Escherichia coli

Infez Med, 2002 Dec, 10(4), 191 - 203
{The gut associated lymphoid tissue, the oral vaccines and the E . coli vaccines: a review}; Aiuti F et al.; The aim of this work was a review of literature with regard to the mucosal immunity, the oral vaccines and the bacterial lysates . The Gut Associated Lymphoid Tissue (GALT) include effector and inductive sites and is constituted by organized and diffuse tissues . GALT defends the integrity of the gut, inhibits the development of allergy and autoimmunity and induce a mucosal and systemic immune response against enteric antigens . Bacterial lysates are innocuous and can reduce the frequency and the seriousness of diarrhoea, mucosal infections and diverticulitis; they induce the production and the biologic activity of secretory IgA and cytokines . The DNA vaccines are able to induce a strong immune response; the oral vaccines formulated with bacterial adhesins can inhibit the entry of pathogens and oral antigens across the gut . The use of adjuvants can amplify the activity of the oral vaccines; an objective of the research is the discovery of potent adjuvants without remarkable toxicity.

Di Yi Jun Yi Da Xue Xue Bao, 2003 May, 23(5), 491 - 3
{Preparation of egg yolk immunoglobulin (IgY) against p53 protein expressed in E.coli}; Wu M et al.; OBJECTIVE: To isolate immunoglobulin (IgY) from egg yolk of p53 protein-immunized hens and to study its reactivity to the antigens . METHOD: Immunization of egg-laying hens was performed for 3 times at the interval of 14 days with purified p53 protein that had been expressed in E.coli . The eggs laid by these immunized hens were then collected during the whole period of the experiment to prepare IgY from egg yolk by ammonium sulfate precipitation . Final purification and identification of the IgY were performed using SDS-PAGE, enzyme-linked immunosorbent assay and Western blotting . RESULT: All immunized hens developed specific antibodies to p53 protein in contrast to the control ones . The highest titers of the IgY occurred 4 weeks after the first immunization and reached 1:10(6), which remained stable for up to 3 months . CONCLUSION: IgY technology is less costly, non-invasive, fast, simple and highly efficient to generate polyclonal antibodies.

Planta, 2003 Sep, 217(5), 813 - 9 Epub 2003 May 13.
A 10-kDa class-CI sHsp protects E . coli from oxidative and high-temperature stress; Jofre A et al.; We report on a new cDNA clone (Qshsp10.4-CI) of a Quercus suber L . class-CI small heat-shock protein (sHsp) obtained from cork (phellem), a highly oxidatively stressed plant tissue . The deduced gene product lacks the C-terminal extension and the consensus I region of the alpha-crystallin domain, being the most C-terminally truncated sHsp reported to date . In an attempt to prove that a protective function is possible for such a truncated sHsp, we overexpressed in Escherichia coli three recombinant sHsp-CIs, one (rQsHsp10.4-CI) showing the same truncation as Qshsp10.4-CI, a second (rN49) lacking the whole alpha-crystallin domain, and a third (rN153) consisting of a full-length sHsp-CI . The overexpression of rN153 and, remarkably, rQsHsp10.4-CI but not rN49 enhanced cell viability under high temperature and, interestingly, under oxidative stress . These results show that the C-terminal extension and the consensus I region of the alpha-crystallin domain are dispensable, but amino acids 1-41 of the alpha-crystallin domain (including the consensus II region) are essential for the protective activity of sHsp-CIs . On the other hand, two-dimensional immunodetection patterns showed accumulation of ca . 10-kDa sHsp-CI immunorelated polypeptides in cork and other oxidatively stressed tissues but not in control and heat-stressed tissues . We discuss the possible role of highly truncated sHsps in relation to oxidative stress.

J Am Chem Soc, 2003 May 14, 125(19), 5964 - 72
Comparison of formation of reactive conformers (NACs) for the Claisen rearrangement of chorismate to prephenate in water and in the E . coli mutase: the efficiency of the enzyme catalysis; Hur S et al.; The Claisen rearrangements of chorismate (CHOR) in water and at the active site of E . coli chorismate mutase (EcCM) have been compared . From a total of 33 ns molecular dynamics simulation of chorismate in water solvent, seven diaxial conformers I-VII were identified . Most of the time (approximately 99%), the side chain carboxylate of the chorismate is positioned away from the ring due to the electrostatic repulsion from the carboxylate in the ring . Proximity of the two carboxylates, as seen in conformer I, is a requirement for the formation of a near attack conformer (NAC) that can proceed to the transition state (TS) . In the EcCM.CHOR complex, the two carboxylates of CHOR are tightly held by Arg28 of one subunit and Arg11* of the other subunit, resulting in the side chain C16 being positioned adjacent to C5 with their motions restricted by van der Waals contacts with methyl groups of Val35 and Ile81 . With the definition of NAC as the C5...C16 distance < or =3.7 A and the attack angle < or =30 degrees, it was estimated from our MD trajectories that the free energy of NAC formation is approximately 8.4 kcal/mol above the total ground state in water, whereas in the enzyme it is only 0.6 kcal/mol above the average of the Michaelis complex EcCM.CHOR . The experimentally measured difference in the activation free energies of the water and enzymatic reactions (Delta Delta G(++)) is 9 kcal/mol . It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared to the water reaction . Comparison of the EcCM.TSA (transition state analogue) and EcCM.NAC simulations suggests that the experimentally measured 100 fold tighter binding of TSA compared to CHOR does not originate from the difference between NAC and the TS binding affinities, but might be due to the free energy cost to bring the two carboxylates of CHOR together to interact with Arg28 and Arg11* at the active site . The two carboxylates of TSA are fixed by a bicyclic structure . The remaining approximately 10% of Delta Delta G(++) may be attributed to a preferential interaction of Lys39-NH(3)(+) with O13 ether oxygen in the TS.

Toxicon, 2003 May, 41(6), 713 - 21
Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E . coli; Yang WL et al.; Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study . In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E . coli . The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX . The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C . The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase . The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained . The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme . As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect . Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.

Environ Mol Mutagen, 2003, 41(4), 237 - 42
E . coli BW535, a triple mutant for the DNA repair genes xth, nth, and nfo, chronically induces the SOS response; Janion C et al.; A strong chronic induction of the SOS response system occurs in E . coli BW535, a strain defective in nth, nfo and xth genes, and hence severely deficient in the repair of abasic sites in DNA . This was shown here by visualization of filamentous growth of the BW535 strain and by measuring the level of beta-galactosidase expressed in BW535/pSK1002 in comparison to the AB1157/pSK1002 strain . The plasmid pSK1002 bears an umuC::lacZ fusion in which lacZ is under the control of the umuC promoter and regulated under the SOS regulon . Increases in the expression of beta-galactosidase occur in BW535 without any exogenous SOS inducer . Chronic induction of the SOS response was observed previously in E . coli strains bearing mutations in certain genes that have mutator activity and BW535 is a moderate mutator strain . However, not all mutators show this property, since chronic induction of SOS was not observed in mutT or mutY mutators . MutT and MutY proteins, when active, protect bacteria from mutations induced by 8-oxoG lesions in DNA . This suggests that accumulation of abasic sites, but not 8-oxoG residues in DNA, induce the SOS response .

DNA Repair (Amst), 2003 May 13, 2(5), 471 - 82
The expression of Exonuclease III from E . coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress; Shokolenko IN et al.; The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment . One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA) . The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage . Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells . Successful repair of DNA is achieved through a precise and highly regulated multistep process . Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency . To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231 . Following transfection, specific exonuclease activity was found in mitochondrial extracts . In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine . mtDNA repair was evaluated using quantitative Southern blot analysis . The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS . This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.

J Photochem Photobiol B, 2003 Mar, 69(3), 161 - 7
Irradiance dependence of the He-Ne laser-induced protection against UVC radiation in E . coli strains; Kohli R et al.; He-Ne laser pre-irradiation-induced protection against UVC damage was investigated in wild-type E . coli K12 strain AB1157 and its isogenic DNA repair mutant strains . At a dose of 7 kJ/m(2), pre-irradiation was observed to induce protection in recA proficient strains (AB1157 and uvrA(-) AB1886) at both the irradiances investigated (2 and 100 W/m(2)) . However, at the same dose (7 kJ/m(2)), while no protection was observed at 100 W/m(2) in the recA(-) strain, some protection appeared to be there at 2 W/m(2) . Mechanistic studies carried out on these strains at the two irradiances suggest that, whereas the protection observed at 100 W/m(2) is mediated by singlet oxygen, that observed at 2 W/m(2) is not . Further, the fact that protection at 100 W/m(2) was observed only in recA proficient strains suggests that it may arise due to the induction of DNA repair processes controlled by the recA gene . The latter may arise due to the oxidative stress produced by singlet oxygen generated by He-Ne laser irradiation . In contrast, the protection observed at 2 W/m(2) appears to be independent of the DNA repair proficiency of the strain.

Biochemistry (Mosc), 2003 Jan, 68(1), 86 - 98
Molecular cloning and heterologous expression in E . coli of cytochrome P45017alpha . Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species; Gilep AA et al.; To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C(19)-steroids . Recombinant cytochromes P45017alpha were expressed in E . coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography . Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC . It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture . The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone.

Luminescence, 2003 Mar-Apr, 18(2), 107 - 12
Simple and rapid bioluminescent detection of two verotoxin genes using allele-specific PCR of E . coli O157: H7; Imamura O et al.; Allele-specific PCR for E . coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2) . VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide . We developed a simple, rapid bioluminescent detection method for E . coli O157 . The method is based on the determination of pyrophosphoric acid (PPi) released during allele-specific PCR . Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction . As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method . This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly . Additionally, this methodology can be used as a high-throughput approach for measuring PCR products .

J Bioenerg Biomembr, 2002 Dec, 34(6), 423 - 31
Expression and characterization of recombinant human cytochrome c in E . coli; Jeng WY et al.; Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress . Here we expressed human cytochrome c in E . coli and purified it to homogeneity with a yield of 10-15 mg/L . The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry . This is similar to that of horse cytochrome c with a value of 0.249 V . The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy . On the basis of our NMR studies, the recombinant human cytochrome c produced in E . coli exhibits the same tertiary fold as horse cytochrome c . These results provide evidence that human cytochrome c expressed in E . coli possesses a similar function and structure to that of the horse protein . It is known that cytochrome c plays a role in many human diseases . This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.

Cell Microbiol, 2003 Apr, 5(4), 245 - 52
The K1 capsule modulates trafficking of E . coli-containing vacuoles and enhances intracellular bacterial survival in human brain microvascular endothelial cells; Kim KJ et al.; Escherichia coli K1 has been shown to invade human brain microvascular endothelial cells (HBMEC) in vitro and translocate the blood-brain barrier in vivo, but it is unclear how E . coli K1 traverses HBMEC . We have previously shown that internalized E . coli K1 is localized within membrane-bound vacuole in HBMEC . The present study was carried out to understand intracellular trafficking of E . coli K1 containing vacuoles (ECVs) in HBMEC . ECVs initially acquired two early endosomal marker proteins, EEA1 and transferrin receptor . Rab7 and Lamp-1, markers for late endosome and late endosome/lysosome, respectively, were subsequently recruited on the ECVs, which was confirmed with flow cytometry analysis of ECVs . However, ECVs did not obtain cathepsin D, a lysosomal enzyme, even after 120 min incubation, suggesting that E . coli K1 avoids lysosomal fusion . In contrast, isogenic K1 capsule-deletion mutant obtained early and late endosomal markers on vacuolar membranes and allowed lysosomal fusion with subsequent degradation inside vacuoles . This observation was consistent with the decreased intracellular survival of K1 capsule-deletion mutant, even though the binding and internalization rates of the mutant were higher than those of the parent E . coli K1 strain . This is the first demonstration that E . coli K1, via the K1 capsule on the bacterial surface, modulates the maturation process of ECVs and prevents fusion with lysosomes, which is an event necessary for traversal of the blood-brain barrier as live bacteria.

Biotechniques, 2003 Mar, 34(3), 524 - 6, 528, 530
Increasing the yield of soluble recombinant protein expressed in E . coli by induction during late log phase; Galloway CA et al.; Recombinant mammalian proteins expressed in E . coli can be difficult to purify in high yield in a soluble and functional form . Various techniques have been described to prevent proteolysis of expressed proteins and/or their sequestering as insoluble aggregates within inclusion bodies . We report conditions for expressing recombinant proteins from E . coli that significantly enhanced the yield of soluble and functional protein . We demonstrate high-yield recovery of a native, high-molecular-weight RNA binding protein without the aid of fusion protein sequence . The principle factor that increased protein yield was the induction of protein expression in a late log phase culture, although reduced temperature during the induction and a low IPTG concentration also contributed to a higher yield.

J Anim Sci, 2003 Feb, 81(2), 474 - 83
Efficacy of an E . coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs; Augspurger NI et al.; Four chick trials and one pig trial were conducted to investigate the phosphorus-releasing efficacy oftwo commercial phytase enzymes (Natuphos and Ronozyme) and an experimental E . coli phytase enzyme (ECP) when added to corn-soybean meal diets containing no supplemental inorganic P (iP) . In the 13- or 14-d chick trials, three or four graded levels of iP (0, 0.05,0.10,0.15%) from KH2PO4 were added to the basal diet to construct standard curves from which bioavailable P release could be calculated for the phytase treatments . In all cases, phytase supplementation levels were based on an assessment of phytase premix activity (i.e., P release from Na phytate at pH 5.5) . Linear (P < 0.01) responses in tibia ash and weight gain resulted from iP supplementation in all assays . In the first chick trial, supplementation of 500 phytase units (FTU)/kg of ECP resulted in superior (P < 0.01) weight gain and tibia ash values compared with 500 FTU/kg of Natuphos . Results of the second chick trial revealed P-release values of 0.032 and 0.028% for 500 FTU/kg Natuphos and Ronozyme, respectively, and these were lower (P < 0.01) than the 0.125% P-release value for 500 FTU/kg of ECP . Tibia ash responded quadratically (P < 0.05) in response to graded levels of ECP up to 1,500 FTU/kg in the third chick trial . Combining Natuphos with either Ronozyme or ECP in Chick Trial 4 revealed no synergism between phytases with different initiation sites of P removal . The pig trial involved 10 individually fed weanling pigs per diet, and and phytase enzymes were supplemented to provide 400 FTU/kg in diets containing 0.60% Ca . Based on the linear regression of fibula ash on supplemental iP intake (r2 = 0.87), P-release values were 0.081% for Natuphos, 0.043% for Ronozyme, and 0.108% for ECP . These trials revealed an advantage of the E . coli phytase over the commercial phytases in young chicks.

Aliment Pharmacol Ther, 2003 Mar 1, 17(5), 695 - 701
Role of immunosuppression in the development of quinolone-resistant Escherichia coli spontaneous bacterial peritonitis and in the mortality of E . coli spontaneous bacterial peritonitis; Cereto F et al.; BACKGROUND: Norfloxacin decreases the incidence of spontaneous bacterial peritonitis in cirrhotics, but promotes the appearance of quinolone-resistant Escherichia coli . AIM: : To define the characteristics of quinolone-resistant E . coli spontaneous bacterial peritonitis . METHODS: E . coli-positive ascitic fluid cultures were identified during a 6-year period . Data on quinolone-sensitive and quinolone-resistant E . coli spontaneous bacterial peritonitis were compared . RESULTS: One hundred and two E . coli-positive ascitic fluid cultures were detected . Cirrhotics accounted for 67 cases . Spontaneous bacterial peritonitis was found in 47 of the 67 (70%) cases {35 (74%) caused by quinolone-sensitive and 12 (26%) caused by quinolone-resistant E . coli} . Norfloxacin prophylaxis was higher in the quinolone-resistant group (92% vs . 6%, P < 0.001) . Compared with patients with quinolone-sensitive E . coli spontaneous bacterial peritonitis, those with quinolone-resistant E . coli spontaneous bacterial peritonitis showed a higher prevalence of associated immunosuppressive factors (immunosuppressive drugs, human immunodeficiency virus infection or cancer) (92% vs . 20%, P < 0.001) . Steroid therapy was independently associated with quinolone-resistant E . coli spontaneous bacterial peritonitis (odds ratio, 49; 95% confidence interval, 3.4-699; P = 0.004) . The Child-Pugh score (P = 0.03), immunosuppression (P = 0.02) and renal failure (P = 0.01) were independent predictors of E . coli spontaneous bacterial peritonitis-related mortality . CONCLUSIONS: Associated immunosuppression is an important co-factor for the development of quinolone-resistant E . coli spontaneous bacterial peritonitis and for E . coli spontaneous bacterial peritonitis-related mortality.

FEBS Lett, 2003 Mar 13, 538(1-3), 139 - 44
Functional EF-Tu with large C-terminal extensions in an E coli strain with a precise deletion of both chromosomal tuf genes; Schnell R et al.; An Escherichia coli strain was constructed in which both chromosomal genes encoding elongation factor (EF)-Tu (tufA and tufB) have been inactivated with precise coding sequence replacements . A tufA gene in an expression vector is supplied as the sole EF-Tu source . By using plasmid replacement, based on plasmid incompatibility, mutant EF-Tu variants with a large C'-terminal extension up to 270 amino acids were studied and proved to be functional in a strain lacking the chromosomal tufA and tufB genes.

Euro Surveill, 1997 Dec, 2(12), 91 - 96
Surveillance of enterohaemorrhagic E . coli (EHEC) infections and haemolytic uraemic syndrome (HUS) in Europe; Ammon A; Since they were first described, Escherichia coliO157: H7 and other related enterohaemorrhagic E . coli(EHEC) have become known as a major infectious cause of bloody diarrhoea . These E . coliproduce one or more shiga-toxins (stx) or Vero cytotoxins . Strictl

Microb Pathog, 2003 Mar, 34(3), 155 - 9
Mutations in hns reduce the adherence of Shiga toxin-producing E . coli 091:H21 strain B2F1 to human colonic epithelial cells and increase the production of hemolysin; Scott ME et al.; Shiga toxin-producing Escherichia coli (STEC) 091:H21 strain B2F1, an isolate from a patient with the hemolytic uremic syndrome (HUS), produces elastase-activatable Shiga toxin (Stx) type 2d and adheres well to human colonic epithelial T84 cells . This adherence phenotype occurs even though B2F1 does not contain the locus of enterocyte effacement (LEE) that encodes the primary adhesin for E . coli O157:H7 . To attempt to identify genes involved in binding of B2F1 to T84 cells a bank of mini-Tn5phoACm(r) transposon mutants of this strain was generated . Several of these mutants exhibited a reduced adherence phenotype, but none of the insertions in these mutants were within putative adhesin genes . Rather, insertional mutations within hns resulted in the loss of adherence . Moreover, the hns mutant also displayed an increase in the production of hemolysin and alkaline phosphatase and a loss of motility with no change in Stx2d-activatable expression levels . When B2F1 was cured of the large plasmid that encodes the hemolysin, the resulting strain adhered well to T84 cells . However, an hns mutant of the plasmid-cured B2F1 strain exhibited a reduction in adherence to T84 cells . Taken together, these results indicate that H-NS regulates the expression of several genes and some potential virulence factors in the intimin-negative B2F1 STEC strain and that the large plasmid is not required for T84 cell colonization.

J Appl Microbiol, 2003, 94(4), 580 - 6
Improved methods of cultivation and production of deuteriated proteins from E . coli strains grown on fully deuteriated minimal medium; Paliy O et al.; AIMS: The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria . This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance . METHODS AND RESULTS: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium . A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures . Three E . coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium . This is the first report of JM109 being adapted to deuteriated minimal media . The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids . Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1) . We also show that all E . coli cells are inherently capable of growth on deuteriated media . CONCLUSIONS: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E . coli strains . Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein . We suggest that E . coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance . Thus plating and selection of colonies leads to highly deuterium-tolerant strains . SIGNIFICANCE AND IMPACT OF STUDY: An understanding of the mechanism of adaptation of E . coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules . This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.

J Mol Biol, 2003 Mar 21, 327(2), 431 - 43
Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E.coli enzymes; Chmiel NH et al.; The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair . The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches . Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene . Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D . These positions are highly conserved in MutY across phylogeny . The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli . In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes . In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G . Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N . Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context . If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene . The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.

Structure (Camb), 2003 Mar, 11(3), 323 - 8
Crystal structure of the E . coli Hsp100 ClpB N-terminal domain; Li J et al.; E . coli Hsp100 ClpB can disaggregate denatured polypeptides by employing ATP hydrolysis . The ClpB N-terminal domain (ClpBN) has been proposed to play important roles in ClpB molecular chaperone activities . We have determined the crystal structure of ClpBN to 1.95 A resolution by MAD methods . The ClpBN monomer contains two subdomains that have similar folds . The crystal structure revealed a hydrophobic groove on the molecular surface . We have constructed ClpB mutants in which the hydrophobic residues within the putative peptide binding groove were replaced by glutamine . These ClpB mutants exhibited severe defects in molecular chaperone activity but retained the wild-type ATPase activity.

Mol Cell, 2003 Feb, 11(2), 315 - 27
Mechanism of the E . coli tau processivity switch during lagging-strand synthesis; Leu FP et al.; The E . coli replication machinery employs a beta clamp that tethers the polymerase to DNA, thus ensuring high processivity . The replicase also contains a processivity switch that dissociates the polymerase from its beta clamp . The switch requires the tau subunit of the clamp loader and is regulated by different DNA structures . At a primed site, the switch is "off." When the replicase reaches the downstream primer to form a nick, the switch is flipped, and tau ejects the polymerase from beta . This switch has high fidelity for completed synthesis, remaining "off" until just prior to incorporation of the last nucleotide and turning "on" only after addition of the last dNTP . These actions of tau are confined to its C-terminal region, which is located outside the clamp loading apparatus . Thus, this highly processive replication machine has evolved a mechanism to specifically counteract processivity at a defined time in the lagging-strand cycle.

Curr Opin Microbiol, 2003 Feb, 6(1), 82 - 90
Tails of two Tirs: actin pedestal formation by enteropathogenic E . coli and enterohemorrhagic E . coli O157:H7; Campellone KG et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals . Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells . Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria . Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable . For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly . In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.

Kansenshogaku Zasshi, 2002 Dec, 76(12), 988 - 94
{A rapid screening method for the detection of E . coli O157 by an automated enzyme-linked fluorescent immunoassay}; Hamada Y et al.; VIDAS ECO is useful as a rapid method for detecting E . coli O157 from food samples, because one can obtain the results within 1 hour by examining the enrichment broth after a 18 hour incubation . In addition, one can handle a large amount of sample, owing to its simplicity . No false negative were recognized in the present experiment and diffuse outbreak samples cases, which confirmed the usefulness of ECO as a screening method . Besides, regarding ECO positive samples, we could confirm by the following day, that they were false positive, by a combined test using an isolation medium after a bead-enzyme-linked immunosorbent assay.

Hua Xi Yi Ke Da Xue Xue Bao, 2002 Jan, 33(1), 94 - 7
{Expression of human nerve growth factor gene in E . coli}; He X et al.; OBJECTIVE: To investigate the structure and function of human nerve growth factor (beta-NGF) and the gene encoding beta-NGF . METHODS: A pair of specific primers (29 mer) for the sequence encoding human beta-NGF was designed and synthesized . A 380 bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM-T Easy vector . The identified insert fragment from the recombinant pGEM-T-NGF was directionally ligated with linearized pGEX-5T with the compatible termini . E . coli JM 109 was transformed with the expression recombinant p5TNGF and induced by IPTG . RESULTS: The cloned DNA fragment was identified as the full-length sequence encoding human beta-NGF by restriction analysis and DNA sequencing . SDS-PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5 x 10(3) u) in the cells transformed by p5TNGF . The soluble fusion protein was determined to be 503.2 mg/L, accounting for 6.8% of the total soluble protein (7.4 g/L) of bacterial cells . This fusion protein was found to have antigenic activities of NGF . CONCLUSION: The clone containing the full-length sequence encoding human beta-NGF is obtained and successfully expressed in E . coli to be of use for studying the biological functions of human beta-NGF gene.

