sensitizers were analyzed by monitoring changes in the physiological state of irradiated cells . Impairment of the cellular functions was more severe in the case of UV radiations . Radiation caused cellular damage in the absence of oxygen . PAR, UV-A, and UV-B radiation induced photobiological and photodynamic reactions mediated by endogenous sensitizers, which significantly shortened the T90 (time needed to reduce a cellular parameter by 90%) based on the growth ability of the cells . In addition, when exogenous sensitizers were present, the photodynamic reactions also had a negative effect on the operation of the electron transport chains . The presence of oxygen might enhance photoinactivation, affecting both the growth ability and the electron transport chains . Endogenous sensitizers were responsible for the noxious action of oxygen . The presence of dissolved organic material played a protective role against the oxygen by absorbing the incident radiation, thereby reducing the energy that reached the endogenous sensitizers.

Nature, 2002 Oct 10, 419(6907), 638 - 41
Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase; Ha T et al.; Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases . Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated . Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase . Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding . DNA unwinding initiates only if a functional helicase is formed via additional protein binding . Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase . These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex . We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13471 - 6 Epub 2002 Oct 08.
Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis; Tani TH et al.; Bacterial adaptation to nutrient limitation and increased population densities is central to survival and virulence . Surprisingly, <3% of Escherichia coli genes are known to play roles specific to the stationary phase . There is evidence that the leucine-responsive regulatory protein (Lrp) may play an important role in stationary phase, so this study used microarrays representing >98% of E . coli genes to more comprehensively identify those controlled by Lrp . The primary analysis compared isogenic Lrp(+) and Lrp(-) strains in cells growing in steady state in glucose minimal medium, either in the presence or absence of leucine . More than 400 genes were significantly Lrp-responsive under the conditions used . Transcription of 147 genes was lower in Lrp(+) than in Lrp(-) cells whether or not leucine was present; most of these genes were tightly coregulated under several conditions, including a burst of synthesis on transition to stationary phase . This cluster includes 56 of 115 genes already known to play roles in stationary phase . Our results suggest that the actual number of genes induced on entrance into stationary phase is closer to 200 and that Lrp affects nearly three-quarters of them, including genes involved in response to nutrient limitation, high concentrations of organic acids, and osmotic stress.

J Bacteriol, 2002 Nov, 184(21), 6065 - 8
The gene yjfQ encodes the repressor of the yjfR-X regulon (ula), which is involved in L-ascorbate metabolism in Escherichia coli; Campos E et al.; Mutations in yjfQ allowed us to identify this gene as the regulator of the operon yjfS-X (ula operon), reported to be involved in L-ascorbate metabolism . Inactivation of this gene renders constitutive the expression of the ula operon, indicating that YjfQ acts as a repressor . We also demonstrate that this repressor regulates the nearby yjfR gene, which in this way constitutes a regulon with the ula operon.

J Bacteriol, 2002 Nov, 184(21), 5999 - 6006
L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO(2) fixation; Herter S et al.; The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea) . The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate . In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate . The putative L-malyl-CoA lyase gene of C . aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied . Beta-methylmalyl-CoA lyase was purified from cell extracts of C . aurantiacus and characterized . We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase . Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction) . The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway . In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.

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