Hunan Yi Ke Da Xue Xue Bao, 2002 Feb 28, 27(1), 1 - 3
{Expression of c-terminal of Parkin in E . coli and the preparation of antiserum}; Ou Yang J et al.; OBJECTIVE: To clone and express 3'-terminal of Parkin gene in E . coli, and prepare its antiserum for further study . METHODS: The glutathion-sulfate-transferase (GST) fusion expression plasmid of 3'-terminal of Parkin gene (937-1959 bp) was constructed and transferred to JM 105 . After being treated with Triton-100 (1%) and Tween-20 (1%) and purified with affinity chromatograph, GST-Parkin C was used to immunize New Zealand rabbits to acquire antiserum . Antiserum was analysed with immunoblot . RESULTS: The GST-Parkin C protein was expressed in JM 105, existing in the form of inclusion body with a molecular weight of around 42 kD; The purity of GST-Parkin C was up to 95%; the titer of antiserum was 1:64; Immunoblotting showed that the prepared antiserum could react specifically with 51.6 kD protein extracted from the mouse brain . CONCLUSION: A high level of expression of GST-Parkin C is obtained in JM 105, and its antiserum can be prepared successfully.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999 Jun 30, 13(2), 130 - 2
{Expression of hepatitis E virus structural gene in E . coli}; Zhang M et al.; OBJECTIVE: To obtain recombinant antigen for development of vaccine against hepatitis E virus . METHODS: Amplified the structural gene (5,816-7,126 nt) by PCR . The upstream primer was 5'-CCATATGAATTCAATAACCTC-3' and the downstream primer was 5'-GGGATCCTATAACTCCCGAGT-3' . Cut the PCR product with Nde I and BamHI, then inserted this fragment into the plasmid pET-11 where a cut was by the same restriction endonucleases . The expression plasmid named pEa47 was transformed into E . Coli BL21 . The recombinant strains were grown at 37 degrees C and induced by IPTG . The recombinant protein was confirmed by Western blot analysis using serum from hepatitis E patient . RESULTS: The structural gene of hepatitis E virus from open reading frame 2,224-660 aa, was expressed in E . Coli BL21 . Western blot assay showed that the expressed 50,000 recombinant protein specifically reacted with the serum antibody from the hepatitis E patient . CONCLUSION: The protein might be useful to develop vaccine against hepatitis E virus infection.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1999 Feb, 21(1), 31 - 6
{Modification of N-terminal cDNA of hGM-CSF and high-expression in E . coli.}; Yin J et al.; OBJECTIVE: N-terminal cDNA of human granulococyte-macrophage colony stimulating factor(GM-CSF) was designed to be modified and highly-expressed in E . coli . METHODS: A pair of oligo-nucleotide primers were used to modify the mature N-terminal cDNA sequence of hGM-CSF with the method of PCR . the modified cDNA of hGM-CSF was cloned into E . coli . expressive vector PBV220 and expressed in E . coli DH5 alpha strain, the biological activities of the recombinant protein was identified by means of cell colony formation and TF-1 cell growth assay in vitro . RESULTS: The expression level of modified hGM-CSF cDNA was higher than that of unmodified native type . SDS-PAGE revealed that expressed protein of hGM-CSF accounted for about 25% of total bacterial cell protein . The biological activity of recombinant protein was about 1.5 x 10(7) U/mg protein . The sequence of 1-16 amino acid of N-terminal of the recombinant protein was same with native hGM-CSF . CONCLUSIONS: Modification of the N-terminal cDNA of hGM-CSF could dramatically enhance the expression level in E . coli system.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2000, 18(1), 37 - 9
{Cloning and expression of Cysticercus cellulosae antigen cC1 in E . coli}; Chen RW et al.; OBJECTIVE: To clone and express Cysticercus Cellulosae antigen cC1 in E . coli . METHODS: cC1 cDNA fragment was cloned to BamHI and PstI sites of pGEM-3Z vector . After alteration of the restriction sites, the fragment was cloned to EcoRI and XhoI sites of pGEX-5T with a synthetic linker to construct recombinant expression vector pGEX-5T-cC1 . RESULTS: The clone produced the largest yield of cC1 protein expression when incubated in 2YT culture medium for 3 h or induced by IPTG for 6 h . Detected by scanning optical densitometry, cC1 constituted 57% of the total bacterial proteins . Western blotting analysis revealed that the GST-cC1 fusion protein exhibited a specific reactive band . CONCLUSION: High level expression of Cysticercus cellulosae antigen cC1 was obtained in E . coli.

Int J Biol Macromol, 2002 Dec 20, 31(1-3), 63 - 9
Synthesis of PHB by recombinant E . coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209; Ramachander TV et al.; An approximately 5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and introduced into Escherichia coli . The recombinant E . coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions . The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85x10(5) . Purified PHB granules were spherical with an average size of 1.1 microm and of stable configuration . DSC thermogram suggested high crystalline nature of the polymer . Maximum thermal degradation of the biopolymer occurred between 250 and 340 degrees C . Recombinant E . coli cells preferentially utilized glycerol as the carbon source and accumulated 25-28 times more PHB than the native S . aureofaciens.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 400 - 5
{Fusion expression of PLA2 gene from Lapemis hardwickii in E . coli}; Yang W et al.; The gene encoding PLA2(PLA2-9) from Lapemis hardwickii Gray venom was cloned to the 3' and of the thioredoxin gene (HP-trxA and trxA) in plasmid pthioHisC and pTRX to construct the pThioHisC-PLA2, and pTRX-PLA2 fusion expression vector . The fusion protein of PLA2 can be expressed in the two different systems induced by IPTG at 25 degrees C, but the expression level and the solubility of the fusion protein in pTRX were better than that in pThioHisC . The expressed product in the two systems were purified by immobilized metal-chelate affinity chromatography . The Trx-PLA2 fusion protein with over 85% purity was obtained and HP-Trx-PLA2 fusion protein can not be purified since it dose not exhibit affinity to the medium . So, the pTRX-PLA2 vector system was established for the large-scale expression and purification.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 311 - 5
{Cloning of IL-1 beta gene and expression in E . coli}; Chang S et al.; IL-1 beta cDNA was obtained by RT-PCR with the template of the total RNA extracted from leukocytes which was separated from human peripheral blood . 5' and 3' primers were synthesized according to literaturees reported sequence of IL-1 beta . IL-1 beta gene was highly expressed in E . coli and the expression level reached to about 40% of total bacteria proteins . Separation, purification and bioactivity analysis of the expressed products was performed . The purity of the final products reach more than 98%, and the culture solution of EL-4 cells induced by hIL-1 beta can promote the proliferation of CTLL-2 cells obviously.

Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 272 - 4
{Studies on preparation of L-phenylalanine from phenylpyruvic acid by E . coli EP8-10}; Xu H et al.; E . coli EP8-10 was selected from the soil . It was able to produce the transaminase with high activity when it was cultivated on the medium containing peptone and beef extract . Optimum conditions of enzyme reaction was: phenylpyruvic acid's concentration of 0.3-0.5 mol/L, L-Asptaric acid used as amino donor, pH 8.5 37 degrees C . When phenylpyruvic acid was 0.3 mol/L, 48 g/L L-phenylalanine was produced after 6 h with 97% conversion rate.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 567 - 72
{Studies on in vitro expression for gI gene of Marek's disease in E . coli by pGEX vector}; Ding J et al.; Glycoprotein I Gene(gI) was amplified from genomic DNA of Marek's disease virus (MDV) 648 strain by polymerase chain reaction(PCR) . PCR product was cloned into pGEX-6p-1 according to the right open reading frame(ORF) . The expression of GST-gI fusion protein was studied in detail on many factors including temperature, timing and IPTG . The curve for OD600 and the growing time of the recombinant bacteria is also established., which is helpful to find the optimal inducing time . GST-gI fusion protein was identified by SDS-PAGE and Western-blotting., and the result shows that the best concentration of IPTG is 0.2-0.5 mmol/L and inducing time have great effects on expression while temperature has little . The fusion protein was injected into mouse five times to identify its antigenicity and the result is positive in indirect fluorescent assay IFA.

Wei Sheng Wu Xue Bao, 1998 Aug, 38(4), 269 - 75
{Subcloning and expression of coding region for cellulase binding domain of CBH I from P . janthinellum in E . coli}; Wang T et al.; The in vitro DNA manipulations, included the nested deletions, of cbh1 from P . janthinellum inserted into pUC18-181 were carried out . The two ends of fragments were modified into blunt ends and the fragments were self-ligated . Then, the encircled plasmids were transformed to E . coli JM109 . Utilizing the characterization of CBD binding to crystalline cellulose, one catalytic domain deletion transformant producing active LacZ-CBD fusion protein was isolated from 24 transformants randomly picked from 400 transformants . The molecular weight of the LacZ-CBD fusion protein is 21 kD . The plasmid was designated pUC 18C . The LacZ-CBD fusion protein produced by JM109(pUC18C) was able to be purified by procedure of adsorption-desorption to cellulose . The pNPC activity of crude enzyme solution of JM109(pUC18C) induced by IPTG were zero, identified the JM109(pUC18C) has no CBHI activity.

Wei Sheng Wu Xue Bao, 1998 Apr, 38(2), 81 - 5
{Expression of the polyketide ketoreductase gene from midecamycin producing strain in E . coli}; Xia H et al.; Two primers were designed and synthesized according to the polyketide ketoreductase from midecamycin producing strain gene(MKR) sequence data which has been reported, MKR gene was amplified using PCR technique . The amplified MKR gene was subcloned into NdeI and BamHI sites of the T7RNA polymerase-dependent pT7-7 expression vector, and introduced into E . coli K38/pGP1-2 . Proteins were isolated from transformant . The result of exclusive labeling by L-35S-methionine of plasmid-encoded proteins SDS-PAGE analysis showed that the MKR was produced in E . coli . The expressed MKR has bioactivity.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 16 - 24
{Construction of recombinant E . coli with high glutathione biosynthetic activity and the biosynthetic process}; Li H et al.; A recombinant strain E . coli II-1, which exhibited high glutathione (GSH) biosynthetic activity and high stability, was constructed by transforming plasmid pGH501 which contains gene gsh I and gsh II into a wild type strain E . coli II . 4 g/L GSH accumulated extracellularly by using toluene-treated cell . In GSH biosynthetic system, GSH production was improved by increasing the concentration of L-glutamate, while inhibited by L-cysteine if it's concentration was beyond 20 mmol/L . In GSH biosynthetic reaction, the apparent little consumption of L-glutamate and glycine was concluded experimentally to be that toluene-treated E . coli II-1 cells still contained high concentration of L-glutamate and glycine . According to the change of energy cofactor in the GSH biosynthetic process, a possible GSH biosynthetic mechanism controlled by E . coli II-1, was proposed: the energy donator of reaction catalyzed by glutathione synthetase (GSH-II) was ADP but not ATP, the reaction was rate-limited step within the whole GSH biosynthetic process, high concentration of ADP might inhibit the activity of GSH-II . Further degradation of GSH was prevented by the addition of 100 mmol/L L-serine and potassium borate mixture . In such case, 23.0 mmol/L (about 7.1 g/L) GSH accumulated at 3 h.

Shi Yan Sheng Wu Xue Bao, 2001 Jun, 34(2), 131 - 5
{High-level expression of human calmodulin in E . coli and its effects on cell proliferation}; Li XJ et al.; The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template . After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220) . The recombinant plasmid was then transformed into E . coli DH5 alpha . After heat induction, a high level expression of CaM protein was obtained . SDS-PAGE analysis showed that the recombinant E . coli could express a 17 kD protein which accounted for about 20% of the total cellular protein . Western blot analysis showed that anti-CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product . rhCaM was purified by Phenyl-sepharose CL-4B affinity chromatography from recombinant bacterial lysate . 3-4 mg of the purified protein were obtained from 1 liter of bacterial culture . The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma) . K562 cells and SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h with rhCaM and CaM-antagonist trifluoperazine(TFP) . Cell proliferation rates was determined by MTT assay . There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates . CaM-antagonist TFP had an inhibitory effect on cell proliferation rate . The inhibition could be corrected by the addition of extracellular rhCaM.

Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 586 - 90
{Cloning and expression of caiDE genes in E . coli BL21(DE3)}; Jiang J et al.; The caiDE genes for carnitine racemase and its relative factor were cloned into pUC18 from the chromosomal DNA of E . coli MC4100 by shot-gun approach and have been verified by sequence analysis . The target plasmid was named as pJX393 and subcloned to form a transcription plasmid pDSW2 . After its transformation into BL21(DE3) strain and induction by IPTG, the recombinant strain containing pDSW2 expressed two apparent protein bands on SDS-PAGE, whose molecular weight were 30 kD and 24 kD individually.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 223 - 8
{Screening and characteristics of mutants of E . coli resistant to acetate inhibition}; Li Z et al.; Acetate is accumulated in the aerobic high cell density culture of Escherichia coli, Which seriously inhibits cell growth and expression of recombinant proteins . To alleviate acetate inhibition, mutants of E . coli JM101 were generated by 60Co radiation and then enriched in continuous culture with acetate as the selective pressure . One of the mutants isolated, JL3, showed obvious increased tolerance toward acetate and maintained phenotypic stability on slant without acetate . In MA medium containing 10 g/L of sodium acetate, the specific growth rate and the glucose consumption rate of JL3 were higher than those of JM101.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 167 - 72
{Expression of two truncated enhancin gene from Helicoverpa armigera granulosis virus in E . coli and its preliminary bioassay}; Liu X et al.; The plasmids pET-30a-Ben and pET-30a-Den which included 1.7 kd and 2.2 kb fragments of 5'-terminal of HaGV enhancin gene were obtained by cutting recombinant plasmid pET-30a-En with Bal I and Dra I respectively . Two fragments were expressed in E . coli successfully and the products were named Ben and Den respectively . The enhancement, which Ben and Den enhance the infectivity of HaNPV and Bt in 3rd larvae of Helicoverpa armigera, was studied . The results indicated that there was increase of the mortality of 10.5%-26.5% and the LT50 decrease of 0.9 d causes by adding Ben, while Den could increase the mortality by 10.2%-33.0% and decrease the LT50 by 1.2 d-1.9 d . The preliminary bioassay on Bt against Helicoverpa armigera indicated the recombinant enhancin could increase the mortality of larvae by 20.7%-35.4%, Den by 16.7%-31.5%, Ben by 11.7%-27.4%.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 323 - 6
{Cloning and expression of the gene encoding novel alpha-amylase from Sulfolobus shibatae in E . coli}; Liu L et al.; A novel alpha-amylase gene was amplified from Sulfolobus shibatae by using PCR technique . The amplified 1.7 kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM . The novel alpha-amylase gene in pSBAM was expressed in E . coli . The production of the novel alpha-amylase activity reached over 8 units/100 mL of the culture . The molecular weight of this enzyme was about 61 kD by SDS-PAGE . The expressed novel alpha-amylase protein in E . coli DH5 alpha accounted for about 20% of the total protein in the recombinant cell . The cooperative action of the novel alpha-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 379 - 83
{Molecular cloning of enhancin gene from Helicoverpa armigera granulosis virus and its expression in E . coli}; Liu X et al.; In order to provide recombinant enhancin for studying its mechanism of increasing the mortality of larvae infected by HaNPV and creating new insecticide, enhancin gene from Helicoverpa armigera granulosis virus was amplified by PCR technique . 2.7 kb fragment of enhancin gene was cloned into EcoRI/XbaI site of plasmid pBluescript KS . Sequence analysis revealed that enhancin gene was similar with that reported in the literature except eight nucleotides and five amino acids . Thereby enhancin gene was inserted into vector pET-30a and expressed successfully in Escherichia coli BL21(DE3) . The preliminary bioassay of expressed product and HaNPV indicated that mortality of larvae increased 31.7%-34.1% in 7 post-infection days and the LT50 decreased at least 1.5-2.1 days.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 57 - 61
{Cloning and expression of the gene encoding maltoologosyl trehalose synthase from Sulfolobus shibatae in E . coli}; Chen W et al.; 2.2 kb DNA fragment encoding a novel enzyme, maltooligosyl trehalose synthase (MTSase) was amplified from Sulfolobus shibatae by using PCR technique . The amplified 2.2 kb DNA fragment was inserted into an expression vector, pBV220, to yield the recombinant plasmid pSBGT1 . MTSase gene in pBSGT1 was expressed in E . coli . The molecular weight of expressed MTSase detected by SDS-PAGE was about 74 kD, which is conformed with that deduced from nucleotide sequence . The expressed MTSase protein accounted for about 4.4% of the total cell protein . The MTSase from transformants containing pBSGT1 is capable of decreasing DE value, forming non-reducing or less-reducing saccharides when allowed to act on reducing partial starch hydrolysates.

Wei Sheng Wu Xue Bao, 2000 Oct, 40(5), 470 - 4
{Cloning and expression of otsA gene in E . coli}; Wang Y et al.; 1.5 kb of otsA gene encoding trehalose synthase has been cloned by PCR amplification . The DNA fragment was ligated to multi-copy vector and transformed to otsBA deleted and otsA deficient strains of E . coli separately . The transformants exhibited growth as well as the otsBA+ wild type on medium containing 0.5 mol/L NaCl . Trehalose was synthesized and accumulated in the transformed cells under osmotic pressure, which was determined by thin layer chromatograph . The results confirmed that otsA gene was functionally expressed in the recipient strains . These studies suggested that engineering otsA gene and trehalose accumulation into crop plants may improve drought and salinity tolerance.

Se Pu, 2001 Jul, 19(4), 301 - 3
{Simultaneous purification and renaturation of recombinant human interferon-alpha expreessed by E . coli by high-performance hydrophobic interaction chromatography}; Guo LA; One-step simultaneous purification and renaturation of interferon-alpha from the inclusion body of E . coli expreessed by high-performance hydrophobic interaction chromatography(HPHIC) are presented . The inclusion body was dissolved by 8 mol/L guanidine hydrochloride . The sample lysis was centrifuged and the solution was renaturated by three different methods, i.e . hydrophobic interaction chromatography, dilution and dialysis . The total activity yield renaturation by hydrophobic interaction chromatography was 2 times and 2.6 times as much as that by dilution and by dialysis . The purity of the target product was 95% according to high-performance size exclusion chromatography and the specific activity was 1.8 x 10(8) IU/mg . The method developed is simpler and more efficient than others.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 167 - 71
{Secretory Expression of the Superactive {Lys(17,18),Glu(21)}-Glucagon in E . coli}; Wen CW et al.; One of the most important findings in structure-function studies on glucagon by means of chemical synthesis is the discovery that {Lys(17,18),Glu(21)}-glucagon had higher biological activity than native glucagon . This mutant of glucagon was called superactive glucagon (SA-glucagon) . In the present work, the possibility to obtain SA-glucagon by means of genetic engineering was studied . The gene of SA-glucagon (SAG) was obtained by PCR from a constructed recombinant glucagon plasmid, pAGluT . A secretory expression vector harboring SAG, pBLSG7, containing P(L) promoter and the gene of phoA signal peptide was constructed . In expression studies after transformation of pBLSG7 into E . coli BL21, it was found that the expression yield of SA-glucagon reached 3.65 mg/L(A(600)=1), about 19.5% of total proteins in the culture medium under shaken flask conditions . In addition, the influence of induction temperature and of E . coli strain on the expression yield of SA-glucagon was also studied.

Hua Xi Yi Ke Da Xue Xue Bao, 2001 Sep, 32(3), 341 - 3, 448
{Molecular cloning and expression in E . coli of the surface-exposed lipoprotein LipL41 gene of Leptospira lai}; Li X et al.; OBJECTIVE: To construct a recombinant expressive plasmid using LipL41 gene of leptospira lai for further research of subunit vaccine of leptospira . METHODS: A pair of oligonucleotide primers were designed based on the sequence of LipL41 of leptospira kirschneri RM 52 in Genbank . With genomic DNA of leptospira lai 017 as template, a fragment was amplified by PCR and DNA sequencing analysis showed this fragment to be the gene that encodes LipL41 . A recombinant vector was constructed using plasmid pGEX1 lambda T and the expression of LipL41 gene was tested . RESULTS: The production of PCR was LipL41 gene . The recombinant plasmid was constructed and testified by nuclease digestion and PCR, and LipL41 gene could express in E . coli . CONCLUSION: The recombinant plasmid has been constructed successfully and LipL41 gene can express stably at high level.

Mol Cell, 2003 Jan, 11(1), 189 - 201
Temporal regulation of topoisomerase IV activity in E . coli; Espeli O et al.; We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication . This phenotype results from a defect in daughter chromosome decatenation during rapid growth . In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected . In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete . These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.

DNA Repair (Amst), 2002 Oct 1, 1(10), 821 - 31
DNA damage-processing in E . coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation; Burger A et al.; UV irradiation of E . coli produces photoproducts in the DNA genome . In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells . However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA . We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR) . At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation . We used excision-defective E . coli B/r cells and worked under yellow light to avoid background PR . During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively . In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed . When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed) . Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective . We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

Radiats Biol Radioecol, 2002 Nov-Dec, 42(6), 636 - 8
{Regularities of excising transposon Tn10 in rec-mutant E . coli cells exposed to gamma radiation}; Zhuravel' DV et al.; The regularities of gamma-induced excision of transposon Tn10 in different rec-strains of E . coli cells after gamma-irradiation have been studied . The survival of cells and relative frequency of the Tn10 elimination as a function of the 137Cs gamma-radiation doses were investigated . RecN and recA-mutants of E . coli were used for study of the role of rec-genes in the gamma-induced transposon excision . It was shown that the induced excision in the recN mutant was reduced . The transposon excision in the recA mutant was not revealed . The obtained results let to conclude that recA, and recN genes are involved not only in DNA repair processes but also in the gamma-induced transposon excision in bacteria.

Microgravity Sci Technol, 2002, 13(4), 24 - 9
Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E . coli; Brown RB et al.; Cultures of Escherichia coli grown in space reached a 25% higher average final cell population than those in comparably matched ground controls (p<0.05) . However, both groups consumed the same quantity of glucose, which suggests that space flight not only stimulated bacterial growth as has been previously reported, but also resulted in a 25% more efficient utilization of the available nutrients . Supporting experiments performed in "simulated weightlessness" under clinorotation produced similar trends of increased growth and efficiency, but to a lesser extent in absolute values . These experiments resulted in increases of 12% and 9% in average final cell population (p<0.05), while the efficiency of substrate utilization improved by 6% and 9% relative to static controls (p=0.12 and p<0.05, respectively) . In contrast, hypergravity, produced by centrifugation, predictably resulted in the opposite effect--a decrease of 33% to 40% in final cell numbers with corresponding 29% to 40% lower net growth efficiencies (p<0.01) . Collectively, these findings support the hypothesis that the increased bacterial growth observed in weightlessness is a result of reduced extracellular mass transport that occurs in the absence of sedimentation and buoyancy-driven convection, which consequently also improves substrate utilization efficiency in suspended cultures.

Mutat Res, 2003 Jan 28, 522(1-2), 145 - 56
In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E . coli: a feasible part of UV-mutagenesis; Burger A et al.; We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or PyPy) in UV-irradiated E . coli deficient in uracil DNA glycosylase . The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated . The deamination rate at TC photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF(PR)) that developed as UV-irradiated cells were held before PR . Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E . coli B/r strain . With a K12 strain, where the targeting apparently is principally by CPD and not (6-4) photoproducts, a larger rate of k = 0.0091 min(-1) at 42 degrees C resulted . The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD . In addition, we used a strain constructed by Cupples and Miller in which beta-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac(+) only when replaced by -GAG- or -GAA- for glutamate . CC photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD . MF(PR) for Lac(+) mutations increased and then decreased as a function of deamination time (at six temperatures 36-48 degrees C) . Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac(+) at a rate about eight-fold less than a second deamination restoring the Lac(-) phenotype . We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations .

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Mar, 12(1), 26 - 8
{Expression of secreted ligand domain of vaccinia growth factor encoded by vaccinia virus strain tian tan in E . coli and insect cells}; Jin Q et al.; Vaccinia virus growth factor (VGF) is encoded by an early gene of vaccinia virus strain TianTan located near the border between the terminal inverted repeats and the internal region of the viral genome as one copy . Its relationship to members of epidermal growth factor superfamily was discovered when computer searches of protein sequences revealed that VGF has strong sequence homology with these growth factors . To further get insight into the biological activity of VGF, we cloned and expressed the secreted ligand domain of VGF (VGFsl) encoded by vaccinia virus strain TianTan in E . coli and insect cells . The results of biological activity test indicated that expressed products is capable of binding to the EGF receptor, and stimulating EGF receptor tyrosin autophosphorylation to a level greater than that of EGF.

DNA Repair (Amst), 2002 Sep 4, 1(9), 703 - 8
Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E . coli cells; Becherel OJ et al.; The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions . Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication . By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer . Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis . Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif . Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.

DNA Repair (Amst), 2003 Jan 2, 2(1), 73 - 89
DNA mismatch repair and acquired cisplatin resistance in E . coli and human ovarian carcinoma cells; Massey A et al.; The contribution of defective DNA mismatch repair (MMR) to acquired resistance to cis-diamminedichloroplatinum(II) (cisplatin) has been investigated in two model systems: E coli dam mutants and the A2780 ovarian carcinoma cell line . Inactivation of MMR-as indicated by the acquisition of an elevated spontaneous mutator phenotype-was observed frequently among survivors of cisplatin-treated dam mutants . These survivors exhibited a stable resistance to further cisplatin treatment . In contrast, none of twelve independent clones of A2780 that had survived cisplatin exposure and acquired stable drug resistance were repair defective . None exhibited the hallmark methylation tolerant phenotype associated with a MMR defect, mRNAs encoding five MMR proteins were easily detectable in all twelve variants, and the levels of four key MMR proteins were similar to those in the repair proficient parental cells . Further analysis indicated two different mechanisms of acquired resistance in A2780 . The first was a protective effect that reduced the level of DNA platination . The second was observed as a reduced sensitivity to cell cycle arrest after cisplatin treatment and a consequent reduced apoptosis . The data suggest that although loss of MMR is a significant mechanism of acquired drug resistance in dam bacteria, alterations related to DNA protection or cell cycle progression after drug damage appear to be more probable than abrogation of MMR as resistance modulators in human cells.

DNA Repair (Amst), 2002 Feb 28, 1(2), 159 - 67
Genetics of mutagenesis in E . coli: various combinations of translesion polymerases (Pol II, IV and V) deal with lesion/sequence context diversity; Wagner J et al.; The biochemistry and genetics of translesion synthesis (TLS) and, as a consequence, of mutagenesis has recently received much attention in view of the discovery of novel DNA polymerases, most of which belong to the Y family . These distributive and low fidelity enzymes assist the progression of the high fidelity replication complex in the bypass of DNA lesions that normally hinder its progression . The present paper extends our previous observation that in Escherichia coli all three SOS-inducible DNA polymerases (Pol II, IV and V) are involved in TLS and mutagenesis . The genetic control of frameshift mutation pathways induced by N-2-acetylaminofluorene (AAF) adducts or by oxidative lesions induced by methylene blue and visible light is investigated . The data show various examples of mutation pathways with an absolute requirement for a specific combination of DNA polymerases and, in contrast, other examples where two DNA polymerases exhibit functional redundancy within the same pathway . We suggest that cells respond to the challenge of replicating DNA templates potentially containing a large diversity of DNA lesions by using a pool of accessory DNA polymerases with relaxed specificities that assist the high fidelity replicase.

DNA Repair (Amst), 2002 Jul 17, 1(7), 507 - 16
Alkylation resistance of E . coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG); Bonanno K et al.; The alkyladenine DNA glycosylase (AAG) has been cloned from mouse and humans . AAG knock out mouse cells are sensitized to a variety of alkylating and cross-linking agents suggesting AAG is active on a variety of substrates . In humans, two isoforms have been characterized that are generated by alternative splicing and contain either exon 1a or 1b (hAAG1 or hAAG2) . In this study, we examine the ability of the both known isoforms of human AAG (hAAG) to contribute to survival of Escherichia coli from treatments with simple alkylating agents and cross-linking alkylating agents . Our results show that hAAG is effective at repairing methyl lesions when expressed in E . coli, but is unable to afford increased resistance to alkylating agents producing larger alkyl lesions such as ethyl lesions or lesions produced by the cross-linking alkylating agents N,N'-bis-chloroethyl-N-nitrosourea (BCNU), N-(2-chloroethyl)-N-nitrosourea (CNU) or mitomycin C . In the case of CNU, expression of hAAG causes increased sensitivity rather than resistance, suggesting deleterious effects of hAAG activity . We also demonstrate that there are no apparent differences between the two isoforms of hAAG when recovery from damage produced by all alkylating agents is tested .

Biotechniques, 2002 Dec, 33(6), 1256 - 60
Purification of histidine-tagged T4 RNA ligase from E . coli; Wang QS et al.; Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4) . The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column . The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet . A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations . The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.

Med Sci Monit, 2002 Dec, 8(12), BR504 - 14
Characterization of dendritic cells generated in vivo by an E . coli derived chimeric dual receptor agonist; Kahn LE et al.; BACKGROUND: Progenipoietin-4 (ProGP-4) is an E . coli derived chimeric growth factor that activates the human Flt3 and G-CSF receptors . ProGP-4 possesses cross-species activity and treatment of mice with ProGP-4 results in increases in the number of WBC and Class II+/CD11c+ cells in both spleen and peripheral blood . Herein, we report morphologic, phenotypic and functional evaluation of Class II+/CD11c+ cells generated by in vivo administration of ProGP-4 . MATERIAL/METHODS: C57BL/6 mice were injected daily with ProGP for 7 to 18 days . Leukocytes from spleen and peripheral blood were analyzed by flow cytometry to enumerate and characterize changes in DC populations . Spleens from ProGP treated mice were evaluated by immunocytochemistry and enriched CD11c+ populations were functionally assessed in a mixed lymphocyte assay and in an antigen dependent CTL assay . RESULTS: Administration of this dual receptor agonist to mice resulted in dose-dependent increases in the numbers of total white blood cells and Class II+/CD11c+ cells in spleen and peripheral blood . CD11c+ cells from ProGP-4 treated mice co-expressed DEC205 and also expressed CD80, CD86 and CD40, albeit at lower levels as compared to Class II+/CD11c+ cells from untreated animals . Despite lower co-stimulatory molecule expression, ProGP-4-generated Class II+/CD11c+ cells stimulated proliferation of allogeneic T cells and an antigen-specific T cell hybridoma as efficiently as bone marrow derived dendritic cells from untreated mice . CONCLUSIONS: The data presented in this report highlight the ability of E . coli derived ProGP-4 to expand large numbers of functional DC in the peripheral blood and lymphoid organs in vivo using a rodent model of hematopoiesis . E . coli derived chimeric receptor agonists such as ProGP-4 may enable further investigations of immunotherapeutic approaches to the treatment of diseases such as cancer and autoimmunity.

Sheng Li Ke Xue Jin Zhan, 1998 Jul, 29(3), 226 - 30
{Recent advances of heterologous gene expression in E . coli}; Zhao M et al.; In recent years, along with the rapid development of genetic engineering technologies, a vast amount of valuable proteins have been expressed in E . coli with high productivity . The problems concerning the productivity and purity of the heterologous proteins are no longer the major ones, as many expression system and protein purification schemes have been developed and perfected . More attention is being payed to the activity, specific activity, correct folding and intactness of the heterologous proteins . Perhaps it is the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins.

Microb Pathog, 2002 Dec, 33(6), 289 - 98
Identification of genomic differences between Escherichia coli strains pathogenic for poultry and E . coli K-12 MG1655 using suppression subtractive hybridization analysis; Stocki SL et al.; Diseases of poultry caused by Escherichia coli result in significant economic loss every year . Specific virulence factors associated with E . coli strains pathogenic for poultry have been identified, but it is likely that others remain to be identified . To identify unique DNA fragments associated with avian strains we used suppression subtractive hybridization . The genome of E . coli K-12 strain MG1655 was subtracted from the genomes of two avian E . coli strains resulting in the identification of 62 fragments specific to the two avian strains . Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function . Two E . coli collections, a reference collection of diverse strains (ECOR) and a collection of 41 avian isolates, were screened for the presence of 25 of the 62 fragments . We identified nine fragments present in significantly more of the avian strains than of the ECOR strains . Five fragments were in significantly more of the ECOR strains than the avian strains . These results suggested that the nine fragments could play a role in the pathogenesis of E . coli as it relates to diseases of poultry.

Biochemistry, 2002 Dec 17, 41(50), 15025 - 35
E . coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A); Troeberg L et al.; The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2 . Full-length human TIMP-4 was expressed in E . coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography . Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively) . Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)) . K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent . TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2 . Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes . The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2 . Similar models of interaction may apply to TIMP-4 . The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

Scott Med J, 2002 Oct, 47(5), 112 - 4
Three years experience of adults admitted to hospital in north-east Scotland with E . coli O157; Cadwgan AM et al.; To describe the epidemiology, clinical features, treatment and outcomes of adults with E . coli O157 infection presenting to Aberdeen Royal Infirmary over a three year period . METHODS: A retrospective casenote review . RESULTS: Thirty-two confirmed cases of E . coli O157 infection were admitted between 1997 and 2000 . The median age was 58 years (range 16-93) . Ten patients (31%) were from the city of Aberdeen and 22 (69%) from surrounding rural areas . Twenty-seven patients (85%) presented between May and October . The source of infection was unknown or unconfirmed in all cases . Bloody diarrhoea was present in 30 (94%) . Leucocytosis was present in 18 (63%) but only four patients (13%) had a fever . Six of the 32 patients (19%) developed Haemolytic-Uraemic Syndrome (HUS) of whom 2 died . Ten patients received antibiotics of whom two developed HUS . Twenty-seven of the 32 (85%) had made a full recovery by time of discharge, three (9%) had impaired renal function and two (6%) died in hospital . CONCLUSION: E . coli O157 infection tends to occur sporadically in rural areas in North East Scotland . It is not usually associated with fever . Infection occurs more commonly in the summer and autumn . HUS complicates infection in almost one fifth of patients.

Structure (Camb), 2002 Dec, 10(12), 1721 - 30
Crystal structures of C4 form maize and quaternary complex of E . coli phosphoenolpyruvate carboxylases; Matsumura H et al.; Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis . The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution . The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate . The crystal structure of E . coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution . Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site . On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

J Med Microbiol, 2002 Dec, 51(12), 1050 - 4
Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E . coli; Chart H et al.; Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E . coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157 . Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined . These common LPS-core epitopes were also present on strains of E . coli O26 which did not produce verocytotoxin . These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E . coli O157.

Curr Opin Microbiol, 2002 Dec, 5(6), 553 - 7
Assembly of cell division proteins at the E . coli cell center; Buddelmeijer N et al.; Cell division in Escherichia coli requires the coordinated action of at least ten proteins . In recent years, substantial progress has been made in understanding the assembly of these proteins at the cell septum . These findings suggest a largely stepwise appearance of cell division proteins at the centre of the cell.

Vaccine, 2002 Dec 13, 21(3-4), 194 - 201
Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization against Helicobacter pylori; Sougioultzis S et al.; Low dose E . coli heat-labile enterotoxin (LT), delivered orally or enterically, has been used as an adjuvant for Helicobacter pylori (H . pylori) urease in healthy adults . In this study we aim to test the safety and adjuvant efficacy of LT delivered rectally together with recombinant H . pylori urease . Eighteen healthy adults without present or past H . pylori infection were enrolled in a double blind, randomized, ascending dose study to receive either urease (60 mg), or urease (60 mg) + LT (5 or 25 microg) . The immunization preparation was administered per rectum on days 0, 14 and 28 . Serum, stool and saliva anti-urease and anti-LT IgG and IgA antibodies (Abs) were measured and urease-specific and LT-specific antigen secreting cells (ASCs) were counted in peripheral blood at baseline and 7 (ASC counts) or 14 days (antibody levels) after each dosing . Peripheral blood lymphoproliferation assays were also performed at baseline and at the end of the study.Rectally delivered urease and LT were well tolerated . Among the 12 subjects assigned to urease+LT, 2 (16.7%) developed anti-urease IgG Abs, 1 (8.3%) developed anti-urease IgA Abs, and 3 (25%) showed urease-specific IgA(+) ASCs . Immune responses to LT were more vigorous, especially in subjects exposed to 5 microg LT . In the urease+ 5 microg LT group, anti-LT IgG and IgA Abs developed in 60 and 80% of the subjects, respectively, while LT-specific IgG(+) and IgA(+) ASCs were detected in all subjects . The magnitude of the anti-LT response was much higher than the response to urease . No IgA anti-urease or anti-LT Abs were detected in stool or saliva and lymphocyte proliferative responses to urease were unsatisfactory . In conclusion, rectal delivery of 5 microg LT is safe and induces vigorous systemic anti-LT immune responses . Further studies are needed to determine if LT can be an effective adjuvant for rectally delivered antigens.

J Mol Biol, 2002 Nov 29, 324(3), 409 - 28
DNA unwinding step-size of E . coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies; Lucius AL et al.; The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods . Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp . In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD . Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol) . However, additional steps, not linked directly to DNA unwinding are also detected . This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.

Vet Microbiol, 2003 Jan 2, 91(1), 65 - 72
Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E . coli isolates from pigs with diarrhoea; Osek J; The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E . coli strains isolated from pigs with postweaning diarrhoea . Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes . Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods . It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant . The most common EAST1-positive E . coli serotype was O149:K91 and these strains were mostly LTI/STII-positive . A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen . Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.

World J Gastroenterol, 2002 Dec, 8(6), 1134 - 7
Effects of recombinant human growth hormone on rat septic shock with intraabdominal infection by E . coli; Huang Y et al.; AIM: To investigate the therapeutic effects of recombinant human growth hormone (rhGH) on rat septic shock with intraabdominal infection by E . coli and its possible mechanism . METHODS: 76 SD rats were divided into 3 groups randomly: control group (group C, n=16) without any special treatment, septic shock group (group S, n=30) received bolus injection of E.coli (1X10(10) cfu x L(-1), 15 ml x kg(-1), ip), treated group (group T, n=30) received bolus injection of E.coli, and then followed by rhGH injection (2.25 U x k g(-1) x d(-1), im) . Group S and group T were further divided into 1d and 3d subgroups, respectively (n=15 each) . Mean arterial pressure (MAP), levels of plasma TNFalpha and endotoxin and leukocyte count were determined on 1st day and 3rd day after E.coli injection . Another 39 SD rats were divided into groups C, S and T (n=13 each) just for observing survival rate within 1 week . RESULTS: (1) On 1st and 3rd day, MAP in group S decreased markedly, and MAP on 1st day lowered more than that of 3rd day (P<0.01), while MAP in group T just decreased slightly . The survival rate within 1 week was much higher in group T (84.6 %) than in group S (46.2 %) (P<0.01) . (2)On 1st day, plasma TNFalpha and endotoxin elevated significantly in group S and group T (P<0.05), and endotoxin in group S had more increase (P<0.01) . On 3rd day, TNFagr in group S returned to the level of group C (P>0.05),while TNFagr in group T went down below the level of group C(P<0.01) . On 3rd day, endotoxin in group S declined, but was still higher than that of group C (P<0.01), endotoxin in group T returned to the level of group C (P>0.05) . (3) On 1st day, neutrophil ratio in total leukocyte count in both group S and group T increased significantly (P<0.05 vs group C) . CONCLUSION: rhGH showed beneficial effects on rat septic shock . The possible mechanisms may involve the attenuation of bacteria/endotoxin translocation and decreased systemic endotoxin level; inhibition of the production and release of TNFalpha; improved circulatory function; improved systemic host defenses and maintenance of intestinal mucosa barrier.

J Biomol Struct Dyn, 2002 Dec, 20(3), 381 - 7
Exploring the role of amino acid-18 of the leucine binding proteins of E . coli; Salopek-Sondi B et al.; Two periplasmic binding proteins of E . coli, the leucine specific-binding protein (LS) and leucine-isoleucine-valine binding protein (LIV), have high similarity in their structure and function, but show different substrate specificity . A key difference between these proteins is residue 18 in the binding pocket, a tryptophan residue in the LS and a tyrosine residue in the LIV . To examine the role of this residue in binding specificity, we used fluorescence and (19)F NMR to monitor ligand binding to three mutants: LSW18Y, LSW18F and LIVY18W . We observed leucine binding to all proteins . LS binds L-phenylalanine but the mutation from Trp to Tyr or Phe disallows this ligand and expands the binding repertoire to L-isoleucine and L-valine . The LIVY18W mutant still retains the ability to bind L-isoleucine and also binds L-phenylalanine.

Biol Chem, 2002 Sep, 383(9), 1435 - 40
Reconstitution of phospholipid flippase activity from E . coli inner membrane: a test of the protein translocon as a candidate flippase; Watkins WE et al.; Phospholipid flipping in biogenic membranes is a key feature of membrane bilayer assembly . Flipping is facilitated by proteinaceous transporters (flippases) that do not need metabolic energy to function . No flippase has yet been identified . The architecture of the E . coli protein translocon suggests that it could account for the flippase activity in the bacterial inner membrane . To test this possibility, we used E . coli cells depleted of SecYE or YidC to assay flipping in proteoliposomes reconstituted from detergent extracts of their inner membranes . We conclude that the protein translocon contributes minimally, if at all, to phospholipid flippase activity in the inner membrane.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Nov, 22(11), 1005 - 7
{Construction and identification of cDNA library of E.coli mRNA with poly(A) tracts}; Hu ZY et al.; OBJECTIVE: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts . METHODS: The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses . RESULTS: cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced . CONCLUSION: The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Nov, 22(11), 986 - 7
{Efficient and constant expression of cloned human beta2-microglobulin gene in E.coli}; Zhou ZM et al.; OBJECTIVE: To obtain human beta2-microglobulin (beta2m) gene that is to be efficiently expressed in E.coli . METHODS: beta2m cDNA including only the coding region for the protein was amplified from Raji cell line by reverse transcriptase-PCR via the primers 5'-GGTGGTCATATGGCTATCCAGCGTACTCCA-3' and 3'-GGTGGTTGCTCTTCCGACATGTCTCGATCC-3' . The product was subsequently cloned into a modified pBV220 vector after digestion with Nde I/Sap I . The recombinant plasmid pBV220-beta2m was transformed into E.coli BL21 after sequence analysis, and the fusion protein was then expressed via induction at 42 for 5 h at D(600) of 0.55-0.60, followed by purification through chitin beads . RESULTS: The beta2m cDNA was identical with those published in Genbank . The expressed fusion protein was identified in the form of inclusion body at the ratio more than 45 % of the E.coli proteins, and was denatured with 8 mol/L urea, followed by refold and purification to a high purity, displaying a relative molecular mass of 12 000 on 10 % SDS-PAGE gel . CONCLUSION: The human beta2m gene was cloned successfully and expressed efficiently and constantly in E.coli BL21,which lays the ground for engineering MHC-tetramers.

J Endocrinol, 2002 Nov, 175(2), 487 - 98
Inducible ablation of adipocytes in adult transgenic mice expressing the E . coli nitroreductase gene; Felmer R et al.; We describe the use of an enzyme prodrug system based on E . coli nitroreductase (NTR) to achieve the specific ablation of adipose tissue . Transgenic mice expressing the NTR gene specifically in the adipose tissue were generated using the adipocyte specific promoter aP2 . After treatment with the prodrug CB1954 these mice showed extensive cell depletion in all fat depots; this was directly correlated to both the dose of prodrug and the levels of NTR expression . Higher doses of CB1954 resulted in complete disappearance of visible adipose stores in some transgenic mice . These mice exhibited an impaired ability to thermoregulate body temperature . Lower doses of CB1954 resulted in a partial reduction of the adipose tissue leaving non-expressing cells that escape ablation . These animals show normal levels of blood glucose and triglycerides but have reduced leptin levels . After 30 days they were able to regenerate the fat depots and leptin levels returned to normal but, interestingly, no NTR-expressing cells were detectable . The present model provides a new approach to manipulate the number of adipocytes at different stages of mouse development and provides a new system for the study of fat metabolism especially in abnormal conditions such as obesity and its modulation through manipulation of the target cell population.

Biochimie, 2002 May-Jun, 84(5-6), 423 - 32
Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein; de Zamaroczy M et al.; A major group of colicins comprises molecules that possess nuclease activity and kill sensitive cells by cleaving RNA or DNA . Recent data open the possibility that the tRNase colicin D, the rRNase colicin E3 and the DNase colicin E7 undergo proteolytic processing, such that only the C-terminal domain of the molecule, carrying the nuclease activity, enters the cytoplasm . The proteases responsible for the proteolytic processing remain unidentified . In the case of colicin D, the characterization of a colicin D-resistant mutant shows that the inner membrane protease LepB is involved in colicin D toxicity, but is not solely responsible for the cleavage of colicin D . The lepB mutant resistant to colicin D remains sensitive to other colicins tested (B, E1, E3 and E2), and the mutant protease retains activity towards its normal substrates . The cleavage of colicin D observed in vitro releases a C-terminal fragment retaining tRNase activity, and occurs in a region of the amino acid sequence that is conserved in other nuclease colicins, suggesting that they may also require a processing step for their cytotoxicity . The immunity proteins of both colicins D and E3 appear to have a dual role, protecting the colicin molecule against proteolytic cleavage and inhibiting the nuclease activity of the colicin . The possibility that processing is an essential step common to cell killing by all nuclease colicins, and that the immunity protein must be removed from the colicin prior to processing, is discussed.

Mol Cell, 2002 Oct, 10(4), 917 - 24
Error-free recombinational repair predominates over mutagenic translesion replication in E . coli; Berdichevsky A et al.; Tolerance mechanisms are important in the ability of cells to cope with DNA damage . In E . coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR) . Here we show that RR effectively repairs gaps opposite DNA lesions . When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events . This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR . Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR . This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 800 - 3
{Expression of histone-based fusion protein HNHG in E.coli}; Dai FH et al.; Histone H1 contributes to condense nucleosome into super-structure during the transformation of chromatin into chromosome . It is shown in this rep or t that the fusion protein HNHG with the core of C-terminus of histone H1(0) expressed in BL21 (DE3) could also condense the plasmid DNA, just as histones did in nucleus . Under electron microscope, plasmid DNA condensed and supercoiled after t he addition of HNHG, in contrast to plasmid DNA control . This specific ability of the fusion protein HNHG of binding and condensing plasmids could be utilized to construct novel exogenous gene delivery systems . HNHG would be a promising candidate for gene delivery.

Int J Med Microbiol, 2002 Sep, 292(3-4), 185 - 93
Adherent-invasive Escherichia coli: a putative new E . coli pathotype associated with Crohn's disease; Darfeuille-Michaud A; Crohn's disease (CD) is an inflammatory bowel disease (IBD) of unknown aetiology . Genetically engineered rodent models showed that IBDs are immunologically mediated and that luminal bacteria play an essential role in the development of the inflammation . Various bacterial pathogens have been incriminated but results have been conflicting . A new pathovar of E . coli, designated adherent-invasive Escherichia coli (AIEC) may be associated with CD . AIEC strains colonize the intestinal mucosa by adhering to intestinal epithelial cells . They are also true invasive pathogens, able to invade intestinal epithelial cells via a macropinocytosis-like process, and to survive and replicate intracellularly after lysis of the endocytic vacuole . Within macrophages, AIEC strains survive and replicate extensively without inducing host cell death and induce the release of high amounts of TNFalpha . All these virulence properties designate AIEC as a possible pathogen potentially able to induce persistent intestinal inflammation, by crossing and breaching the intestinal barrier, moving to deep tissues, and continuously activating macrophages.

Radiats Biol Radioecol, 2002 Jul-Aug, 42(4), 395 - 8
{Radioprotective effect of E . coli endotoxin on hematopoietic stem cells is partially suppressed by inhibiting production of nitric oxide by administering N-omega-nitro-L-arginine}; Konopliannikov AG et al.; Lipopolysaccharide of E . coli (LPS) injected to mice one day before the total gamma-irradiation caused a substantial increase in the level of endogeneous colonies formed in the spleen . It is known that this type of endotoxin may result in considerable production of nitric oxide from macrophages in different tissues . Therefore it is possible that the activation of hemopoietic stem cells after LPS-treatment was completely or partially stimulated by nitric oxide, which is the most important physiological mediator, or by action some other mediators (cytokines and growth factors) produced by hemopoietic microenvironment elements in response to the action of nitric oxide . This assumption was checked in experience with combined treatment of mice by LPS and a nonspecific inhibitor of nitric oxide production--N omega-nitro-1-arginine (NA) . NA used in high dose (250 mg/kg) reduced partially (approximately by 30%) the LPS-increased level of spleen endogeneous colonies . When LPS was injected to mice 15 minutes after gamma-irradiation, this led to a slight increase in level of spleen colonies . In case when LPS was used together with NA after gamma-irradiation, this increase was still found.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Mar, 22(3), 203 - 5
Cloning E . coli mRNAs with poly (A) tails by restrction digest polymerase chain reaction; Hu ZY et al.; OBJECTIVE: To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli . METHODS: mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters . Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors . RESULTS: More than 100 gene fragments were cloned, 30 of which were sequenced . CONCLUSION: Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.

Nat Struct Biol, 2002 Nov, 9(11), 839 - 43
Insights into negative modulation of E . coli replication initiation from the structure of SeqA-hemimethylated DNA complex; Guarne A et al.; The SeqA protein binds clusters of fully methylated or hemimethylated GATC sequences at oriC and negatively modulates the initiation of DNA replication . We find that SeqA can be proteolytically cleaved into an N-terminal multimerization and a C-terminal DNA-binding domain and have determined the crystal structure of the C-terminal domain in complex with a hemimethylated GATC site . SeqA makes direct hydrogen bonds and van der Waals contacts with the hemimethylated A-T base pair in addition to interactions with the surrounding bases and DNA backbone . The tetrameric protein-DNA complex found in the crystal suggests that SeqA binds multiple GATC sites on separate DNA duplexes, altering the overall DNA topology and sequestering oriC from replication initiation.

Structure (Camb), 2002 Oct, 10(10), 1425 - 33
Structural and functional analysis of the kid toxin protein from E . coli plasmid R1; Hargreaves D et al.; We have determined the structure of Kid toxin protein from E . coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells . Kid forms a two-component system with its antagonist, Kis antitoxin . Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E . coli F plasmid despite the lack of any notable sequence similarity . Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB . A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 1060 - 7
Using a CFD model to understand the fluid dynamics promoting E . coli breakage in a high-pressure homogenizer; Miller J et al.; A Computational Fluid Dynamic (CFD) model of flow in a high-pressure homogenizing valve (APV Gaulin model 30CD) was developed with the Fluent software . The 2D model consists of an unstructured hexagonal mesh, dense in the regions of high gradients . The flow (single-phase) was modeled as laminar upstream of and in the channel (gap) and turbulent downstream of the channel exit . Applying a realizable kappa-epsilon turbulence model, the CFD model accurately predicted the effect of gap space on fluid dynamic conditions upstream (inlet pressure and pressure gradient) and downstream (impact pressure) of the channel for a valve with a standard (CD-0) impact distance (0.25 mm) and a 1 cP fluid . This CFD model was then used to estimate the magnitude of the fluid dynamic parameters (except cavitation effects) presumed to be responsible for cell breakage, as a function of gap space, impact distance and fluid viscosity . The CFD models predicted that for a given volumetric flowrate the upstream fluid conditions (inlet pressure gradient, maximum channel strain rate) and the maximum energy dissipation rate in the post-gap jet depend only on the gap space and the fluid viscosity and not on the impact distance . The impact pressure however depends on the gap spacing, the fluid viscosity and especially the impact distance . Experimental results indicate that higher inlet pressures are required to break cells, if the impact distance is increased . By conducting experiments to isolate individual cell breakage mechanisms for a single pass, threshold values were identified for breaking Escherichia coli cells: pressure gradient, 1.2 x 10(12) Pa/m; energy dissipation rate, 1.0 x 10(10) m(3)/s(2); and impact pressure, 160 psig . By isolating the wall impact as the sole mechanism responsible for breaking the E . coli cells between 3000 and 6000 psig inlet pressure, a relationship between E . coli cell breakage rate and maximum wall impact pressure was established (eq 5).

J Biochem Mol Biol, 2002 Sep 30, 35(5), 508 - 12
Expression of enzymatically-active phospholipase Cgamma2 in E . coli; Ozdener F et al.; Phospholipase C-gamma-2 (PLCgamma2) activation is a key signaling event for many cell functions . In order to delineate the pathways that lead to PLCgamma2 activation, we devised a quick method for obtaining sufficient PLCgamma2 . We obtained the full-length cDNA for human PLCgamma2 and expressed it in E . coli using the expression vector pT5T . To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons . The protein expression was detected in a Western blot analysis by both anti-PLCgamma2 antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal . Crude lysates that were prepared from bacteria that express PLCgamma2 were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate . Similar to previous reports on PLCgamma2 that is isolated from mammalian tissue, the recombinant enzyme was Ca2+ dependent with optimal activity at 1-10 microM Ca2+.

J Biochem (Tokyo), 2002 Oct, 132(4), 643 - 8
Comparative study on the biological properties of 2',5'-oligoadenylate derivatives with purified human RNase L expressed in E . coli; Yoshimura A et al.; An endoribonuclease, RNase L, which is activated in the presence of 2',5'-linked oligoadenylates, p(1-3)A(2'p5'A)(>2), is the terminal factor of the anti-viral action of interferon . Activation of RNase L results in inhibition of viral proliferation along with induction of apoptosis . Attempts to acquire more effective activators, 2-5A derivatives, have been made for the development of antiviral or anticancer agents . However, the ability of 2-5A derivatives to activate RNase L could not simply be compared due to the diversity of the assay methods used . We have now developed a facile method for assaying the activity of RNase L involving the use of non-fusion RNase L expressed in Escherichia coli and yeast 5S ribosomal RNA as a substrate . Using this method, several 2-5A derivative species have been revaluated . The results suggest that 2-5A molecules modified at the 8-position of the third (from the 5' terminus) adenine ring cause effective dimerization of RNase L and thus increase the ability of RNase L activation.

Reprod Domest Anim, 2002 Oct, 37(5), 269 - 74
Postovulatory effect of intravenous administration of lipopolysaccharide (E . coli, O55:B5) on the contractile activity of the oviduct, ova transport, binding of accessory spermatozoa to the zona pellucida and embryo development in sows; Mwanza AM et al.; The effect of lipopolysaccharide (LPS) (E . coli, O55:B5), administered 18 h after ovulation in the second oestrus after weaning, on the contractile activity of the oviduct, ova transport, sperm binding to zona pellucida (ZP) and embryo development, was studied in 14 Swedish crossbred (Landrace Yorkshire) multiparous sows . The endotoxin group (E-group) sows were administered with 300 ng/kg of LPS while the control group (C-group) sows were administered with 5 ml of saline i.v . via an indwelling jugular cannula . Immediately after evidence of standing oestrus, a Millar pressure transducer was placed intraluminally about 3 cm into the mid-isthmus, via laparotomy . Pressure recordings of the oviduct were collected from all conscious sows until slaughter . After slaughter, the genital tract opposite to the side with the transducer was retrieved, and three equal isthmic segments and the first third of the uterine horn part adjacent to the utero-tubal-junction (UTJ) were flushed separately to recover the ova . The intervals (mean+/-SD) from ovulation to slaughter (OS) and insemination to ovulation (IO) were not different between the E-group (44.5 +/- 5.7 h; 13.3 +/- 6.5 h) and the C-group (42.7 +/- 5.9 h; 14.8 +/- 4.1 h), respectively . Ova recovery rate (RR) in the E-group (80.2 +/- 22.9%) did not differ from that in the C-group (85.2 +/- 4.5%) . The frequency distribution of ova recovered in the different segments did not significantly (p > 0.05) differ between the groups . The E-group showed higher cleavage rate than controls . A higher proportion of spermatozoa bound to the ZP was also found in the E-group compared with controls . The isthmic intraluminal pressure slightly increased (p = 0.07) 18 h after ovulation and immediately following LPS in the E-group, compared with the C-group . The frequencies of phasic pressure fluctuations were significantly (p < 0.05) lower at 30 and 38 h after ovulation in the E- than in the C-group . It can be concluded from the present study that a single i.v . administration of LPS (300 ng/kg body weight) to sows, 18 h after ovulation might be associated with changes in isthmic pressure and the frequency of phasic pressure fluctuations, increased numbers of spermatozoa attached to the ZP and an enhanced embryo development but not with ova transport rates.

J Biochem Mol Biol, 2002 Mar 31, 35(2), 213 - 9
Rat malonyl-CoA decarboxylase; cloning, expression in E . coli and its biochemical characterization; Lee GY et al.; Malonyl-CoA decarboxylase (E.C.4.1.1.9) catalyzes the conversion of malonyl-CoA to acetyl-CoA . Although the metabolic role of this enzyme has not been fully defined, it has been reported that its deficiency is associated with mild mental retardation, seizures, hypotonia, cadiomyopathy, developmental delay, vomiting, hypoglycemia, metabolic acidosis, and malonic aciduria . Here, we isolated a cDNA clone for malonyl CoA decarboxylase from a rat brain cDNA library, expressed it in E . coli, and characterized its biochemical properties . The full-length cDNA contained a single open-reading frame that encoded 491 amino acid residues with a calculated molecular weight of 54, 762 Da . Its deduced amino acid sequence revealed a 65.6% identity to that from the goose uropigial gland . The sequence of the first 38 amino acids represents a putative mitochondrial targeting sequence, and the last 3 amino acid sequences (SKL) represent peroxisomal targeting ones . The expression of malonyl CoA decarboxylase was observed over a wide range of tissues as a single transcript of 2.0 kb in size . The recombinant protein that was expressed in E . coli was used to characterize the biochemical properties, which showed a typical Michaelis-Menten substrate saturation pattern . The Km and Vmax were calculated to be 68 microM and 42.6 micromol/min/mg, respectively.

J Mol Biol, 2002 Sep 27, 322(4), 663 - 7
Atomic resolution structure of a succinimide intermediate in E.coli CheY; Simonovic M et al.; Isomerization of aspartate to isoaspartate occurs spontaneously in proteins, causes changes in protein structures, and correlates positively with the aging processes of many organisms, including Alzheimer disease in humans . Aspartate isomerization proceeds through an unstable cyclic succinimide intermediate . There are few protein structure determinations that have characterized the intermediates and products of this isomerization reaction . Here we report the discovery of an unusually stabilized succinimide ring in the 1.1A structure of the Escherichia coli CheY protein, as determined from a crystal eight years old . The ring is formed by the side-chain of aspartate 75 and the backbone nitrogen of glycine 76 in an exposed loop of the molecule . Stabilization of the succinimide is through interaction of a sulfate ion oxygen atom with the imide nitrogen atom . Formation of the ring caused conformational changes in the loop, but did not alter the overall structure of the protein.

Biochemistry, 2002 Oct 1, 41(39), 11857 - 67
Communications between the high-affinity cyclic nucleotide binding sites in E . coli cyclic AMP receptor protein: effect of single site mutations; Lin SH et al.; The binding of adenosine 3',5'-cyclic monophosphate (cAMP) and its nonfunctional analogue, guanosine 3',5'-cyclic monophosphate (cGMP), to the adenosine 3',5'-cyclic monophosphate receptor protein (CRP) from Escherichia coli was investigated by means of fluorescence and isothermal titration calorimetry (ITC) at pH 7.8 and 25 degrees C . A biphasic fluorescence titration curve was observed, confirming the previous observation reported by this laboratory (Heyduk and Lee (1989) Biochemistry 28, 6914-6924) . However, the triphasic titration curve obtained from the ITC study suggests that the cAMP binding to CRP is more complicated than the previous conclusion that CRP binds sequentially two molecules of cAMP with negative cooperativity . The binding data can best be represented by a model for two identical interactive high-affinity sites and one low-affinity binding site . Unlike cAMP, the binding of cGMP to CRP exhibits no cooperativity between the high-affinity sites . The effects of mutations on the bindings of cAMP and cGMP to CRP were also investigated . The eight CRP mutants studied were K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L . These sites are located neither in the ligand binding site nor at the subunit interface . The binding was monitored by fluorescence . Although these mutations are at a variety of locations in CRP, the basic mechanism of CRP binding to cyclic nucleotides has not been affected . Two cyclic nucleotide molecules bind to the high-affinity sites of CRP . The cooperativity of cAMP binding is affected by mutation . It ranges from negative to positive cooperativity . The binding of cGMP shows none . With the exception of the T127L mutant, the free energy change for DNA-CRP complex formation increases linearly with increasing free energy change associated with the cooperativity of cAMP binding . This linear relationship implies that the protein molecule modulates the signal in the binding of cAMP, even though the mutation is not directly involved in cAMP or DNA binding . In addition, the significant differences in the amplitude of fluorescent signal indicate that the mutations also affect the surface characteristics of CRP . These results imply that these mutations are not perturbing specific pathways of signal transmission . Instead, these results are more consistent with the concept that CRP exists as an ensemble of native states, the distribution of which can be altered by these mutations.

Semin Thromb Hemost, 2002 Aug, 28(4), 383 - 92
Inhibition of B16-BL6 melanoma lung colonies by semisynthetic sulfaminoheparosan sulfates from E . coli K5 polysaccharide; Poggi A et al.; Heparin (H), heparan sulfate (HS), and related glycosaminoglycans can inhibit cancer cell invasion, possibly due to their ability to interact with vascular growth factors, adhesion molecules, endoglycosidases, and signaling proteins, in addition to the well-known effects on the clotting system . We evaluated the antitumor activity of a series of semisynthetic sulfaminoheparosan sulfates (SAHSs) with different degree and distribution of sulfates, obtained by chemical modifications of the E . coli K5 polysaccharide, namely type A, B, and C compounds . B16-BL6 melanoma cells (10 5 cells/mouse) were injected intravenously (i.v.) in a lateral tail vein of C57BL6 mice at a dose of 0.5 mg/ mouse together with test compounds . Tumor lung nodules were significantly reduced as compared with controls only by H (95.5 +/- 1.0% inhibition), SAHS-2 (84.2 +/- 5.0% inhibition), and SAHS-4 (91.1 +/- 4.2% inhibition), among compounds tested . SAHS-2 and SAHS-4 are type B compounds, with a sulfate/carboxylate ratio similar to that of H . A typical mammalian HS showed only 54.8% inhibition . Supersulfated low-molecular-weight heparin and heparan sulfate (ssLMWH and ssLMWHS) showed an activity similar to that of unfractionated compounds . H and SAHS-4 inhibited dose dependently B16-BL6 lung colonies, with IC-50 values of 0.05 and 0.1 mg/mouse, respectively . The relationship with ex vivo anticoagulant potency was evaluated by activated partial thromboplastin time (aPTT) on mouse plasma at different time intervals after i.v . injection (0.1 to 0.5 mg/mouse) of the compound . H showed a dose-dependent anticoagulant activity lasting up to 2 hours, whereas SAHS-4 showed a potent anticoagulant effect only at a dose of 0.5 mg/mouse . Accordingly, H but not SAHS-4 consistently inhibited B16-BL6 lung colonies when given 1 hour before tumor cells . SAHS-4 derivatives, with different size and/or affinity depleted of AT binding sites, showed an inhibitory effect on B16-BL6 melanoma similar to that of SAHS-4, suggesting that the greater antitumor effect of H was not due to AT-mediated inhibition of blood clotting . Interactions with other blood inhibitors, such as heparin cofactor II or tissue factor pathway inhibitory protein cannot be ruled out . The better effect of H may be due to persistence in the circulation and/or ability to inhibit tumor neoangiogenesis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(1), 49 - 55
Overexpression of a Novel Highly Active Tumor Necrosis Factor Mutant in E . coli; Gao CS et al.; A synthetic gene of human tumor necrosis factor alpha (hTNF-alpha) was mutated by polymerase chain reaction using a multi-site mutated primer with the Arg-2 codon (CGT) replaced by a lysine codon (AAA) . The mutated gene was inserted into vector pSB-92 at the downstream of P(L) promoter to give a recombinant plasmid pBS-TNF-K2 and expressed in E . coli . Soluble {Lys(2)} hTNF-alpha mutein was obtained in high yield, accounting for more than 60% of the total cellular protein and easy to purify . Compared to the wild type hTNF-alpha, purified {Lys(2)} hTNF-alpha has slightly less toxicity but shows tens to hundreds times greater inhibitory activity to several human tumor cell lines.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(1), 8 - 14
Expression of C-terminal Segment of Bone Morphogenetic Protein in E . coli; Lin S et al.; Two recombinant C-terminal segments of human bone morphogenetic protein 2A (BMP2A), designated as BMP23 (173 aa) and BMP24 (134 aa), were expressed under the control of P(L) promoter in E . coli . SDS-PAGE analysis showed two induced protein bands at 20 kD and 15.5 kD, which were about 10% and 20% of the total bacterial protein, respectively . Both the expressed proteins were insoluble aggregates, which reached 80% purity after rapid preparation of the products as the inclusion body . The results of N-terminal amino acid sequencing showed that the first 15amino acids at the N-terminal were identical to those encoded by the cDNA genes of both BMP24 . After refolding both BMP23 and BMP24 formed disulfide-linked dimeric protein bands in SDS-PAGE . Bioassay in vivo revealed that BMP23 induced chondrocytes differentiation and BMP24 stimulated collagen synthesis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(3), 265 - 271
Cloning of Human Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) cDNA and Its Expression in E . coli; Yao J et al.; Human GM-CSF cDNA fragment encoding the mature GM-CSF was obtained by using RT-PCR method with total RNA extracted from induced human fetal lung cells HFL . The sequence of the h GM-CSF cDNA thus obtained was the same as those reported . In order to get expression of a high level in, the 5' terminal nucleotide sequence of hGM-CSF cDNA was modified by using PCR . The modified h GM-CSF cDNA was inserted into plasmid pET-11d containing T7 promoter, with the resulted expression of plasmid pETC-5 . E . coli BL21(DE3) was transformed with pETC-5 and an expressed strain BLEC4 was selected . SDS-PAGE analysis revealed the rhGM-CSF was produced and accumulated up to 16% of the total cellular protein in the form of inclusion body in BLEC$ cells after induced by 0.5 mM IPTG for 2 h . ELISA and TF-1 cell culture assay showed that the biological activity of the partially purified and renatured rhGM-CSF was similar to that of the natural human GM-CSF.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(3), 257 - 264
The Cloning and Construction of a Murine-human Chimeric Antibody with Specificity for HBsAg and the Expression of the Chimeric Heavy Chain in E . coli; Chen YH et al.; The genes encoding the heavy and light chain variable regions (V(H) and V(K)) of the murine monoclonal antibody with the specificity for hepatitis B virus surface antigen (HBsAg) have been clone by using RT-PCR . They were combined to the Human C(gamma)(3) and C(K) genes respectively to construct the murine human chimeric antibody genes The chimeric heavy chain was expressed in E . coli, and characterized by SDS-PAGE, Western-blot, indirect ELISA Assay to demonstrate the expression product has the HBsAg-binding ability.

Bioseparation, 2001, 10(4-5), 189 - 96
Refolding of protein inclusion bodies directly from E . coli homogenate using expanded bed adsorption chromatography; Cho TH et al.; To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography . The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment . It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield . To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E . coli cell homogenate . This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 492 - 498
The Spectroscopic Study of E . coli Arginyl-tRNA Synthetase (ArgRS) and its Mutants; Gu WL et al.; The conformation of native enzyme ArgRS and its mutants ArgRS306KA, ArgRS306KR and ArgRS381KA was studied by absorbance spectrum, Difference spectrum with solvent perturbation, fluorescence spectrum and CD spectrum . The results showed that the chromophores of ArgRS306KR and ArgRS306KA had shifted to different micro environments in comparison to the native enzyme . Compared to the native enzyme, ArgRS306KA had and even bigger conformational change than ArgRS306KR, while the conformation difference between ArgRS381KA and ArgRS was not big enough to be perceivable in the spectrum . The analysis of CD spectrum showed the less the percentage of beta-turn in the ArgRS mutants, the lower the activity of the mutants . The conclusion can be drawn that the positive charge of Lys306 is very important to maintain the conformation of ArgRS, the change of conformation might be mainly responsible for the loss of enzyme of the mutants, while the substitution of Lys381 seems to cause no perceivable change of the enzyme conformation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(6), 583 - 589
Expression of the Variable Region Genes of the Monoclonal Antibodies Against Metal-bound Tetrapeptide in E . coli; Han LY et al.; The variable region genes of the light and heavy chains obtained from three stems of McAb against metal-bound tetrapeptide were joined into a single chain by a linker . A 39 bp fragment of the N-terminal of CGRP was joined to the C-terminal of the heavy chain to constitute the Lv-linker-Hv-CGRP single chain gene which was cloned into the vector pTC01 and expressed in E . coli 71/18 . The molecular weight of the expressed product was approximately 26 kD as shown by SDS-PAGE . Its expression level was about 20%-30% to he total cellular proteins . The product was a soluble protein and showed binding activity with its hapten by indirect ELISA assay.

Insect Mol Biol, 2002 Oct, 11(5), 487 - 96
Drosophila yolk protein produced in E . coli is accumulated by mosquito ovaries; Bownes M et al.; Despite similar functions, the yolk proteins of the higher dipteran flies and the vitellogenins found in other insects are unrelated at the sequence level and have evolved from different genes . Both are selectively endocytosed into the ovary via receptors belonging to the LDLR receptor subfamily . We cloned the Drosophila yp1 gene into an E . coli expression vector and showed that the yolk protein produced by E . coli is taken up into ovaries of both Drosophila melanogaster and the malaria mosquito Anopheles gambiae, which normally uses vitellogenin.

Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1500 - 14
Recognition of a cysteine substrate by E . coli gamma-glutamylcysteine synthetase probed by sulfoximine-based transition-state analogue inhibitors; Hiratake J et al.; A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E . coli gamma-glutamylcysteine synthetase (gamma-GCS) . The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition . As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution . The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO) . The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.

Structure (Camb), 2002 Sep, 10(9), 1211 - 23
E . coli dihydroorotate dehydrogenase reveals structural and functional distinctions between different classes of dihydroorotate dehydrogenases; Norager S et al.; The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP . Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus . The structure of class 2 E . coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain . The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs . Significant differences between the two classes of DHODs were identified by comparison of the E . coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 122 - 7
The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E . coli; Bizouarn T et al.; Proton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP(+) by NADH . Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel . Domains I and III have been separately expressed and characterized structurally by, e.g . X-ray crystallography and NMR . These domains catalyze transhydrogenation in the absence of domain II . However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme . Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme . In order to resolve the structure-function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III . This review describes some relevant recent results in this field of research.

Biochemistry, 2002 Sep 10, 41(36), 10985 - 93
Parallel symmetric immobile DNA junctions as substrates for E . coli RuvC Holliday junction resolvase; Sha R et al.; RuvC is a well-characterized Holliday junction resolvase from E . coli . The presence of symmetry in its preferred recognition sequence leads to ambiguity in the position of the crossover point in the junction, because a symmetric junction can undergo branch migration . Symmetric immobile junctions are junctions that contain such symmetric sites, but are prevented from migrating by their physical characteristics . RuvC activity had been analyzed previously by traditional symmetric immobile junctions, in which the helix axes are held antiparallel in a double-crossover motif . Bowtie junctions are branched four-arm molecules containing 5',5' and 3',3' linkages at their crossover points . A new type of symmetric immobile junction can be made by flanking the crossover point of a Bowtie junction with a symmetric sequence . The junction is immobile because mobility would lead to pairing between parallel, rather than antiparallel, nucleotide pairs . In contrast to conventional Holliday junctions and their analogues, the Bowtie junction assumes a parallel, rather than antiparallel, helical domain conformation, offering a new type of substrate for Holliday junction resolvases . Here, we report the digestion of Bowtie junctions by RuvC . We demonstrate that Bowtie junctions can function as symmetric immobile junctions in this system . We also show that RuvC cleaves antiparallel junctions much more efficiently than parallel junctions, where the protein can bind (and cleave) only one site at a time . These data suggest that the presence of two binding sites leads to communication between the two subunits of the enzyme to increase its activity.

Virus Genes, 2002, 25(1), 5 - 13
High-level expression of the C-terminal hydrophobic region of HCV E2 protein ectodomain in E . coli; Liu J et al.; High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult . The extreme hydrophobicity of this region has been suspected to be detrimental to the host . In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E . coli . Nonetheless, when the central region (aa 484-622) of E2 was deleted, full-length protein was efficiently produced . C-terminal region aa 567-700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein . However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein . Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568-571 of E2 . C-terminal region of E2 ectodomain (aa 567-730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level . The protein was highly insoluble and was purified under denaturing conditions . The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it . These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E . coli host and has no dominative adverse effect on its bacterial expression . Other nucleotide and/or amino acid sequence properties seem to play a more important role . This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(4), 383 - 388
His(6) Fusion Expression of Myristoyl-CoA: Protein N-myristoyltransferase in E . coli and its Purification; Xu ZP et al.; Saccharomyces cerevisiae Myristoyl-CoA: protein N-myristoyltransferase (NMT) gene was cloned into a His(6)-fusion expression vector pMFHT . After transforming into E . coli BL21 (DE3), His(6)-NMT was induced to express at 37 degrees by IPTG . SDS-PAGE analysis showed an induced expression product band of about 54 kD which constituted about 10% of the total bacterial proteins . The analysis of product solubility revealed that His(6)-NMT was predominantly soluble . On the basis of these results, His(6)-NMT was purified in one-step to 95% of purity from bacterial lysates using immobilized metal (Ni(2+)) chelation affinity chromatography . The in vitro labelling experiment demonstrated that His(6)-NMT had an activity similar to that of mature NMT, suggesting that the His(6)-tag did not affect the enzyme activity . His(6)-tag in the N-terminal of NMT makes it be possible to immobilized simply on Ni(2+)-IDA Sepharose 6B resin, which can be used to screen the peptide inhibitors of NMT from Phage Display random peptide library.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(6), 613 - 616
Cloning and High Expression in E.coli of a Chimeric Gene Coding for Rice Fructose-l,6-bisphosphate Aldolase; Chen HB et al.; A chimeric gene (l 104 bp) coding for rice fructose-l,6-bisphosphate aldolase has been constructed by DNA recombination of a synthetic 5'- fragment (-24 to 272) and an RT-PCR amplified product at restriction site S fu I . The synthetic fragment was assembled from six oligonucleotides by T4 DNA ligase reaction according to a single-stranded DNA method (Chen H-B et al, Nucleic Acids Res 1990, 18, 871-878), the PCR amplified fragment (217 - 1 080) was obtained by carrying out a PCR in the presence of rice cDNA as the template and both the 5'- and the 3'- primers . The whole gene was successively cloned into plasmids pWR13 and pPLc2833, and highly expressed in E . coli to produce the expected product . After purification through stepwise precipitation and cation-exchange column chromatography, the recombinant aldolase showed an enzyme activity as high as (1l.0 +/- 0.3) units/mg with the turnover number = 27s(-1) and K(m) (FBP)=4.2 &mgr;M by two different methods.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(6), 591 - 596
The Overproduction of Leucyl-tRNA Synthetase in E . coli and Its Purification; Li T et al.; The 3.2 kb gene (leuS) encoding for leucyl-tRNA synthetase (LeuRS) has been cloned from E.coli K-12, and overexpressed 35 times more than that in the host strain TG1 . In order to further increase the production of LeuRS, two types of leuS with different length of the 3' flanking region: leuS1 has an additional 130 bp over that of leuS2 were ligated into pKK-233-2 and pTrc-99B, respectively . In E . coli TG1 transformant harboring the recombination plasmid pTrc-99B with leuS2, the yield of LeuRS was 135 times that in TG1 strain . The purified enzyme that showed one band on SDS-PAGE was obtained after two steps of column chromatography . During the construction of plasmid, a substitute of G for C was introduced at position of base 4 in the coding region of leuS, so that Gln2 of LeuRS was changed to Glu . This enzyme was designated LeuRS2E . The kinetic constants of LeuRS2E showed that the substitution has no effect on the enzyme activity and could be used in the studies of LeuRS as the native enzyme.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 249 - 52
{High-level expression of human acidic fibroblast growth factor in E . coli and its purification}; Li X et al.; A reconstructed human acidic fibroblast growth factor (haFGF) cDNA was cloned into the expression vector pkk223-3, and the expression in Escherichia coli . JM109 was induced by IPTG induction; the expression level of the recombinant haFGF was about 80 mg/L . The expressed haFGF was purified to identity by heparin affinity chromatography and the recovery rate of haFGF was 65% . The specific activity of the purified haFGF was ED50 4.6 ng/ml . The characters of recombinant haFGF was identical to that of natural one.

Arch Virol, 2002 Sep, 147(9), 1733 - 45
Expression of immunoreactive forms of the hepatitis C NS5A protein in E . coli and their use for diagnostic assays; Kalamvoki M et al.; In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E . coli as fusion proteins with the MBP or the GST protein . The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays . A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A . Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein . None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens . With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen . The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV.

Biochim Biophys Acta, 2002 Aug 15, 1572(1), 101 - 6
Cell-to-cell communication in response of E . coli cells at different phases of growth to low-intensity microwaves; Shcheglov VS et al.; Effects of millimeter waves (MMW) at the frequency of 51.755 GHz were studied in logarithmic and stationary E . coli cells at various cell densities . The changes in the genome conformational state (GCS) were analyzed by the method of anomalous viscosity time dependence (AVTD) . Before lysis, the cells were adjusted to the cell density of 4x10(7) cells/ml and all AVTD measurements were run at this cell density . Stationary cells responded to MMW by increase in AVTD, while the same MMW exposure decreased AVTD in logarithmic cells . MMW effects depended on cell density during exposure and were stronger for stationary cells . The observed dependence on cell density suggested a cell-to-cell communication between cells during exposure to microwaves . Decrease in power density (PD) resulted in more striking differences between responses at different cell densities . The data provided evidence that intercellular communication in response to MMW depended on cell status and PD of microwaves . The MMW effects were studied in more detail at low intensity of 10(-17) W/cm(2) in the range of cell densities 4x10(7) to 8x10(8) cells/ml . The obtained sigmoid-like dependence of MMW effect on cell density saturated at approximately 5x10(8) cells/ml . The dependence of MMW effect on cell density was very similar in this study and in previous studies with weak extremely low frequency (ELF) electromagnetic fields (EMF) . The data suggested that cell-to-cell communication might be involved in response of cells to weak EMF of various frequency ranges.

Radiat Environ Biophys, 2002 Jun, 41(2), 145 - 8
Radiation sensitivity of N . pharaonis in comparison with E . coli K12 strains; Quint RM et al.; To investigate the radiation sensitivity of the natronobacterium Natronomonas pharaonis in comparison with Escherichia coli strains (N . pharaonis DSM 2160T, E . coli strains AB1157 and K12 lambda s) were exposed to gamma-radiation (60Co-gamma-source, 100 Gy min-1) in the presence of oxygen (air) and under strongly reduced oxygen conditions (argon-saturated medium) . After irradiation, the colony-forming ability (dose-survival curves) and the D37 dose were determined . The oxygen content of the solutions containing high NaCl concentrations was measured with an oxygen electrode (Clark electrode) . It was found that N . pharaonis can tolerate a remarkably higher irradiation dose than the two E . coli strains (approx . 1.5-fold of K12 lambda s and approx . 4-fold of AB1157) . The oxygen enhancement ratio (OER) is 2.8 for N . pharaonis and 2.6 for both E . coli strains . Therefore the higher radiation resistance of the N . pharaonis is not due to the low oxygen content of the cell solution (high salt concentration) but is probably related to the higher DNA repair ability of this archaebacteria strain.

Bioorg Khim, 2002 Jul-Aug, 28(4), 332 - 40
{Cleavage of RNA in a hybrid duplex by E . coli ribonuclease H . III . Substrate characteristics of hybrid duplexes of RNA and oligodeoxyribonucleotides containing a bleomycin A5 residue}; Vorob'ev PE et al.; The effect of the bleomycin A5 residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E . coli RNase H . The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides . The RNA in the bleomycin-containing hybrid duplexes is cleaved by the E . coli RNase H; however, the initial hydrolysis rate (v0) is 2.6-3.4-fold reduced for the continuous duplexes . In the case of tandem hybrid complexes, the effect of bleomycin on v0 was less pronounced . We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes . Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties . The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol . 28, no . 4; see also http://www.maik.ru.

Biochemistry, 2002 Sep 3, 41(35), 10834 - 48
Mutational, kinetic, and NMR studies of the mechanism of E . coli GDP-mannose mannosyl hydrolase, an unusual Nudix enzyme; Legler PM et al.; GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P . M., Massiah, M . A., Bessman, M . J., and Mildvan, A . S . (2000) Biochemistry 39, 8603-8608) . Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations . Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10(2.2)-fold, increased the dissociation constant of Mn2+ from the ternary E-Mn2+-GDP complex 3-fold, increased the K(m)Mg2+ 20-fold, and decreased the paramagnetic effect of Mn2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn2+ . In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn2+ on its side chain -NH2 group . With wild-type GDPMH, the effect of pH on log(kcat/K(m)GDPmann) at 37 degrees C showed an ascending limb of unit slope, followed by a plateau yielding a pK(a) of 6.4, which increased to 6.7 +/- 0.1 in the pH dependence of log(kcat) . The general base catalyst was identified as a neutral His residue by the DeltaH(ionization) = 7.0 +/- 0.7 kcal/mol, by the increase in pK(a) with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln . Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10(3.4)-fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role . The H88Q mutant showed a 10(2.3)-fold decrease in kcat, a 4.4-fold increase in K(m)GDPmann, and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis . One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole HdeltaC, H(epsilon)C, N(delta), and N(epsilon) resonances of the four histidines and defined their protonation states . The pK(a) of His-124 (6.94 +/- 0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/- 0.20) . The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5 . Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys . The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold . The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state . Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state . From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.

Rev Med Chil, 2002 Jun, 130(6), 603 - 9
{Evaluation of molecular and immunoenzymatic assays for detecting Enterohemorrhagic E coli in food borne outbreaks}; Vidal M et al.; BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC), is an emergent pathogen that causes sporadic infections and outbreaks of gastroenteritis associated with consumption of contaminated food products . Because detection of EHEC in diarrhea patients is not routinely performed, infection is most probably underestimated . AIM: To compare three techniques to detect EHEC: Colony hybridization with DNA probes, polymerase chain reaction (PCR) for the detection of stx1 and stx2 genes and immunoenzymatic detection by ELISA (Premier EHEC) of Stx1 and Stx2 toxins . MATERIAL AND METHODS: Four outbreaks of food-borne gastroenteritis were studied including 16 patients and 78 strains of E coli . Twenty-one (26.9%) strains, hybridized with the stx1 probe, 1 (1.3%) hybridized only with the stx2 probe and 36 (46.1%) with both probes . PCR amplification for cytotoxin genes was observed in 6 strains (7.7%) from the second outbreak studied . The immunoenzymatic assay detected the cytotoxins in 18 (23.0%), of the 78 studied strains . Agreement between probes and ELISA was 44.8%, between PCR and probes 34.7% and 82.4% between ELISA and PCR . CONCLUSIONS: These results indicate a variable yield among different EHEC detection techniques . Considering PCR as the gold standard, ELISA technique showed a better sensitivity and specificity than probes.

Med Dosw Mikrobiol, 2002, 54(2), 119 - 27
{Evaluation of practical usefulness of selected phenotypic and genotypic markers for pathogenicity of enterotoxic and entero-hemorrhagic strains of E.coli . }; Karakulska J; This study included a description of enterotoxic and verocytotoxic activity of thirty strains of E . coli and their ability to produce beta-haemolysis in a ram blood medium . Enterotoxic and verocytoxic activity was determined by using RPLA test . The synthesis of enterotoxin LT was observed in 10 strains and the production of E . coli shiga toxins type 1--Stx1 (1 strain) or type 2 (2 strains) was observed in 3 strains of serotype O157: H7 . The beta-haemolytic characteristics in vitro were demonstrated by 17 strains (57%) isolated from pigs . Among them 16 (94%) were found to possess the genes that determine the enterotoxins or enterotoxins and E . coli shiga toxin Stx2v synthesis . Using the PCR technique, 21 strains (70%) were found to possess the genetic determinants of enterotoxins LT, STa and/or STb synthesis or enterotoxins and E . coli shiga toxin Stx2v synthesis . Further genetic study showed that the strains possessed the genes elt and estB were predominant (33%) among the toxic strains of E . coli isolated from piglets and calves.

Protein Expr Purif, 2002 Aug, 25(3), 430 - 6
Efficient expression and secretion of recombinant hirudin III in E . coli using the L-asparaginase II signal sequence; Tan S et al.; One of the hirudin variants HV3 was efficiently expressed in Escherichia coli using the L-asparaginase II signal sequence and the product was secreted into the culture medium . For the secretory manufacture of HV3, the L-asparaginase II signal sequence containing a single NheI restriction site at its 3' end was designed using the degenerate codons and PCR-amplified from E . coli chromosomal DNA . The synthetic HV3 coding sequence was fused to the signal sequence in-frame by its 5' NheI restriction site . The above signal-HV3 fusion gene was inserted into an expression vector pTA, which was derived from pkk223-3 such that its expression was under the control of the tac promotor . The resulting HV3 secretion expression vector pTASH thus constructed was introduced into an E . coli host cell AS1.357 with high L-asparaginase II producing level . After inducing with IPTG, the expression product was efficiently secreted into the culture medium and shake-flask culturing gave a yield of approximately 5 x 10(5)ATU/L (approximately 60mg/L) . The secreted HV3 was easily purified from culture supernatant using ultrafiltration, ion-exchange column chromatography, and FPLC reverse-phase chromatography . The purified rHV3 from the culture supernatant had the expected N-terminal amino sequence and strong antithrombin activity, suggesting that the signal sequence was completely removed and the product was processed accurately during the secretion process.

PDA J Pharm Sci Technol, 2002 Jul-Aug, 56(4), 220 - 7
Wet heat inactivation of lipopolysaccharide from E . coli serotype 055:B5; Fujii S et al.; Wet heat inactivation of lipopolysaccharide (LPS) from E . coli Serotype 055:B5 was investigated . The LPS solutions were heated at study temperatures ranging from 78 degrees C to 175 degrees C, and were assayed using the Limulus Amebocyte Lysate (LAL) test . Plots of the log of the amount of endotoxin remaining versus heating time showed biphasic decreases . The initial slopes are associated with a faster rate of decrease to about a 0.5-log unit reduction, and were followed by a slower, linear rate of decline in the secondary slopes . The curves were applied to the biexponential model expressed by the equation (Et = E1e-klt + E2e-k2t) . The LPS inactivation rates (k1 and k2) each conformed to their own Arrhenius equation . Therefore, the processes required to achieve the desired level of LPS inactivation can be obtained by mathematical means.

Lett Appl Microbiol, 2002, 35(3), 218 - 22
Isolation of Shiga toxin-producing Escherichia coli O103 from sheep using automated immunomagnetic separation (AIMS) and AIMS-ELISA: sheep as the source of a clinical E . coli O103 case?
Urdahl AM, Cudjoe K, Wahl E, Heir E, Wasteson Y.
AIMS: To investigate whether a sheep flock was the original reservoir of a Shiga toxin-producing Escherichia coli (STEC) O103 strain causing a clinical human case and to compare the two diagnostic methods automated immunomagnetic separation (AIMS) and AIMS-ELISA . METHODS AND RESULTS : AIMS detected Escherichia coli O103 in 36.5% of the samples and AIMS-ELISA detected E . coli O103 in 52.1% of the samples . Polymerase chain reaction detected stx1 and eae in three of 109 E . coli O103 isolates . Pulsed field gel electrophoresis showed that the sheep and human STEC O103 were characterized by distinctly different profiles . CONCLUSIONS: The sheep flock was shown to carry STEC O103, although an association between the sheep flock and the clinical human case could neither be proven nor eliminated . Substantial agreement was found between AIMS and AIMS-ELISA, but AIMS-ELISA was less time consuming and resulted in a higher recovery of E . coli O103 . SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that sheep may be carriers of STEC that are associated with human disease and that the methods described can be used to increase the sensitivity of STEC detection.

Blood, 2002 Sep 1, 100(5), 1869 - 77
Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation; Mollnes TE et al.; Complement plays an essential role in inflammation and tissue damage . However, it is largely unknown to what extent the system acts as a primary inducer of secondary mediator systems in the inflammatory network of human whole blood . Here we describe a novel in vitro model using the thrombin-specific hirudin analog lepirudin as anticoagulant, which, in contrast to heparin, did not interfere with complement activation . The model was used to study the role of complement in Escherichia coli-induced inflammatory responses . Granulocyte and monocyte oxidative burst was complement dependent as it was reduced by 85% and 70%, respectively, by the C3 {corrected} binding peptide compstatin . A similar reduction was found by inhibition of C5, C5a, and C5a receptor (C5aR) . Furthermore, anti-CR3 antibodies were as efficient as the C5aR antagonist in reducing granulocyte oxidative burst, whereas blocking CD14 or C3aR had no effect . Up-regulation of granulocyte CR3 was virtually abolished by a C5aR antagonist . Opsonization and phagocytosis was completely inhibited by blocking of C5aR or CR3, whereas blocking of the FcgammaRs (CD16, CD32, CD64) had no effect . In contrast to oxidative burst and phagocytosis, cytokine secretion was largely complement independent . Thus, anti-CD14 abolished tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 secretion, whereas IL-8 was equally inhibited by anti-CD14 and compstatin . In conclusion, the present model is particularly useful for studying complement as part of the inflammatory network . The results emphasize a crucial role for C5a-C5aR interaction in E coli-induced up-regulation of CR3 and the subsequent oxidative burst and phagocytosis . Complement inhibition may have therapeutic implications in oxidative burst-induced tissue damage.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(3), 288 - 292
High Expression of a cDNA Coding for Human Lymphotoxin alpha Derivative in E . coli and Purification of Its Protein Product; Li P et al.; Using PCR, a cDNA coding for human lymphotoxin alpha derivative (hLTalphaDa) lacking 27 amino acids at N-terminal of natural hLT was constructed . The expression construct was expressed in E . coli BL21 (DE3) . The product of expression was in the form of inclusion bodies, and accounted for 60%-80% of total bacterial proteins . The product protein was purified to over 95% by treatments of inclusion bodies . The specific activity of hLTalphaDa was above 2x10(8) IU(per mg protein) . Its cytotoxic activity can be neutralized by monoclone antibody against hLT . The anti-tumor effects of hLTalphaDa were also tested in vitro and in vivo.

EMBO J, 2002 Aug 15, 21(16), 4357 - 67
Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal; Jin H et al.; An UGA stop codon context which is inefficient because of the 3'-flanking context and the last two amino acids in the gene protein product has a negative effect on gene expression, as shown using a model protein A' gene . This is particularly true at low mRNA levels, corresponding to a high intracellular ribosome/mRNA ratio . The negative effect is smaller if this ratio is decreased, or if the distance between the initiation and termination signals is increased . The results suggest that an inefficient termination codon can cause ribosomal pausing and queuing along the upstream mRNA region, thus blocking translation initiation of short genes . This cis control effect is dependent on the stop codon context, including the C-terminal amino acids in the gene product, the translation initiation signal strength, the ribosome/mRNA ratio and the size of the mRNA coding region . A large proportion of poorly expressed natural Escherichia coli genes are small, and the weak termination codon UGA is under-represented in small, highly expressed E.coli genes as compared with the efficient stop codon UAA.

Bioinformatics, 2002, 18 Suppl 1, S241 - 8
Modelling regulatory pathways in E . coli from time series expression profiles; Ong IM et al.; MOTIVATION: Cells continuously reprogram their gene expression network as they move through the cell cycle or sense changes in their environment . In order to understand the regulation of cells, time series expression profiles provide a more complete picture than single time point expression profiles . Few analysis techniques, however, are well suited to modelling such time series data . RESULTS: We describe an approach that naturally handles time series data with the capabilities of modelling causality, feedback loops, and environmental or hidden variables using a Dynamic Bayesian network . We also present a novel way of combining prior biological knowledge and current observations to improve the quality of analysis and to model interactions between sets of genes rather than individual genes . Our approach is evaluated on time series expression data measured in response to physiological changes that affect tryptophan metabolism in E . coli . Results indicate that this approach is capable of finding correlations between sets of related genes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 387 - 392
Molecular Cloning of Human Interleukin-5 cDNA and Its Expression in E . coli; Mei JJ et al.; hIL-5 cDNA was amplified through reverse transcription-polymerase chain reaction from peripheral blood lymphocytes induced with PMA and calcium ionophore A23187 . The hIL-5 fragment was cloned into pUC18 and its sequence was identified to be hIL-5 cDNA sequence . The fragment which encodes hIL-5 mature polypeptide was amplified and cloned into pBV220 to express hIL-5 recombinant protein in E . coli, but there was no expected recombinant protein expressed . Only one clone expressed a 10kD peptide at high level . DNA sequencing showed that this clone had an adenosine deletion at 86th codon in hIL-5 cDNA and expressed a polypeptide of 93 amino acids at high level, and hIL-5 cDNA had not yet been expressed in E.coli successfully . These results suggested that two consecutive rare codons after 86th codon in hIL-5 cDNA could block polypeptide synthesis in E . coli . Through recombinant PCR technology the rare codons after 86th codon in hIL-5 cDNA were mutated into corresponding codons preferentially used in E . coli, and the mutated hIL-5 cDNA was highly expressed in pBVhIL5-H/DH5alpha to approximately 15% TCP after thermal induction . hIL-5 recombinant protein existed as inclusion bodies in E . coli . ELISA with a cross-reacting rat anti-mIL-5 monoclonal antibody confirmed the expressed product was hIL-5 recombinant protein.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 369 - 374
Construction of Single-chain Antibody to Carcinoembryonic Antigen and Its High Expression in E . coli; Li B et al.; The heavy and light chain variable region (V(H) and V(K)) genes encoding the murine monoclonal antibody E(7)B(10) to carcinoembryonic antigen were linked into single-chain antibody gene with 36 oligonucleotide (Gly(3)Ser)(3) by using recombinant PCR . The construct was highly expressed in E . coli as inclusion bodies (IB), accounting for 20% of the total bacteria proteins, and was characterized by SDS-PAGE and Western blot . When the content of inclusion bodies was denatured and followed by renaturation, it was shown to possess abiliby to kind to its specific antigen CEA by using RIA and competitive ELISA.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(6), 611 - 617
Effect of Deletion of 245 and 252 Arginines in E . coli Arginyl-tRNA Synthetase on Its Structure and Function; Wu JF et al.; In order to study structure and function of E.coli arginyl-tRNA synthetase(ArgRS), deletion of R245 and 252 in this enzyme was carried out by site-directed mutagenesis of gene coding for this enzyme, respectively . The genes, argSdeltar245 and argSdeltar252-encoding ArgRS mutant, ArgRSdeltaR245 and ArgRSdeltaR252 were obtained and cloned into pUC18 or pTrc99B, respectively . The expression of argSdeltar245 and the properties of the mutant ArgRSdeltaR245 were studied . The gene encoding ArgRS, argS was expressed as a soluble protein; however, ArgRS deleted R245 formed inclusion body during the expression of argSdeltar245 in E . coli . After dissolution of the inclusion body by 8 mol/L urea or 7 mol/L guandidine chloride, followed by renaturation by the methods of gel filtration or dilution, the specific activity of the mutant enzyme, ArgRSdeltaR245 was about 40 unit/mg, that is only 0.3% activity of the native enzyme . By detection of ultraviolet absorbance and difference spectroscopy and titration of DTNB, the conformations of the native and mutant enzymes were compared . It was shown that the tertiary structure of ArgRSdeltaR245 was more relaxed than that of ArgRS, and the chromophoric groups in the mutant enzyme were more exposed than that of the native enzyme . It may be the main reason of forming inclusion body during the mutant gene expression and losing activity of enzyme.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(6), 593 - 596
Cloning and Expression of aroG Gene of E . coli and Its Co-expression with pheA and tyrB Genes; Jiang PH et al.; 3-Deoxy-D-arabino-heptulonate-7-phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway . In E . coli, DAHP is encoded by aroG Gene . In this work, aroG was cloned from an E . coli mutant strain resistant to m-fluro-L-phenylalanine (mPF) and p-fluro-L-phenylalanine (pPF) by PCR . The gene was expressed under the control of lambda phage promoter p(R) in P2392 strain of E . coli . Distinct band was detected as the product of aroG on SDS-PAGE . The specific activity in crude extract of DAHP was raised to 1.7-fold . Based on the cloning and expression of pheA (encoding both chorsmate mutase CM and prephenate dehydratase PD) and tyrB (encoding phenylalanine aminotransferase PAT) genes, aroG, pheA and tyrB genes were constructed and expressed in P2392 . The results showed that the specific activities of DAPH, CM/PD and PAT in crude extracts were increased by 1.7, 13.9/7.8 and 2.3-fold, respectively.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(6), 544 - 549
A New Strategy for Optimizing the Translation Initiation Rate of Foreign Gene Expression in E . coli; Wang YL et al.; The translation initiation rate is greatly affected by the secondary structure of the translation initiation region (TIR) of mRNA . A novel system was established for improving the translation initiation rate of a foreign gene in E . coli . As a model, the 5' 114 bp coding sequence (38 amino acids from the start codon) of human proliferating cell nuclear antigen (PCNA) gene was fused with the lacZ' gene at its 5' end in vector pTZ19R . A Shine/Dalgarno (SD) sequence GAGGT was inserted to the -8 position of AUG by site-directed mutation . Then the flanking sequences of SD, which were the 6 nucleotides upstream the SD and the 7 nucleotides between SD and AUG, were randomly changed by PCR using a synthetic primer with partially random sequences . This random mutation led to potential variations in the secondary structure of the TIR of mRNA through base pairing with the 5' coding sequence . The 5' PCNA-LacZ' mRNA could be efficiently and specifically transcribed by inducible T7 RNA polymerase in E . coli strain JM 109 (DE3) . There were 269 clones of 5' PCNA-LacZ' fusion plasmid selected first by the blue color on X-gal plate and then by hybridization with a 5' PCNA probe . Eight clones with different blue colors among the 269 clones were chosen for beta-galactosidase activity assay . The results showed that the difference between their enzyme activities was more than 20 fold, but there seemed no apparent difference at the transcription level as assayed by RNA dot hybridization, which suggested that the difference in the expression of the fusion protein was due to the different rate of translation initiation . Thus, by this strategy, an effective translation initiation region for the high expression of human PCNA gene in E . coli was obtained.

Genes Cells, 2002 Aug, 7(8), 755 - 68
Transcription termination and anti-termination in E . coli; Nudler E et al.; Transcription termination in Escherichia coli is controlled by many factors . The sequence of the DNA template, the structure of the transcript, and the actions of auxiliary proteins all play a role in determining the efficiency of the process . Termination is regulated and can be enhanced or suppressed by host and phage proteins . This complex reaction is rapidly yielding to biochemical and structural analysis of the interacting factors . Below we review and attempt to unify into basic principles the remarkable recent progress in understanding transcription termination and anti-termination.

Presse Med, 2002 Jun 22, 31(22), 1021 - 3
{Pulmonary strongyloidiasis complicated by E . coli meningitis in a HIV-1 and HTLV-1 positive patient}; Hovette P et al.; INTRODUCTION: Disseminated strongyloidiasis occurs in immunodepressed patients, notably those infected by retroviruses . OBSERVATION: A pulmonary strongyloidiasis, complicated by an Escherichia coli meningitis, occurred in a patient exhibiting seropositivity HIV1 for the past year . The status of cell immunity, with 354 lymphocytes T CD4+/mm3, could not explain this severe complication . This led to the diagnosis of an HTLV1 infection . The strongyloidiasis was treated with two cycles of ivermectine, which cured the patient . COMMENTS: In HIV-infected patients exhibiting severe strongyloidiasis, research for an HTLV co-infection is recommended.

Biochim Biophys Acta, 2002 Jul 29, 1598(1-2), 147 - 55
Cloning, expression, secondary structure characterization of HMG box 1 of hUBF from E . coli and its binding to DNA; Yang W et al.; Human upstream binding factor (hUBF) belonging to a family of protein containing DNA binding domain-HMG box, is important in the activation of rRNA gene transcription . It contains six tandemly arranged HMG box domains, each of which is thought to be as a basic architectural unit in the interaction of DNA and protein . Here the DNA binding domain of hUBF HMG box 1 was cloned and heterologously expressed in Escherichia coli . Through a single purification step using a Ni2+-chelating column, the highly purified recombinant protein could be obtained . This recombinant protein contains 99 amino acids with a hexahistidine tag added to the C-terminus . It was expressed as a monomer, which was determined by gel filtration . Circular dischroism studies show that it comprises approximately 54.3% alpha-helix and 43.6% random coil at pH 7 . This result is in good agreement with that of FTIR, which are 59.9% alpha-helix and 40.1% random coil . There is no obvious change for the secondary structure of the recombinant protein as increasing pH from 5.0 to 12.0 . But denaturation occurs at pH 3.0 . Like many HMG box domains that were found in other proteins, it could bind to four-way DNA junction, a putative intermediate in DNA recombination, in a structure-specific manner . Magnesium ion has no effect on this binding activity, which is determined by both gel mobility shift assays and surface plasmon resonance (SPR) . Since Mg2+ is present in the nucleus and RNA polymerase I is Mg2+-stimulated, we believe that this property is relevant for hUBF in vivo . SPR research shows that the recombinant hUBF HMG box 1 also has a strong binding ability to a GC-rich fragment within the rRNA gene core promoter.

Auton Neurosci, 2002 Jun 28, 98(1-2), 99 - 101
NO-ergic mechanisms are implicated in a disturbed cardiac rhythm after systemic application of lipopolysaccharide E . coli to rats; Koulchitsky SV et al.; In acute experiments on nembutal-urethan-anaesthetized rats, a slow infusion of subseptic dose of lipopolysaccharide (LPS) Escherichia coli (1 mg/ml) via the right jugular vein immediately led to bradycardia and extrasystoles . Preliminary administration of 20 mg/kg N(G)-nitro-L-arginine methyl ester (L-NAME) or 30 mg/kg aminoguanidine hydrochloride prevented the LPS-induced extrasystoles but did not affect the pattern of bradycardia . We conclude that nitric oxide (NO)-ergic mechanisms are involved in provoking electrical instability of the heart in conditions of endotoxemia.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(2), 163 - 166
Cloning and Expression of Annexin V cDNA in E.coli; Sun JX et al.; The cDNA encoding the mature annexin V was isolated by using RT-PCR method from total RNAs of fresh human placenta . The result of sequencing indicated that the sequence of isolated annexin V cDNA was the same as the reported nucleotide sequence of annexin V . The annexin V cDNA was cloned into expression plasmid pET24a(+) under T7 promoter and then transformed into E.coli BL21(DE3) . SDS-PAGE analysis revealed that the human annexin V was highly expressed and accumulated up to 38% of total bacterial proteins in soluble form after the induction by 1 mmol/L IPTG . The purified human annexin V can significantly prolong activated partial thromboplastin time (APTT).

Zhonghua Yi Xue Za Zhi, 2002 May 10, 82(9), 593 - 6
{Cloning expression and characterization of a multifunctional anticoagulated peptide gene in E . Coli}; Mu R et al.; OBJECTIVE: To investigate the feasibility about the cloning, expression and characterization of multifunctional anticoagulated peptide . METHODS: We designed a construct using glutathione S-transferase (GST) as a protein vector, fused via a cleavable linker to an antithrombotic peptide of 31 amino acids . The peptide was designed to include three inhibitory regions: (1) the Lys-Gly-Asp (KGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin . The amino acid sequence of the 31 amino acid peptide was reverse translated, and the gene was chemically synthesized and cloned into an expression vector pGEX-5X-3 as a 3'fusion to the GST gene . Gene expression was induced in E . coli DH5 alpha cells and the fusion protein was purified using affinity chromatography . RESULTS: The purified fusion protein significantly lengthened the activated partial thromboplastin time (74.7 s tested in 80 micromol/L) and thrombin time (102.3 s tested in 80 micromol/L) and inhibited the amidolytic activity of thrombin 11% activity compared with the control tested in 100 micromol/L . The ADP-induced platelet aggregation was markedly inhibited by the purified fusion protein . The study has also shown that GST exhibits relevant activity of antiplatelet weaker than the purified fusion protein . 20.7% activity compared with the control tested in 100 micromol/L . CONCLUSION: Our results confirm that it is feasible to design a hybrid multifunctional protein that targets various components of the haemostatic process.

J Mol Biol, 2002 Jul 26, 320(5), 963 - 78
Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?
Serganov A, Ennifar E, Portier C, Ehresmann B, Ehresmann C.
Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA . Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start . The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA . To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling . The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity . This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature . The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides . Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area . It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner . However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding . Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.

Arch Microbiol, 2002 Aug, 178(2), 141 - 8 Epub 2002 May 29.
A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E . coli KdpE and activates kdp expression in E . coli; Ballal A et al.; The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality . Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE . From a nitrogen-fixing cyanobacterium, Anabaena sp . strain L-31, a kdpDgene was cloned (GenBank accession no . AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E . coli KdpD . A chimeric kdpD gene was constructed and expressed in E . coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E . coli KdpD were replaced by those from Anabaena KdpD . In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E . coli KdpE, which closely resemble those of the E . coli wild-type KdpD . Cells of E . coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock . The data demonstrate that Anabaena KdpD can interact with the E . coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(4), 459 - 462
Expression, Purification and Characterization of Regulatory Subunit of Type I cAMP-dependent Protein Kinase in E.coli; Liu XL et al.; Regulatory subunit of type I cAMP-dependent protein kinase (RI) possesses two cAMP-binding sites with high affinity for cAMP . In the current study, human RI cDNA fragment coding for residue 93-381 was cloned from neuronal cells . After subcloning into pET30a, the recombinant RI was expressed in E.coli BL21 . Considerable amount of soluble RI subunit was produced in BL21 upon induction of IPTG . RI was purified to homogeneity over 95% after His-bind metal chelation chromatography . Approximately 8 mg pure RI was obtained from 0.1 liter of BL21 culture . Human RI prepared with this method binds to cAMP specifically and therefore can be used in cAMP assay.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(4), 405 - 408
Sequence Analysis of SOD Gene of Bombyx mori Nuclear Polyhedrosis Virus and Its Expression in E.coli; Wang WB et al.; Superoxide dismutases scavenge superoxide radicals and protect cells from oxidative stress . SOD gene of Bombyx mori nuclear polyhedrosis virus has been cloned by PCR, and is expressed in E.coli with the activity of SOD being 576.13 u/mL . DNA sequence analysis shows that SOD gene of BmNPV encodes 151 amino acids . The homology between BmNPV and AcNPV of the nucleotide sequences of SOD gene is 97.2%, and 56% between BmNPV and human SOD1.

Epidemiol Infect, 2002 Jun, 128(3), 363 - 71
An outbreak of gastroenteritis in Osaka, Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E . coli heat-stable enterotoxin 1 (EAST1); Zhou Z et al.; In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill . Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%) . Laboratory tests for other bacterial pathogens and viruses were negative . The E . coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner . The organisms did not express known enterotoxigenic E . coli (ETEC) colonization factors . In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E . coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E . coli (EIEC) . Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E . coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E . coli (EAggEC), or diffusely adhering E . coli . However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene . To our knowledge, this is the first report of an outbreak caused by E . coli that did not have well-characterized virulence genes other than EAST1 . The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI . Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already . We propose that diarrhoeal specimens should be examined for E . coli possessing the EAST1 gene.

Cancer Chemother Pharmacol, 2002 Jul, 50(1), 65 - 70 Epub 2002 Jun 08.
Suicide gene therapy using E . coli beta-galactosidase; Farquhar D et al.; PURPOSE: Suicide gene therapy offers the potential to increase the selective toxicity of antitumor agents by intratumoral expression of exogenous enzymes that convert nontoxic prodrugs to toxic products . The use of herpes simplex virus thymidine kinase with ganciclovir, and E . coli cytosine deaminase with 5-fluorocytosine are well-known examples of this approach . The purpose of this study was to investigate a novel suicide gene therapy using E . coli beta-galactosidase (beta-gal) as the prodrug-activating enzyme . Advantages of this approach include: (1) the ability to use prodrugs that are cleaved by beta-gal to agents that are known to possess activity against human solid tumors, and (2) the extensive experience gained with targeting beta-gal to specific tumors in experimental animals and in humans . METHODS: Two different structural types of anthracycline prodrugs, N-{4"-(beta- D-galactopyranosyl)-3"-nitrobenzyloxycarbonyl}daunomycin (Daun02) and N-{(4" R,S)-4"-ethoxy-4"-(1"'- O-beta- D-galactopyranosyl)butyl}daunorubicin (gal-DNC4) were investigated . The prodrugs were evaluated as substrates for beta-gal . Cytotoxicity studies of Daun02 were conducted against a murine tumor (Panc02), two human breast tumors (MCF-7 and T47D), and three human prostate tumors (PC3, DU145 and LNCAP) that had been transduced to express beta-gal . Antitumor studies of Daun02 were conducted against mouse tumor Panc02 xenografts implanted subcutaneously . RESULTS: Daun02 was a good substrate for beta-gal . By comparison, gal-DCN4 was a poor substrate . Except for PC3, the beta-gal-transduced tumors showed 3- to 60-fold increased sensitivity to Daun02 compared with mock-transduced control cells . Daunomycin was formed from Daun02 in tissue culture medium containing beta-gal-transduced cell lines but was not observed in the medium from mock-transduced controls . In vivo therapeutic studies of Daun02 against the Panc02 tumor in athymic mice showed no significant inhibition of tumor growth . Pharmacokinetic studies showed limited distribution of the prodrug beyond the vascular space . CONCLUSIONS: E . coli beta-gal may be useful as a prodrug-activating enzyme in suicide gene therapy and has the potential to increase the selective toxicity of conventional antitumor agents . Although this approach worked well against tumor cells in vitro, it was not effective against a xenograft model in vivo, apparently because of poor drug-tissue distribution.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(1), 49 - 54
An NF-IL6 3'UTR RNA-specific Binding Protein in E . coli Purification and Partial Protein Sequencing; Qing GL et al.; protein was found in E.coli which can specifically bind to NF-IL6 mRNA 3'UTR . After a series of purification steps, the RNA-specific binding protein was directly sequenced on the Porton LF3200 Protein Sequencer . A sequence of 10 amino acids of the purified NF-IL6 3'UTR binding protein was obtained, namely, Ala-Thr-Arg-Ile-Glu-Phe-His-Gly-Cyss(?)-Gly . A BLAST search with the 10 amino acids against NCBI database failed to identify any protein with identical sequence . The significance of identifying an eukaryotic mRNA binding protein in E.coli is discussed.

Toxicol In Vitro, 2002 Aug, 16(4), 421 - 5
Epithelial cells challenged with a Rac-activating E . coli cytotoxin acquire features of professional phagocytes; Falzano L et al.; Activation of Rho, Rac and Cdc42 GTPases by an Escherichia coli cytotoxin (CNF1) has been reported to induce a phagocytic-like activity by epithelial cells in terms of a ruffle-driven capture and ingestion of large material . More recently, it has been reported that treatment with CNF1 induces superoxide anion release by these cells following a phagocytic stimulus . We herein show that in epithelial cells both transfection with the dominant form of Rac (RacV12) and treatment with the Rac-activating epidermal growth factor (EGF) may increase the secretion of superoxide anions on challenge with latex beads . Moreover, exposure to CNF1 induces a significant augmentation of acidic vesicles where the internalized particles were detectable . Our results indicate that (i) Rac is a pivotal GTPase for inducing in epithelial cells superoxide anion generation and (ii) the internalized material travels trough acidic compartments in CNF1-treated epithelial cells . Altogether this suggests a novel role for epithelial cells that, following Rac activation, might share with professional phagocytes the task of eliminating unwanted pathogens.

Prog Urol, 2002 Apr, 12(2), 298 - 302
{E coli spondylitis after uteroscopy for lithiasis}; Hamie F et al.; The authors report a rare complication of ureteroscopy for stones of the lumbar ureter: Escherichia coli spondylitis.

Biochemistry, 2002 Jul 16, 41(28), 8921 - 34
Kinetics of the E . coli replication factor DnaC protein-nucleotide interactions . II . Fluorescence anisotropy and transient, dynamic quenching stopped-flow studies of the reaction intermediates; Galletto R et al.; The nature of the intermediates in the binding of MANT-ATP and MANT-ADP to the E . coli replicative factor DnaC protein (accompanying paper) has been examined using the fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow techniques . Using molar fluorescence intensities of individual intermediates of the reaction, we derived the Stern-Volmer equation that provides a direct method to quantitatively address the quenching of the fluorescence of a transient intermediate by an external, neutral quencher . The data indicate that in the first intermediate, (C)(1), the solvent has full access to the MANT group . Thus, the nucleotide-binding site is located on the surface of the protein, fully open to the solvent . Moreover, formation of the first intermediate does not affect the structure of the binding site . On the other hand, in the second intermediate, (C)(2), the entire binding site changes its conformation, resulting in diminished access of the solvent to the bound nucleotide . The time course of the fluorescence anisotropy in the reaction provides direct, unique insight into the mobility of bound nucleotides in each intermediate . The analysis is facilitated by the fact that the anisotropy can be expressed as a function of the relative molar intensities and steady-state anisotropies of the individual intermediates . The major decrease of the nucleotide mobility occurs in the formation of the first intermediate and reflects the fact that the MANT group is immobilized to a similar extent as the ribose region of the bound nucleotides . Transition to the second intermediate and closing of the binding site leads to only a moderate, additional decrease of nucleotide mobility . The temperature effect on the studied interactions indicates that the formation of individual intermediates is accompanied by very different enthalpy and entropy changes predominantly generated from the structural changes of the protein . Analysis of the salt effect indicates that the net release of a single ion, observed in equilibrium studies, occurs in the formation of the first intermediate . The lack of any salt effect on the (C)(1) <--> (C)(2) transition indicates that the closing of the binding site does not include a net ion release or uptake . Moreover, prior to the nucleotide binding, the conformational transition of the DnaC protein is exclusively controlled by the nucleotide binding and release.

Biochemistry, 2002 Jul 16, 41(28), 8907 - 20
The E . coli replication factor DnaC protein exists in two conformations with different nucleotide binding capabilities . I . Determination of the binding mechanism using ATP and ADP fluorescent analogues; Galletto R et al.; The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E . coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique . The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration . Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein . Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding . The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations, as defined by In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)(2) that binds the nucleotide . The lack of any cofactor binding to the (DnaC)(1) state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein . Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation . The (C)(1) intermediate dominates the distribution of the DnaC protein population in the presence of cofactors . The formation of (C)(1) is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors . Transition to (C)(2) places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity approximately 8-fold higher than that of the free cofactor . The significance of these results for the functioning of the DnaC protein is discussed.

Genes Dev, 2002 Jul 1, 16(13), 1696 - 706
Spot 42 RNA mediates discoordinate expression of the E . coli galactose operon; Moller T et al.; The physiological role of Escherichia coli Spot 42 RNA has remained obscure, even though the 109-nucleotide RNA was discovered almost three decades ago . Structural features of Spot 42 RNA and previous work suggested to us that the RNA might be a regulator of discoordinate gene expression of the galactose operon, a control that is only understood at the phenomenological level . The effects of controlled expression of Spot 42 RNA or deleting the gene (spf) encoding the RNA supported this hypothesis . Down-regulation of galK expression, the third gene in the gal operon, was only observed in the presence of Spot 42 RNA and required growth conditions that caused derepression of the spf gene . Subsequent biochemical studies showed that Spot 42 RNA specifically bound at the galK Shine-Dalgarno region of the galETKM mRNA, thereby blocking ribosome binding . We conclude that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit . Our results reveal an interesting mechanism by which the expression of a promoter distal gene in an operon can be modulated and underline the importance of antisense control in bacterial gene regulation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 502 - 5
Cloning expression in E.coli and biological activity of human thymosin beta(4); Che YK et al.; The cDNA thymosin beta(4) was synthesized by combining of chemical and enzymatic methods . First, two complement fragments of thymosin beta(4) cDNA were synthesized by DNA synthesizer, and then denatured, annealed and extended by DNA polymerase . This fragment of thymosin beta(4) was then inserted into the EcoRV and HindIII restriction endonuclease site of an expression plasmid pLDH4 (a kind of E.coli plasmid) by blunt and cohesive ligations . Finally, the recombinant plasmid which expressed thymosin beta(4) was screened by digestion and DNA sequencing . This recombinant plasmid highly expressed the thymosin beta(4), which accounted for 30% of total bacteria proteins . By salting out and chromatography, a 95% purity of recombinant thymosin beta(4) was obtained . Biological assay indicated that the recombinant thymosin beta(4) could induce lymphocyte proliferation and differentiation.

Nature, 2002 Jul 4, 418(6893), 99 - 103
AID mutates E . coli suggesting a DNA deamination mechanism for antibody diversification; Petersen-Mahrt SK et al.; After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination . All three processes depend on activation-induced cytidine deaminase (AID), a B-cell-specific protein that has been proposed (because of sequence homology) to function by RNA editing . But indications that the three gene diversification processes might be initiated by a common type of DNA lesion, together with the proposal that there is a first phase of hypermutation that targets dC/dG, suggested to us that AID may function directly at dC/dG pairs . Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner . Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA . We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome--that is, hypermutation phases 1 and 2, gene conversion or switch recombination--dependent on the way in which the initiating dU/dG lesion is resolved.

EMBO J, 2002 Jul 1, 21(13), 3347 - 57
Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains; Shih YL et al.; The MinE protein functions as a topological specificity factor in determining the site of septal placement in Escherichia coli . MinE assembles into a membrane-associated ring structure near midcell and directs the localization of MinD and MinC into a membrane- associated polar zone that undergoes a characteristic pole-to-pole oscillation cycle . Single (green fluorescent protein) and double label (yellow fluorescent protein/cyan fluorescent protein) fluorescence labeling experiments showed that mutational alteration of a site on the alpha-face of MinE led to a failure to assemble the MinE ring, associated with loss of the ability to support a normal pattern of division site placement . The absence of the MinE ring did not prevent the assembly and disassembly of the MinD polar zone . Mutant cells lacking the MinE ring were characterized by the growth of MinD polar zones past their normal arrest point near midcell . The results suggested that the MinE ring acts as a stop-growth mechanism to prevent the MinCD polar zone from extending beyond the midcell division site.

Cell, 2002 Jun 14, 109(6), 757 - 67
E . coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation; Park JS et al.; Transcription and DNA repair are coupled in E . coli by the Mfd protein, which dissociates transcription elongation complexes blocked at nonpairing lesions and mediates recruitment of DNA repair proteins . We show that Mfd influences the elongation state of RNA polymerase (RNAP); transcription complexes that have reverse translocated into the backtracked position, a potentially important intermediate in RNA proofreading and repair, are restored to the forward position by the activity of Mfd, and arrested complexes are rescued into productive elongation . Mfd may act through a translocase activity that rewinds upstream DNA, leading either to translocation or to release of RNA polymerase when the enzyme active site cannot continue elongation.

J Biotechnol, 2002 Aug 28, 97(3), 199 - 212
Production of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E . coli strains; Mukhopadhyay UK et al.; The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique . Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence . The high level expression of both the polypeptide hormones was achieved in E . coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system . The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out . A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones . A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed . The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones . The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.

J Mol Biol, 2002 May 17, 318(5), 1175 - 88
Evidence that the KH RNA-binding domains influence the action of the E . coli NusA protein; Zhou Y et al.; The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains . An essential function in E . coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of lambda . Tandem KH domains have been identified downstream of the S1 domain . We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains . The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action . The changes in both KH domains interfere with NusA binding to RNA . A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action . However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein . These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the lambda N protein.

Genes Cells, 2002 Jul, 7(7), 639 - 51
Over-expression of human DNA polymerase lambda in E . coli and characterization of the recombinant enzyme; Shimazaki N et al.; BACKGROUND: DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases in eukaryotic cells . Pol lambda contains a nuclear localization signal (NLS), a BRCA1-C terminal (BRCT) domain, a proline-rich region, helix-hairpin-helix (HhH) and pol X motifs . Since the amino acid sequence for Pol lambda shares a high degree of homology to Pol beta, Pol lambda is considered to have a similar enzymatic nature to Pol beta . RESULTS: Recombinant human Pol lambda was shown to possess template-directed DNA polymerase activity in its monomeric form . Pol lambda required either Mn2+ or Mg2+ as a metal co-factor to catalyse this activity, and optimal activity was detected at pH 8.5-9.0 . Pol lambda was insensitive to aphidicolin, but was sensitive to dideoxynucleoside triphosphates or N-ethylmaleimide . By constructing the truncated Pol lambda, the proline rich region was shown to act in a suppression of its polymerization activity . A chimeric enzyme comprised of the Pol lambda N-terminal region and Pol beta also showed a reduced Pol beta activity . Proliferating cell nuclear antigen (PCNA) directly interacts with Pol lambda through its Pol beta like region in vitro . CONCLUSIONS: Pol lambda possesses similar enzymatic nature to Pol beta; requirements of cations and optimal conditions for pH and NaCl concentration, aside from sensitivity to N-ethylmaleimide and template preference . The proline rich region of Pol lambda functions as a suppressor domain for its polymerization activity (SDPA) . Pol lambda interacts directly with PCNA through its Pol beta like region . The functional consequence of this interaction is the negative regulation of Pol lambda activity.

Nat Struct Biol, 2002 Aug, 9(8), 570 - 5
Structure and catalytic mechanism of the E . coli chemotaxis phosphatase CheZ; Zhao R et al.; The protein CheZ, which has the last unknown structure in the Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of the response regulator CheY by an unknown mechanism . Here we report the co-crystal structure of CheZ with CheY, Mg(2+) and the phosphoryl analog, BeF(3)(-) . The predominant structural feature of the CheZ dimer is a long four-helix bundle composed of two helices from each monomer . The side chain of Gln 147 of CheZ inserts into the CheY active site and is essential to the dephosphorylation activity of CheZ . Gln 147 may orient a water molecule for nucleophilic attack, similar to the role of the conserved Gln residue in the RAS family of GTPases . Similarities between the CheY{bond} CheZ and Spo0F {bond}Spo0B structures suggest a general mode of interaction for modulation of response regulator phosphorylation chemistry.

Int J Food Microbiol, 2002 Jul 25, 77(1-2), 91 - 7
Effects of cutting process on pork meat contamination by verotoxin-producing Escherichia coli (VTEC) and E . coli O157:H7; Bouvet J et al.; The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant . Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day . Potable water was also collected . PCR detection of stx genes was performed on a total of 2042 samples . In addition, a second PCR specific for E . coli O157:H7 detection was carried out on the stx-positive samples . VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped . No E . coli O157:H7 was detected . Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting) . No VTEC isolates harboured eae, ehx and uidA genes . VTEC contamination routes were not clearly identified.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 295 - 298
Expression of Human Epiregulin in E.coli; Xi QS et al.; Human epiregulin cDNA was amplified from the lung cancer cell line A549 using RT-PCR . After adding 6 His codon to its 3' end, it was cloned into a high efficient secretive Escherichia coli system with alkaline phosphatase promoter(phoA promoter)constructed in our lab and induced for expression . The product was purified one-step by Ni-NTA column . Amino acid sequence analysis revealed the identity of our product with that previously reported . The product showed strong proliferative effect on fibroblast cell line Balb/c3T3 and growth inhibitory effect on epithelial carcinoma cell line A431.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 211 - 216
The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli; Yang YG et al.; In order to efficiently recover recombinant proteins, a temperature-sensitive lytic system was constructed on the basis of the feature that T4 lysozyme disrupts the bacteria through cutting specific bond in the peptidoglycan layer of cell wall . This system was evaluated by constructing and introducing a low copy plasmid pSC-lys (pSC101 replication origin) into E.coli . The plasmid contained a temperature sensitive T4 lysozyme (LYS(ts)) gene under the control of three tandem tac promoters and the LacI repressor, which is compatible with other plasmids carrying pMB1, ColE1 replication origins, etc . Under the optimum lysis conditions, 2--5 fold condensed cultures resuspended in buffer A, beta-galactosidase, recombinant chaperone GroEL and ZZ-fusion salmon hexamic calcitonin (Cal6) in E.coli were released simply, rapidly, and quantitatively, as co-expressed with LYS(ts) . The two tested recombinant proteins maintained their significant productions . Instead of other cumbersome lysising methods, this novel lytic system will be useful in recovery of recombinant proteins for further purification in the field of biotechnology.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(4), 397 - 400
Interleukin 18 High Level Expression in E.coli Purification and Renaturation of the Recombinant Protein; Pei DS et al.; Using the total RNA extracted from mitogen-stimulated human peripheral blood mononuclear cells (PBMC) as template, the cDNA of interleukin 18 was amplified by RT-PCR . The cDNA was subsequently cloned into the expression vector pJW2 and sequenced . The recombinant human IL-18 (rhIL-18) was expressed efficiently in inclusion bodies in E.coli with the yield accounting for 20% total bacteria proteins . The inclusion bodies were washed with 2 mol/L urea and rhIL-18 was further purified using Sephadex G-100 column chromatography in 8 mol/L urea . After purification, the purity of rhIL-18 was greater than 90% as judged by SDS-PAGE . The purified rhIL-18 showed significant and dose-dependent IFN-gamma-inducing activity in human PBMC, in the presence of 0.5 mg/L Con A.

Vet Immunol Immunopathol, 2002 Sep 10, 87(3-4), 287 - 90
Oral immunisation of pigs with fimbrial antigens of enterotoxigenic E . coli: an interesting model to study mucosal immune mechanisms; Cox E et al.; The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively . Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens . A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)) . Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection . In F4R(-) pigs such an intestinal mucosal immune response does not occur . However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen . The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.

J Appl Microbiol, 2002, 93(1), 7 - 14
Prevalence of verotoxin-producing Escherichia coli (VTEC) and E . coli O157:H7 in French pork; Bouvet J et al.; AIMS: To determination the prevalence of VTEC in pork products and the surrounding environment of the pork plant (slaughterhouse and cutting plant), and characterization of the VTEC strains isolated (virulence genes and serotype) . METHODS AND RESULTS: Among the 2146 carcass and pork samples and 876 environmental samples (swabs of surfaces or materials), 328 (15%) and 170 (19%) were PCR-positive for stx genes respectively . VTEC strains were recovered from positive samples by colony hybridization or immunoconcentration, serotyped and genetically characterized . Strains of E . coli O157:H7 were not isolated from 3 uidA-positive samples detected by PCR . The VTEC isolates did not harbour eae, ehx and uidA genes . CONCLUSIONS: Pigs and pork meat may contain VTEC strains but characterization of the strains based on virulence factors showed that the potential danger of pork meat appears to be low since although all strains harboured a stx gene, they did not have other virulence genes . SIGNIFICANCE OF THE STUDY: General hygiene measures appear to be sufficient and specific hygiene measures for VTEC are not necessary at this time . The porcine VTEC strains isolated in our study probably do not present a hazard.

Am J Physiol Gastrointest Liver Physiol, 2002 Jul, 283(1), G74 - 86
Release of ATP during host cell killing by enteropathogenic E . coli and its role as a secretory mediator; Crane JK et al.; Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children . We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators . EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit . Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added . In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains . In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine . Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not . The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754 . EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors . These findings provide new insight into how EPEC causes watery diarrhea.

J Mol Biol, 2002 Jun 7, 319(3), 649 - 71
Kinetic studies and structural models of the association of E . coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates; Saecker RM et al.; The kinetics of interaction of Esigma(70) RNA polymerase (R) with the lambdaP(R) promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42 degrees C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA . Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I(2), RP(o)) are single-exponential with first order rate constant beta(CR) . Interpretation of the polymerase concentration dependence of beta(CR) in terms of the three step mechanism of open complex formation yields the equilibrium constant K(1) for formation of the first kinetically significant intermediate (I(1)) and the forward rate constant (k(2)) for the conformational change converting I(1) to the second kinetically significant intermediate I(2): R + P-->(K(1))<--I(1)(k(2))-->I(2) . Use of rapid quench mixing allows K(1) and k(2) to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing . Given the large (>60 bp) interface formed in I(1), its relatively small binding constant K(1) at 37 degrees C at this {salt} (approximately 6 x 10(6) M(-1)) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes . Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k(a){DeltaC(o,++)(a)= -1.5(+/-0.2)kcal K(-1)}, now shown to originate directly from formation of I(1) {DeltaC(o)(1)= -1.4(+/-0.3)kcal K(-1)} rather than from the formation of I(2) as previously proposed . The isomerization I(1)-->I(2) exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy {E(act)(2)= 34(+/-2)kcal} . This kinetic signature suggests that formation of the transition state (I(1)-I(2)++ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water . Structural and biochemical data lead to the following hypotheses to interpret these results . We propose that formation of I(1) involves coupled folding of unstructured regions of polymerase (beta, beta' and sigma(70)) and bending of promoter DNA (in the -10 region) . We propose that interactions with region 2 of sigma(70) and possibly domain 1 of beta induce a kink at the -11/-12 base pairs of the lambdaP(R) promoter which places the downstream DNA (-5 to +20) in the jaws of the beta and beta' subunits of polymerase in I(1) . These early interactions of beta and beta' with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 1127 - 37
Crystal structure of E . coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity; Li J et al.; E . coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology . ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences . NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA) . To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A resolution . The NBD1 monomer structure contains one domain that comprises 11 alpha-helices and six beta-strands . When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded beta-sheet as its core . The N-terminal portion of NBD1 structure has an extra beta-strand flanked by two extra alpha-helices that are not present in other AAA structures . Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do . No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure . Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides . However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule . ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPgammaS molecule . Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity . None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities . On the basis of our structural and mutagenesis data, we proposed a "see-saw" model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 131 - 136
Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli; Tang GL et al.; To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation . As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e . triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc . Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG . Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute . Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator . Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained . This shuttle was used to transform of Cyanobacteria Synechococcus sp . PCC 7942, and a few positive colonies were obtained.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 77 - 81
Barotolerant E.coli Induced by High Hydrostatic Pressure; Gao X et al.; The barotolerant E.coli TG1P, DH5alphaP, HB101P were obtained from TG1,DH5alpha, HB101 after treatment with high hydrostatic pressure, and the survival rates of the formers raised 2--3 magnitude, from 1.06x10(-6), 2.98x10(-4), 3.65x10(-3) to 8.31x10(-3), 3.40x10(-1), 1.69x10(-1),respectively . Comparison of partial proteomes of wild and barotolerant bacteria of TG1 and DH5alpha with two-dimensional electrophoresis showed that three proteins were obviously highly expressed in barotolerant bacteria . The N terminal sequence and molecular weight of one protein was shown to be V(L)EAGEFFMRA and 21 kD, with high identity to a known outmembrane protein in E.coli.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 30 - 34
The Thioredoxin Reductase-deficient E.coli Mutant Enhances Expression into Solution of Recombinant Proteins Containing Cys Residues; Tong Q et al.; A 3D artificial protein, a salmon calcitonin hexa-polymer, a salmon calcitonin octo-polymer and a human prourokinase, was expressed in the cytoplasma of E.coli GJ980(trxB(-)) mutant . These recombinant proteins containedcysteine residues of different length ranging from 12-22 residues . The mutation was mapped to the gene for thioredoxin reductase(trxB) and was found to eliminate the activity of this enzyme, which was thought to contribute to the sulfhydryl reducing potential of the cytoplasm . Recombinant salmon calcitonin hexapolymer, salmon calcitonin octo-polymer and human prourokinase had more soluble form in cytoplasm of GJ980 mutants than in wild-type strain, while 3D-protein, which has nocysteine residue, still remain in insoluble form . Results indicate the GJ980(trxB(-)) strain allowed the formation of disulphide bonds in the cell cytoplasm which is believed to encourage correct folding and soluble expression of the recombinant proteins.

BMC Biochem . 2002 May 29;3(1):13.
The role of the Zn(II) binding domain in the mechanism of E . coli DNA topoisomerase I; Ahumada A et al.; BACKGROUND: Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain) . The 67 kDa N-terminal domain (Top67) has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA . We analyzed the role of the ZD domain in the enzyme mechanism . RESULTS: Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA . The two domains had similar affinities to ssDNA . However, only Top67 could bind dsDNA with high affinity . DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme . DNA rejoining also did not require the presence of the ZD domain . CONCLUSIONS: We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage . However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 238 - 242
Coexpression of DNA Fragmentation Factor Subunits in E.coli by Two Incompatible Plasmids; Yang W et al.; The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD (DFF40/CAD) and 45 kD(DFF45/ICAD) subunits . Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase-activated DFF40 nuclease, which is inhibited by a chaperone-like subunit DFF45 . In this work, the coding regions of human DFF45 and DFF40 mRNAs were amplified from total RNA of HeLa cells by RT-PCR . Two 1 kb DNA fragments were obtained and were cloned into a kanamycin-resistant bacterial expression vector, pET-28a( ), generating pET28a-DFF45 and pET28a-DFF40, which were then used to transform E.coli BL21(DE3), respectively . After induction with IPTG, DFF45 and DFF40 were expressed effectively, accounting for about 56% and 22% of total bacterial proteins, respectively . Since successful expression of properly folded DFF40 requires coexpression with DFF45, the full-length DFF45 cDNA was inserted into an ampicillin resistant expression vector, pET-21a( ), and the recombinant plasmid was designated pET21a-DFF45 . Under screening pressure by ampicillin and kanamycin simultaneously, E.coli BL21(DE3) was cotransformed with pET21a-DFF45 and pET28a-DFF40 . Upon induction with IPTG, DFF45 and DFF40 were coexpressed efficiently and the desired products comprised about 30% and 17% of total cell proteins, respectively . To further study the stability of the two incompatible plasmids'coexistance in E.coli, the cotransformant was cultured in liquid medium containing ampicillin and kanamycin for 14 h, and more than 75% of the cells were found to be resistant to the two antibiotics, that is, they carried both pET21a-DFF45 and pET28a-DFF40 . Thus, a novel method of coexpressing different proteins using two incompatible plasmids was developed.

J Environ Health, 2002 Jun, 64(10), 16 - 20, 26, 25
Laboratory investigation of an E . coli O157:H7 outbreak associated with swimming in Battle Ground Lake, Vancouver, Washington; Samadpour M et al.; Escherichia coli O157:H7 has been associated with a number of waterborne outbreaks, but it has never been recovered from an implicated environment . This paper reports on an August 1999 outbreak of E . coli O157:H7 associated with swimming in Battle Ground Lake in Clark Country, Washington . E . coli O157:H7 was isolated from duck feces, as well as from two water samples . The authors used pulsed-field gel electrophoresis to compare these isolates with patient isolates for genetic homology . All the isolates yielded the same restriction fragment patterns . In addition, using polymerase chain reaction, the authors found patient isolates and environmental isolates to have the same virulence factors (Stx, eaeA, and hly).

J Biomol Struct Dyn, 2002 Jun, 19(6), 999 - 1006
Structure of glutamate decarboxylase from E . coli: spectral studies; Bazhulina NP et al.; Structure of recombinant glutamate decarboxylase (GAD alpha) was studied by optical methods and electron microscopy . The active (pH 4.6) and inert (pH 6.3) holoGAD and apoGAD were investigated . Absorption and CD spectra were recorded in the range of 190 - 500 nm . Visible spectra were resolved into the bands corresponding to individual electron transitions using lognormal curves . The structures of predominant tautomers of internal aldimines were determined as ketoenamine at pH 4.6 and enolimine at pH 6.3 . CD spectra show that holoGAD and apoGAD exhibit a negative band at 204 - 245 nm and a positive band near 190 - 204 nm . The contents of the secondary structure elements were estimated on the basis of the values of the mean residue ellipticity . Evidently, the main difference between the GAD forms studied is in the content of alpha-helix and random coil . HoloGAD has 50% of alpha-helix at pH 4.6 and 67% at pH 6.3, whereas apoGAD - 17 and 27%, respectively . Thus presented data establish the essential role of pyridoxal phosphate (PLP) in the organization of the GAD secondary structure due to tightening its polypeptide chain . It seems possible, that conformational changes induced by PLP binding stabilize the protein structure and promote the assembly of subunits into macromolecule, which was confirmed by electron microscopy.

Shi Yan Sheng Wu Xue Bao, 1998 Dec, 31(4), 333 - 9
{Cleavage activity of ribozymes on a target RNA of tobacco mosaic virus in E . coli}; Zhang QQ et al.; A target cDNA fragment from TMV RNA was inserted to a reporter gene CAT immediately to the 3' end of the translation initiation codon ATG resulting in the formation of a chimeric CAT gene in an in vivo transcription and expression vector . The in vivo activities of various ribozymes on the target sequence were observed by determining the changes of the CAT activities of the chimeric CAT gene expressed in E . coli . The CAT activity was reduced by up to 30% when a specific ribozyme RZ1, RZ1A or RZ1B was transcribed, no change in CAT activity was observed when a non-specific ribozyme RZ3 was expressed . The protein electrophoresis and primer extension experiments indicated that this reduction in CAT activity was due to the specific cleavage of the ribozymes to the chimeric CAT mRNA at the target region hence the decrease in CAT protein synthesis.

Protein Sci, 2002 Jun, 11(6), 1442 - 51
Role of water in the catalytic cycle of E . coli dihydrofolate reductase; Shrimpton P et al.; Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) . Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate . However, in the X-ray structures representing the Michaelis complex of the E . coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5 . In fact, the side chain of Met 20 blocks access to N5 . Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes . The r.m.s . deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 A . We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR . Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access . Protonation of N5 did not increase the accessibility by water . Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 A and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation . However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access . The average time during which water was found in hydrogen bonding distance to N5 was significantly increased . These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation . Such a mechanism is supported by the very close contact between C4 of NADP+ and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.

Biotechniques, 2002 May, 32(5), 1162 - 7
High-sensitivity hybridization assay for quantitation of residual E . coli DNA; Ji X et al.; Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical . For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity . Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds . We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E . coli DNA levels in purified protein drugs by means of a slot-blot hybridization method . The assay utilizes digoxigenin-labeled E . coli DNA probes and SuperSignal chemiluminescent substrate . The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E . coli DNA . The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E . coli or other bacterial expression systems.

Biol Trace Elem Res, 2002 May, 86(2), 167 - 75
Microcalorimetric study of the action of Ce(III) ions on the growth of E . coli; Ruming Z et al.; A microcalorimetric technique based on bacterial heat output was used to evaluate the action of Ce(III) ions on the growth of Escherichia coli . The power-time curves of the growth metabolism of the bacteria were studied in the presence and absence of Ce(III) by means of a LKB-2277 Bioactivity Monitor, by a stopped-flow method at 37 degrees C . For evaluation of the results, the maximum power, Pmax, the growth rate constant k, and the heat effects Qlog, Qstat, and Qtot for the log phase, the stationary phase, and total heat output, respectively, were determined . For comparison, a spectrophotometer was used to estimate the number of cells in the liquid culture . The shape of the bacteria was examined by electron microscopy . We concluded that the presence of cerium ions at concentrations below 350 microg/mL have a stimulatory effect on the growth of E . coli, whereas concentrations at or above 400 microg/mL may have an inhibitory effect.

J Biotechnol, 2002 May 23, 95(3), 257 - 68
Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E . coli; Jose J et al.; Bovine adrenodoxin (Adx) was expressed on the surface of Escherichia coli as a monomeric fusion protein with the translocation unit of the AIDA-I autotransporter . The fusion protein remained anchored in the outer membrane by the beta-barrel of the autotransporter . Dimeric Adx molecules were formed spontaneously on the bacterial surface with high efficiencies . Adx dimers could be activated to biological function by chemical incorporation of the {2Fe-2S} cluster . By adding purified adrenodoxin reductase and P450 CYP11A1, a whole cell biocatalyst system was obtained, which effectively synthesized pregnenolone from cholesterol . Addition of artificial membrane constituents or detergents, which was indispensable before to get functional steroidal P450 enzymes, was not necessary . The whole cell activity (0.21 nmol x h(-1) x nmol(-1) CYP11A1) was in the same range as obtained earlier for reconstitution assays . The whole cell system developed here is an easy to handle, stable tool for the expression of membrane-associated P450 enzymes without the need of microsome preparation or reconstitution of artificial membrane vesicles . Moreover, it is the first report on functional dimer formation of a protein anchored on the surface of E . coli after being transported as a monomer . This seems to be a special feature of the autotransporter translocation unit, containing a beta-barrel, motile in the outer membrane and opens a new dimension for the surface display of multimeric proteins.

Biotechnol Bioeng, 2002 Jun 30, 78(7), 761 - 9
A strategy for in vivo screening of subtilisin E reaction specificity in E . coli periplasm; Sroga GE et al.; We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity . Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures . The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca . eightfold higher as compared to the expression level at 30 degrees C . Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates . Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo . Intact E . coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively . Additionally, the periplasmic fractions were isolated from the three E . coli strains expressing the respective subtilisins and tested for amidase activity . The amidase activity of the three subtilisins was ca . 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions . The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities .

Genes Dev, 2002 May 1, 16(9), 1102 - 15
Initiation of tRNA maturation by RNase E is essential for cell viability in E . coli; Ow MC et al.; RNase E, an essential endoribonuclease in Escherichia coli, is involved in 9S rRNA processing, the degradation of many mRNAs, and the processing of the M1 RNA subunit of RNase P . However, the reason that RNase E is required for cell viability is still not fully understood . In fact, recent experiments have suggested that defects in 9S rRNA processing and mRNA decay are not responsible for the lack of cell growth in RNase E mutants . By using several new rne alleles, we have confirmed these observations and have also ruled out that M1 processing by RNase E is required for cell viability . Rather, our data suggest that the critical in vivo role of RNase E is the initiation of tRNA maturation . Specifically, RNase E catalytic activity starts the processing of both polycistronic operons, such as glyW cysT leuZ, argX hisR leuT proM, and lysT valT lysW valZ lysY lysZ lysQ, as well as monocistronic transcripts like pheU, pheV, asnT, asnU, asnV, and asnW . Cleavage by RNase E within a few nucleotides of the mature 3' CCA terminus is required before RNase P and the various 3' --> 5' exonucleases can complete tRNA maturation . All 59 tRNAs tested involved RNase E processing, although some were cleaved more efficiently than others.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 85 - 7
{Inducible expression of non-structural protein 3 of hepatitis C virus in E . coli}; Cheng J et al.; BACKGROUND: To express recombinant non-structural protein 3 of hepatitis C virus (HCV) in E . coli . METHODS: The non-structural 3 (NS3) region DNA fragment of HCV was amplified by polymerase chain reaction (PCR) and inserted into inducible proeukaryotic expressive vector pET 30C(+)at Bam H1/EcoR1 sites . The competent BL21 (DE3) E.coli was transformed, and then cultured and induced with IPTG . The expressed HCV NS3 protein was confirmed with ELISA and dot blot hybridization using HCV NS3-specific single chain Fv (ScFv) antibody . RESULTS: 1 893 bp DNA fragment of HCV NS3 coding region was amplified by PCR technique . HCV NS3 expressive vector pET-NS3 was constructed . After transformation with pET-NS3 and induction with IPTG, recombinant HCV NS3 protein was expressed and confirmed by specific ELISA and dot blot hybridization . CONCLUSIONS: The recombinant HCV NS3 can be expressed in E . coli.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2001 Dec, 15(4), 317 - 20
{Expression of the human papillomavirus type 58L1 capsid protein in E.coli and preparation of the HPV58 L1 mouse antiserum}; Tian H et al.; BACKGROUND: Human papillomavirus type 58 (HPV58) has been shown to be one of the most important highly-oncogenic, risky HPV types . The present study aimed at investigating the expression of the HPV58 L1 major capsid protein by E.coli and preparation of HPV58 L1 antiserum . METHODS: The full length L1 coden region of HPV58 L1 was amplified by PCR, the cloned, sequenced . The expression vector pRSETB58 L1 was constructed to produce HPV58 L1 protein . The protein expressed was purified by SDS-PAGE, and used to immunize BALB/c mice . RESULTS: HPV58 L1 protein was expressed in E . coli, which has cross reaction with anti-HPV16 L1 antibody . The mouse anti-HPV58 L1 antibody was obtained and used to test the HPV58 L1 protein expressed in insect cell . CONCLUSIONS: HPV58 L1 major capsid protein was efficiently expressed in E . coli . The mouse anti-HPV58 L1 specific antibody was prepared, which can be used to test the HPV58 L1 protein expressed in eukaryotic cell.

J Am Chem Soc, 2002 May 1, 124(17), 4804 - 10
Conformational selection of glycomimetics at enzyme catalytic sites: experimental demonstration of the binding of distinct high-energy distorted conformations of C-, S-, and O-glycosides by E . Coli beta-galactosidases; Garcia-Herrero A et al.; We show that the conformational features of the molecular complexes of E . coli beta-galactosidase and O-glycosides may differ from those formed with closely related compounds in their chemical nature, such as C- and S-glycosyl analogues . In the particular case presented here, NMR and ab initio quantum mechanical results show that the 3D-shapes of the ligand/inhibitor within the enzyme binding site depend on the chemical nature of the compounds . In fact, they depend on the relative size of the stereoelectronic barriers for chair deformation or for rotation around Phi glycosidic linkage.

Jpn J Infect Dis, 2002 Feb, 55(1), 14 - 8
Prevalence of Escherichia coli possessing the eaeA gene of enteropathogenic E . coli (EPEC) or the aggR gene of enteroaggregative E . coli (EAggEC) in traveler's diarrhea diagnosed in those returning to Tama, Tokyo from other Asian countries; Ogata K et al.; To elucidate the importance of enteropathogenic Escherichia coli (EPEC) and enteroaggregative E . coli (EAggEC) as etiological agents in traveler's diarrhea, the detection of the eaeA and aggR gene in E . coli strains isolated from overseas travelers with diarrhea in Tama, Tokyo was carried out using a PCR method . Of 192 travelers who were mostly adults and had visited Asian countries from April 1998 to March 1999, aggR-positive E . coli strains were detected in 26 (13.5%) . These strains represent the second predominant enteropathogen following enterotoxigenic E . coli (ETEC), whereas eaeA-positive E . coli strains were confirmed in seven subjects (3.6%) . In 13 cases with aggR and four cases with eaeA, the organisms were detected in stool samples of patients as the only potential enteric pathogen . The clinical symptoms of these patients were similar to those in patients with ETEC; however, the severity of illness was milder than that associated with ETEC alone . Three strains with eaeA and five strains with aggR were typed as six different kinds of O serogroups, of which four strains belonged to the classical EPEC serogroups (O55, O114, O119, and O127a) . These findings suggest that aggR-positive E . coli (EAggEC) is a significant causative agent in traveler's diarrhea . In addition, it was demonstrated that eaeA-positive E . coli (EPEC) is markedly correlated with diarrhea in adults.

Protein Sci, 2002 May, 11(5), 1074 - 81
Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E . coli aspartate transcarbamoylase; Fetler L et al.; The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states . To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine . The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme . Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme . Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern . In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure . Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution . This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model . Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.

Biotechniques, 2002 Apr, 32(4), 783 - 4, 786, 788
Efficient method to generate homologous recombinant baculovirus genomes in E . coli; Hou S et al.; Here we describe a convenient method to generate homologous recombinant baculoviral genomes in E . coli . The recombination takes place with the aid of recombination enzymes provided by the phage lambda Red system between a bacmid (a baculoviral genome that can replicate in bacteria) and a linear fragment . Proof of concept was provided when the cathepsin gene (v-cath) of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) was replaced by the chloramphenicol resistance gene (CmR) . First, CmR was inserted between the flanking sequences of the HaS-NPV v-cath . Each of the flanking regions was about 1 kb . The fragment was linearized and electroporated into bacteria containing both the HaSNPV bacmid and the lambda Red system . Recombinant bacmids resistant to chloramphenicol were selected . In comparison to the standard co-transfection/plaque assays, this method significantly reduces the time required to construct baculovirus knockout mutants . It may also be useful in the manipulation of other large viral genomes.

Int J Pharm, 2002 Apr 26, 237(1-2), 163 - 70
Effects of polyethylene glycol attachment on physicochemical and biological stability of E . coli L-asparaginase; Soares AL et al.; L-asparaginase obtained from E . coli strains is an important enzyme widely used in leukemia treatment . However, hypersensitivity reactions must be considered a relevant adverse effect of asparaginase therapy . One approach to reduce the hypersensitivity reactions caused by this enzyme is to change its physicochemical and biological properties by means of polyethylene glycol (PEG) conjugation, resulting in a less immunogenic enzyme with much longer half-time of plasmatic life . This work investigated the factors that could interfere in PEG-enzyme's stability . The complexation did not affect the range of pH activity and stability was improved in acid medium remaining stable during 1 h at pH 3.5 . The PEG-enzyme exhibited activity restoration capacity (32% after 60 min) when subjected to temperatures of 65 degrees C in physiological solution . The PEG-enzyme in vitro assays showed a very high stability in a human serum sample, keeping its activity practically unchanged during 40 min (strength to non-specific antibodies or proteases in serum) . An increase of PEG-enzyme catalytic activity during the lyophilization was observed . The process of modification of L-asparaginase with PEG improved both physicochemical and biological stability.

Biochemistry, 2002 Apr 23, 41(16), 5307 - 12
Differential effects of temperature on E . coli and synthetic polyhydroxybutyrate/polyphosphate channels; Das S et al.; Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C . Here we examine the gating and conductance of E . coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C . E . coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures . Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased . The effect was fully reversible . Channel conductance exhibited three distinct phases . Below 25 degrees C, as PHB approached its glass temperature (ca . 10 degrees C), the conductance of both E . coli and synthetic channels remained at about the same level (95-105 pS) . Between 25 degrees C and ca . 40 degrees C, the conductance of E . coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively . Above 40 degrees C, E . coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off . The discontinuities in the temperature curves for E . coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E . coli cells . It is postulated that E . coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.

Mol Cell Biochem, 2002 Feb, 231(1-2), 75 - 82
Action of E . coli endotoxin, IL-1beta and TNF-alpha on antioxidant status of cultured hepatocytes; Catala M et al.; We have previously reported that endotoxin induces in vivo oxidative stress in liver and a significant increase in hepatic and plasma glutathione concentrations during the acute phase of reversible endotoxic shock in rats . In the present study we examined the in vitro effects of E . coli 0111:B4 endotoxin (lipopolysaccharide, LPS), IL-1beta and TNF-alpha on antioxidant status of cultured hepatocytes in order to differentiate between the direct and mediated endotoxin action . LPS increased total glutathione (tGSH) levels after 2 h treatment but decreased oxidized glutathione (GSSG) content which lead to a marked decrease in GSSG/tGSH index . At shorter treatment times a biphasic and dose-dependent behaviour was observed . Cytokines (IL-1beta and TNF-alpha) produced significant decreases in tGSH and GSSG after 30 min treatment . Despite its prooxidant effect, TNF-alpha significantly reduced GSSG/tGSH index . Although no significant effects were observed on glutathione reductase activity, both LPS and cytokines induced an important inhibition of glutathione peroxidase which can justify the lipid peroxidation previously observed both in liver during reversible endotoxic shock and in cultured hepatocytes after treatment with endotoxin . The inhibition of hepatic glutathione peroxidase, besides the stimulation of GSH synthesis by LPS and GSH efflux by cytokines, guarantees the export of hepatic glutathione in its reduced form for other organs, contributing to the interorgan homeostasis . On the other hand, the results presented here support a new role for GSSG/tGSH index different from a mere indicator of oxidative stress.

FEBS Lett, 1970 Feb 25, 6(4), 297 - 301
An initiation factor causing dissociation of E . coli ribosomes; Albrecht J et al.; Purification of crude initiation factors, essential for polypeptide synthesis in cell-free systems of E . coli, yielded a fraction DF which causes dissociation of 70 S ribosomes . Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg(2+) concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration . GTP did not stimulate this dissociating action . 2 &mgr;g of our most purified preparation caused 100% dissociation of 100 &mgr;g of 70 S ribosomes without added GTP . DF-induced dissociation is a very rapid process at 37 degrees C and is temperature-dependent in the range of 0 degrees -37 degrees C . DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl-tRNA and mRNA.

FEBS Lett, 1970 Dec 23, 12(1), 4 - 8
Dissociation of 70 S E . coli ribosomes induced by a ribosomal factor (DF) . Electrophoretic studies of the ribosomal particles; J Talens et al.; The dissociation of purified 70 S.E . coli ribosomes, induced by the dissociation factor DF, has been studied by submitting the reaction mixtures to electrophoresis on polyacrylamide gels . The electrophoretic analysis of the ribosome mixtures revealed a heterogeneity which escaped detection by conventional sucrose gradient centrifugation . Increasing amounts of DF in the reaction mixtures converted 70 S ribosomes to particles (designated 70 S (I)) which migrate slower in the electric field than the original 70 S ribosomes . These 70 S (I) ribosomes still consist of both subunits . They dissociate upon further raising the DF concentration.

J Lab Clin Med, 2002 Mar, 139(3), 155 - 62
Extraintestinal pathogenic Escherichia coli: "the other bad E coli"; Johnson JR et al.; Extraintestinal pathogenic Escherichia coli (ExPEC), the specialized strains of E coli that cause most extraintestinal E coli infections, represent a major but little-appreciated health threat . Although the reasons for their evolution remain mysterious, by virtue of their numerous virulence traits ExPEC clearly possess a unique ability to cause disease outside the host intestinal tract . Broader appreciation of the existence and importance of ExPEC and better understandings of their distinctive virulence mechanisms, reservoirs, and transmission pathways may lead to effective preventive interventions against the morbid and costly infections ExPEC cause.

Acta Vet Hung, 2001, 49(4), 377 - 83
Testing of a Chemiluminescence Enzyme Immunoassay for selective detection of E . Coli O157 from ground beef samples; Kovacs HD et al.; The aim of this study was to evaluate a Chemiluminescence Enzyme Immunoassay (CLIA) developed for the detection of E . coli O157:H7, using different E . coli O157 serotypes . The sensitivity and specificity of the kit were determined from the tenfold dilutions of the 24-hour broth cultures of the test strains . According to the results obtained in this trial, the sensitivity of the kit is 10(3)-10(4) cells ml-1, and it is specific for E . coli O157 . Twenty-five g ground raw beef samples were prepared and inoculated with E . coli O157:H7 at different CFU g-1 . The samples were incubated in 225 ml of modified E . coli broth with novobiocin (mEC + n) at 42 degrees C for 4 h and the immunoassays were performed following the instructions of the manufacturer . According to the results obtained by the CLIA test 10(1)-10(2) E . coli O157 g-1 can be detected from the sample . So this kit seems to be suitable for screening the samples before selective cultivation of E . coli O157:H7.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 331 - 40
Regulation of ribosomal RNA synthesis in E . coli: effects of the global regulator guanosine tetraphosphate (ppGpp); Wagner R; The global regulatory nucleotides (p)ppGpp are major effectors for the control of ribosomal RNA in bacteria . The effector molecules accumulate to different cellular levels at amino acid deprivation or during different growth rates . They change the activity of RNA polymerase to transcribe from sensitive promoters (e.g . ribosomal RNA promoters) . Sensitive promoters are characterized by a GC-rich discriminator element in addition to further structural requirements not completely understood . ppGpp must also be regarded as a mediator for growth rate control although it appears that ppGpp-independent regulatory mechanisms exist . Inhibition occurs at various steps during initiation but also during elongation where RNA polymerase pausing is observed . From the existing data a mechanistic model for the action of ppGpp is suggested considering structural details of RNA polymerase obtained at high resolution.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 217 - 22
Members of the Fur protein family regulate iron and zinc transport in E . coli and characteristics of the Fur-regulated fhuF protein; Hantke K; The regulator Fur represses with Fe2+ as cofactor iron uptake genes . The fhuF gene reacts very sensitive to minor changes of Fe2+ and Fur . It is assumed that FhuF helps in the mobilisation of iron out of the hydroxamate siderophores transported into the cell . Analysis of the protein revealed an unusual {2Fe-2S} cluster bound to a Cys-Cys-X10-Cys-X2-Cys motif in FhuF . suf genes responsible for the synthesis of the iron sulfur center were identified . The Zur protein shows 27% identity to the Fur protein of E . coli . It regulates as a repressor the high affinity uptake system znuACB . Only two additional Zur binding sites in the promoter region of genes with unknown function were found . Properties of Zur and Fur proteins from different bacteria are compared.

Neurochem Int, 2002 Jul, 41(1), 55 - 63
Expression in E . coli and purification of recombinant fragments of wild type and mutant human prion protein; Corsaro A et al.; Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain.The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases.Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases . Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc) . We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP . Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E . coli . The recovery of the purified fusion protein was monitored following the evaluation of the GST activity . The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin.The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody . The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge . The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

Mol Cells, 2002 Feb 28, 13(1), 137 - 43
Characterization of mycoplasma arginine deiminase expressed in E . coli and its inhibitory regulation of nitric oxide synthesis; Noh EJ et al.; We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia . Here, we report the over-expression of recombinant ADI (rADI) in E . coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment . We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E . coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E . coli . The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography . The rADI expressed in E . coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M . arginini . The Km for arginine of rADI was approximately 370+/-50 microM . Its optimal temperature and pH were 41 degrees C and pH 6.4, respectively, and enzyme activity remained > or = 50% for 5 d at physiological temperature and pH . Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS . Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate . These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia . Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

Biochemistry, 2002 Mar 26, 41(12), 3984 - 90
Indirect determination of the rate of iron uptake into the apoprotein of the ribonucleotide reductase of E . coli; Umback NJ et al.; The second-order rate constant k(apo) for uptake of FeII by the apoprotein of Ribonucleotide Reductase R2 has been measured by letting that reaction compete with the uptake of FeII by ferrozine (rate constant k(Fz)) . The rate of the FeII/ferrozine reaction was studied at high ferrozine concentrations, and an effective first-order rate constant k(Fz) for the disappearance of FeII determined in the presence of bovine serum albumin as a viscogen . Solutions of apoprotein and ferrozine in various ratios were mixed with FeII solutions in a stopped-flow apparatus, and the growth of the 562 nm FeII(ferrozine)3 absorbance monitored . Attempts to fit the data to a variety of kinetic schemes imply that uptake of the second FeII by apo is slower than uptake of the first, suggesting that the rate-determining step in the activation of R2 is a conformational change after the uptake of the first iron . The resulting value of k(apo) is 1.8(1) x 10(6) M(-1) x s(-1).

Scand J Immunol, 2002 Feb, 55(2), 204 - 9
Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E . coli strain Nissle 1917; Cukrowska B et al.; The aim of this study was to analyze the influence of oral administration of E . coli Nissle 1917 on the systemic humoral and cellular immunity in premature infants . Thirty-four premature infants were colonized with E . coli Nissle 1917 in a randomized, placebo-controlled blinded clinical trial . Stool samples of infants were analyzed repeatedly for the presence of the administered strain . The proliferative response to bacterial antigens of E . coli origin was measured in whole blood of 34 colonized infants and 27 noncolonized controls . E . coli colonization induced a significant increase in the proliferation of blood cells cultivated with bacterial components of E . coli Nissle 1917 and another E . coli strain in colonized infants as compared with noncolonized controls . Significantly higher amounts of specific anti-E . coli Nissle 1917 antibodies (Ab) of immunoglobulin (Ig)A isotype and nonspecific polyclonal IgM were found in the blood of colonized infants compared to noncolonized placebo controls . We concluded that the oral application of E . coli Nissle 1917 after birth significantly stimulates specific humoral and cellular responses and simultaneously induces nonspecific natural immunity.

EMBO J, 1983, 2(3), 375 - 80
Degrees of relatedness of T-even type E . coli phages using different or the same receptors and topology of serologically cross-reacting sites; Schwarz H et al.; The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated . Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein . Absence of cross-reactivity appears to reflect high degrees of dissimilarity . A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber . Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages . Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.

EMBO J, 1983, 2(1), 105 - 10
The topology of the proton translocating F0 component of the ATP synthase from E . coli K12: studies with proteases; Hoppe J et al.; The accessibility of the three F0 subunits a, b and c from the Escherichia coli K12 ATP synthase to various proteases was studied in F1-depleted inverted membrane vesicles . Subunit b was very sensitive to all applied proteases . Chymotrypsin produced a defined fragment of mol . wt . 15,000 which remained tightly bound to the membrane . The cleavage site was located at the C-terminal region of subunit b . Larger amounts of proteases were necessary to attack subunit a (mol . wt . 30,000) . There was no detectable cleavage of subunit c . It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F1 sector . The F1 sector was found to afford some protection against proteolysis of the b subunit in vitro and in vivo . Protease digestion had no influence on the electro-impelled H+ conduction via F0 but ATP-dependent H+ translocation could not be reconstituted upon binding of F1 . A possible role for subunit b as a linker between catalytic events on the F1 component and the proton pathway across the membrane is discussed.

EMBO J, 1983, 2(9), 1487 - 92
A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E . coli cells; Matthias PD et al.; We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells . This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells . A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells . These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell . The molecules are in most cases unrearranged and can be rescued into E . coli cells by bacterial transformation.

Genome Res, 2002 Mar, 12(3), 515 - 22
BAC library of T . pallidum DNA in E . coli; Smajs D et al.; Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence . A single 15.6-kb region of the T . pallidum chromosome was missing in the BAC library, between bp 248727 and 264323 . In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene . Altogether, 13 predicted T . pallidum ORFs (1.25% of the total) were incomplete or missing in the library . Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert . Of mapped clones, 19 were selected to represent the minimal set of E . coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T . pallidum ORFs . Using this minimal set of clones, at least 12 T . pallidum proteins were shown to react with pooled sera from rabbits immunized with T . pallidum, indicating that at least some T . pallidum genes are transcribed and expressed in E . coli.

Plant J, 2002 Mar, 29(5), 637 - 47
Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E . coli and generation of the active form in stress response in seedlings; Matsuoka D et al.; The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes . MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified . Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs . We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro . The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme . The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins . In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro . Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly . These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.

J Mol Recognit, 2002 Jan-Feb, 15(1), 27 - 32
Selective infection of E . coli as a function of a specific molecular interaction; Nilsson N et al.; Selective infection of phage is when the bacterial infection depends on the specific molecular interaction between an antigen and a phage-displayed protein sequence such as an antibody . Engineering of the normal infection into pathways, directed by a specific protein--protein interaction, has raised several mechanistic questions . Here, we address the type of display and the affinity between the interacting pairs . The deleted phage R408d3 was used for the first time in selective infection and was shown to exhibit a superior performance compared to the VCSM13 phage . Furthermore, the affinity between the interacting pairs also affected the selective infection process and a correlation between affinity and infection efficiency was detected, thus implying that selective infection is the method of choice for selection of rare high-affinity interactions in molecular libraries .

J Am Chem Soc, 2002 Mar 6, 124(9), 1836 - 7
Adding L-3-(2-Naphthyl)alanine to the genetic code of E . coli; Wang L et al.; An unnatural amino acid, L-3-(2-naphthyl)alanine, has been site-specifically incorporated into proteins in Escherichia coli . An orthogonal aminoacyl-tRNA synthetase was evolved that uniquely aminoacylates the unnatural amino acid onto an orthogonal amber suppressor tRNA, which delivers the acylated amino acid in response to an amber nonsense codon with translational fidelity greater than 99% . This result, together with the in vivo site-specific incorporation of O-methyl-L-tyrosine reported previously, demonstrate that this methodology may be applicable to a host of amino acids . The expansion of the genetic code to include amino acids beyond the common 20 would provide an opportunity to better understand and possibly enhance protein (and perhaps organismal) function.

J Mol Biol, 2002 Feb 22, 316(3), 531 - 46
Thermodynamics of E . coli cytidine repressor interactions with DNA: distinct modes of binding to different operators suggests a role in differential gene regulation; Tretyachenko-Ladokhina V et al.; Interactions between the Escherichia coli cytidine repressor protein (CytR) and its operator sites at the different promoters that comprise the CytR regulon, play an important role in the regulation of these promoters . The natural operators are palindromes separated by variable length central spacers (0-9 bp) . We have suggested that this variability affects the flexibility of CytR-DNA contacts, thereby affecting the critical protein-protein interactions between CytR and the cAMP receptor protein (CRP) that underlie differential repression and activation of CytR-regulated genes . To assess this hypothesis, we investigated the thermodynamics of CytR binding to the natural operator sequences found in udpP and deoP2 . To separate effects due to spacing from effects due to the differing sequences of the recognition half-sites of these two operators, we also investigated CytR binding to artificial hybrid operators, in which the half-site sequences of udpP and deoP2 were exchanged . Thermodynamic parameters, DeltaS(o), DeltaH(o) and DeltaC(o)(p), were determined by van't Hoff analysis of CytR binding, monitored by changes in the steady-state fluorescence anisotropy of dye-conjugated, operator-containing oligonucleotides . Large differences in thermodynamics were observed that depend primarily on the central spacer rather than the sequences of the recognition half-sites . Binding to operators with deoP2 spacing results in a very large, negative DeltaC(o)(p) . Association is strongly favored enthalpically and strongly disfavored entropically at ambient temperature . By contrast, binding to operators with udpP spacing results in a small, negative DeltaC(o)(p) . Association is weakly favored both enthalpically and entropically at ambient temperature . A difference of such magnitude in DeltaDeltaC(o)(p) has not been reported previously for specific binding of a transcription factor to different sites . The identical salt dependence of CytR binding to deoP2 and udpP operators indicates that ion-dependent processes do not contribute significantly to this difference . Thus, the different thermodynamic effects appear to reflect distinctly different modes of site-specific DNA binding . We discuss similarities to operator binding by CytR homologs among LacI family repressors, and we consider how different CytR binding modes might affect interactions with other components of the gene regulatory machinery that contribute to differential gene regulation .

Biochemistry, 2002 Mar 5, 41(9), 2946 - 55
Ability of E . coli cyclic AMP receptor protein to differentiate cyclic nucelotides: effects of single site mutations; Lin SH et al.; Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes . Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences . Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP . Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides . In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition . For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied . cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants . The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity . To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications . Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface . Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication . Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.

Biotechniques, 2002 Feb, 32(2), 402, 404, 406 - 9
Generation of von Willebrand factor epitope libraries expressed in E . coli; Jumilly AL et al.; The von Willebrand factor (VWF) subunit is composed of several domains, often coinciding with structural regions, characterized through their specific interaction with a ligand . Since several monoclonal antibodies (MoAbs) have been shown to functionally interfere with one of the specific interactions, we have created libraries of bacterial clones expressing peptidic sequences of VWF to map antibodies directed against this protein . Randomly cleaved fragments of VWF cDNA have been cloned in a plasmid designed for the expression of small peptides as part of larger fusion proteins . The NovaTope system is a useful procedure for protein analysis, allowing screening of epitopes composed of contiguous amino acid residues . To map MoAbs with conformational discontinuous epitopes displayed on small as well as large peptidic domains, this technique had to be widely modified to obtain two VWF peptide libraries expressing two ranges of peptide length (15-70 and 100-300 amino acids) . Screening with six MoAbs with an epitope in a known region was performed to control both libraries . Four MoAbs were mapped through the characterization of overlapping sequences for 5-10 different positively expressed clones respectively . Two of these mapped MoAbs had no known inhibitory effect and bind reduced VWF only . The fact that the two other MoAbs mapped VWF functional interactions with ligands, platelet GPIIb/IIIa and Factor VIII, respectively, demonstrate that our libraries are valuable tools to determine conformational epitopes.

Protein Sci, 2002 Mar, 11(3), 558 - 70
Binding specificity and the ligand dissociation process in the E . coli biotin holoenzyme synthetase; Kwon K et al.; The binding of the Escherichia coli biotin holoenzyme synthetase to the two ligands, biotin and bio-5'-AMP, is coupled to disorder-to-order transitions in the protein . In the structure of the biotin complex, a "glycine-rich" loop that is disordered in the apo-enzyme is folded over the ligand . Mutations in three residues in this loop result in significant changes in the affinity of the enzyme for both biotin and bio-5'-AMP . The kinetic basis of these losses in the affinity resides primarily in changes in the unimolecular rates of dissociation of the complexes . In this work, isothermal titration calorimetry has been employed to examine the detailed thermodynamics of binding of three loop mutants to biotin and bio-5'-AMP . The energetic features of dissociation of the protein*ligand complexes also have been probed by measuring the temperature dependencies of the unimolecular dissociation rates . Analysis of the data using the Eyring formalism yielded entropic and enthalpic contributions to the energetic barrier to dissociation . The thermodynamic results coupled with the known structures of the apo-enzyme and biotin complex have been used to formulate a model for progression from the ground-state complex to the transition state in biotin dissociation . In this model, the transition-state is characterized by both partial disruption of noncovalent bonds and acquisition of some of the disorder that characterizes the glycine-rich loop in the absence of ligand.

Dig Dis Sci, 2002 Jan, 47(1), 190 - 200
Ileal luminal nitric oxide synthase inhibitors and E . coli lipopolysaccharide effects in the anesthetized rat; Mailman D; Some of the effects of bacterial toxins are mediated through the local production of nitric oxide (NO) or its products . This study examined if NO inhibition in the intestinal mucosa had effects on the responses to intravenous lipopolysaccharide (LPS) in anesthetized rats . Aminoguanidine (AMGU, 500 microM), a relatively selective inducible NO synthase (iNOS) inhibitor, or N(G)-nitro-L-arginine (NOLARG, 50 or 500 microM), a nonselective inhibitor of iNOS and constitutive NOS (cNOS), were perfused through the ileal lumen during intravenous LPS (17 mg/kg) or saline administration . Intestinal H20 transport, NO3- + NO2- (NOx) secretion, absorptive site mucosal blood flow (ASBF), blood pressure, plasma {NOx}, tissue damage, and blood leukocytes were measured for 4 hr . LPS increased luminal NOx secretion . At 50 microM, luminal NOLARG attenuated the LPS-induced NOx secretion and increased blood pressure . There were no significant changes in lethality, plasma {NOx} or other parameters . At 500 microM, luminal NOLARG converted a nonlethal dose of LPS into a lethal dose, but AMGU did not increase lethality . The LPS-induced luminal NOx secretion was blocked by 500 microM intraluminal AMGU and NOLARG . Luminal NOx secretion also increased in control animals . This increase was blocked by 500 microM NOLARG but not AMGU . Luminal 500 microM NOLARG increased blood pressure, but AMGU did not . Luminal 500 microM NOLARG prevented the LPS-induced increase in plasma {NOx} and the decrease in leukocytes, but AMGU did not . Tissue damage occurred with intravenous LPS plus intraluminal 500 microM NOLARG . It was concluded that luminal AMGU inhibited mucosal iNOS . Luminal NOLARG inhibited mucosal cNOS and iNOS, and cNOS inhibition primed a lethal LPS effect . NOLARG, but not AMGU, was absorbed from the intestine and had systemic effects.

Chembiochem, 2001 Sep 3, 2(9), 695 - 701
Functional display of active bovine adrenodoxin on the surface of E . coli by chemical incorporation of the {2Fe-2S} cluster; Jose J et al.; The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications . Up to now, however, cellular surface display was merely restricted to simple polypeptide chains . Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli . For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway . Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions . The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell . Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.

J Mol Biol, 2002 Feb 1, 315(5), 1199 - 208
Analysis of the E . coli NifS CsdB protein at 2.0 A reveals the structural basis for perselenide and persulfide intermediate formation; Lima CD; The Escherichia coli NifS CsdB protein is a member of the homodimeric pyridoxal 5'-phosphate (PLP)-dependent family of enzymes . These enzymes are capable of decomposing cysteine or selenocysteine into L-alanine and sulfur or selenium, respectively . E . coli NifS CsdB has a high specificity for L-selenocysteine in comparison to l-cysteine, suggesting a role for this enzyme is selenium metabolism . The 2.0 A crystal structure of E . coli NifS CsdB reveals a high-resolution view of the active site of this enzyme in apo-, persulfide, perselenide, and selenocysteine-bound intermediates, suggesting a mechanism for the stabilization of the enzyme persulfide and perselenide intermediates during catalysis, a necessary intermediate in the formation of sulfur and selenium containing metabolites .

Antonie Van Leeuwenhoek, 2001 Dec, 80(3-4), 297 - 300
A convenient and rapid method for genetic transformation of E . coli with plasmids; Chen X et al.; A convenient and rapid method for the genetic transformation of Escherichia coli with plasmids is proposed . By mixing the recipient cells and plasmid DNA and spreading them directly on selective medium plates containing Ca2+, the so-called 'plate transformation' could achieve almost the same transformation efficiency as the classical transformation method with calcium . The whole protocol takes only about 2 min, its simplicity compared favorably, not only to the usual protocol, but also to all other documented modifications.

Biosci Biotechnol Biochem, 2001 Dec, 65(12), 2798 - 801
Human tyrosine tRNA is also internally cleavable by E . coli ribonuclease P RNA ribozyme in vitro; Ando T et al.; Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure . Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P) . Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs . This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E . coli RNase P RNA . This tRNA is the first example for hyperprocessed non-Drosophila tRNAs . The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.

World J Gastroenterol, 1998 Apr, 4(2), 165 - 168
Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli; Zhu FL et al.; AIM:To obtain greater antigenicity of HCV NS3 protein.METHODS:The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients.The DNA sequence was determined by dideoxy-mediated chain termination method using T7 polymerase.HCV NS3 protein was expressed in E . coli.RESULTS:Sequence analysis indicated that the HCV isolate of this study belongs to HCV-II; SDS-PAGE demonstrated an M(r) 23800 and an M(r) 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately.Western blotting and ELISA showed NS3 protein possessed greater antigenicity.CONCLUSION:Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.

J Cell Sci, 2002 Jan 1, 115(Pt 1), 91 - 8
Cell-surface attachment of pedestal-forming enteropathogenic E . coli induces a clustering of raft components and a recruitment of annexin 2; Zobiack N et al.; Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes . To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC) . We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment . Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites . Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10 . EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact . This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 249 - 55
Prevalence of verotoxin-producing Escherichia coli and E . coli O157:H7 in pig carcasses from three French slaughterhouses; Bouvet J et al.; Verotoxin-producing Escherichia coli (VTEC) are important food-borne pathogens in humans . Several studies have demonstrated that cattle are a major reservoir of VTEC but few data are available about the occurrence of VTEC in other species . In France, there is no data about pigs and pork meat . The main purpose of this study was to evaluate the prevalence of E . coli O157:H7 and other VTEC in pork carcasses . The second aim of the study was to get a picture of pork carcass contamination by VTEC . Pork carcasses from three French slaughterhouses (50 carcasses per slaughterhouse) were tested for the presence of VTEC and E . coli O157:H7 . For each carcass, both internal and external sites were investigated (five on pig skin and three on muscles) and samples were collected by cutting out a surface of 25 cm2 . A total of 1200 samples were analyzed by the polymerase chain reaction (PCR) after an enrichment step . Primers used were degenerate-sequences which allowed amplification of various types of verotoxin genes (stx) . In addition, a second PCR which specifically detected E . coli O157:H7 was carried out on the stx-positive samples . The percentage of stx-positive PCR samples and carcasses was 12.7% (152/1200) and 50% (75/150), respectively . No E . coli O157:H7 was detected . The prevalence for each slaughterhouse was not significantly different . Skin samples of belly, leg and shoulder allowed detection of more than 80% of the VTEC positive carcasses.

J Mol Biol, 2002 Jan 18, 315(3), 351 - 71
Open and closed conformation of the E . coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism; Koellner G et al.; The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution . The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations . The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other . With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation) . By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation) . Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site . This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism . In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation . Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form . The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204 . The mechanism explains the broad specificity of E . coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates . The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor .

J Mol Biol, 2002 Jan 11, 315(2), 155 - 70
A novel view of domain flexibility in E . coli adenylate kinase based on structural mode-coupling (15)N NMR relaxation; Tugarinov V et al.; Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd and LID, catalyzes the reaction AMP+ATP-->2ADP . In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis . Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for (15)N relaxation . SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond . The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling . AKeco diffuses globally with correlation time tau(m)=15.1 ns, while locally two different dynamic cases prevail . The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S(2), of 0.8-0.9 and local motional correlation times, tau, mainly between 5-130 ps . This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations . The domains AMPbd and LID feature small parallel (Z(M)) ordering of S(2)=0.2-0.5 which can be reinterpreted as high perpendicular (Y(M)) ordering . M denotes the local ordering/local diffusion frame . Local motion about Z(M) is given by tau( parallel) approximately 5 ps and local motion of the effective Z(M) axis about Y(M) by tau( perpendicular)=6-11 ns . Z(M) is tilted at approximately 20 degrees from the N-H bond . The orientation of the Y(M) axis may be considered parallel to the C(alpha)(i-1)-C(alpha)(i) axis . The tau( perpendicular) mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion . A powerful new model of protein flexibility/domain motion has been established . Conformational exchange (R(ex)) processes accompany the tau( perpendicular) mode . The SRLS analysis is compared with the conventional model-free analysis .

Zhonghua Zhong Liu Za Zhi, 2000 May, 22(3), 198 - 200
{Expression in E . coli of protein encoded by human esophageal cancer related gene-1}; Wang Y et al.; OBJECTIVE: To express esophageal cancer related gene-1 (ECRG-1)--in E . coli . METHODS: The human ECRG-1 was cloned by RT-PCR . Expression plasmid of the ECRG-1/GST pi fusion gene was constructed and introduced into E . Coli . The fusion protein was induced to express by ITPG . The recombinant protein was identified by Western blot . BALB/C mice were immunized with the protein purified by SDS-PAGE for the preparation of polyclonal antibody . RESULTS: DNA sequencing confirmed that the sequence of ECRG-1 was identical to that of the previously obtained by 5'-RACE technique . Expression of the protein in E . coli was verified by Western blot . The polyclonal antibody obtained from immunized BALB/C mice had a title of 1:3,200 . CONCLUSION: The antibody available is useful for the further study of structure and function of the esophageal cancer related protein.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 Nov, 22(11), 645 - 7
{High-level expression of Mycobacterium tuberculosis 16,000 antigen in E . coli}; Xiong Z et al.; OBJECTIVE: To gain the recombinant protein antigen 16,000 of Mycobacterium tuberculosis highly expressed in E . coli and study its immunological characteristics . METHODS: DNA fragments code for the protein were obtained by PCR, then cloned into the pET plasmid vector to gain recombinant E . coli . Cells were cultured and induced to produce recombinant protein, whose molecular size and present form were analyzed by SDS-PAGE, and its immunological characteristics were analyzed by Western-blotting and ELISA technology . RESULTS: The clone was analyzed at the nucleotide lever and shown the same DNA sequence coding for natural 16,000 protein . Analyzed by SDS-PAGE and Western-blotting, it was found to produce immunoreactive proteins with mobilities very similar to those of the 16,000 protein antigen, and the recombinant protein amounted to 40% of total cell proteins . ELISA results indicated that the purified recombinant protein could distinguish sera from tuberculosis patients with anti-PPD antibody and those from tuberculin positive contacts . CONCLUSIONS: Recombinant 16,000 protein antigen highly expressed in the form of solution in E . coli was gained and this antigen was located in cell plasma . This recombinant protein showed specific immunogenicity.

Biochemistry, 2002 Jan 8, 41(1), 236 - 43
E . coli MEP synthase: steady-state kinetic analysis and substrate binding; Koppisch AT et al.; 2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids . Recombinant E . coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM . The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH . The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors . Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP . Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP . These results are consistent with an ordered mechanism where NADPH binds before DXP.

Prikl Biokhim Mikrobiol, 2001 Nov-Dec, 37(6), 742 - 6
{Interaction of E . coli cells with ascorbic acid and sodium nitrite studied by ESR method}; Zhumbaeva TT et al.; Ascorbic acid, an effective modulator and regulator of cell metabolism, was shown to induce the production of nitric oxide in E . coli cells . This process was detected by EPR spectroscopy as the generation of a spectral signal typical of nitrosyl-iron-sulfur centers (Fe-S-NO) under anaerobic conditions . Incubation of E . coli cells in the presence of ascorbic acid under aerobic conditions was shown to be accompanied by sodium nitrite formation . It is suggested that ascorbic acid is capable of supporting the system of energy supply to cells in hypoxia caused by reduced oxygen content or treatment with sodium nitrite.

Vopr Med Khim, 2001 Sep-Oct, 47(5), 477 - 82
{Sensitivity of E . coli and C . freundii strains to 5-azacytidine}; Lebenka AIu et al.; The sensitivity of E . coli and C . freundii strains to 5-azacytidine and restrictase activity of partially purified cell-free extracts were investigated . Restrictase activity was found only in 5-azacytidine-sensitive strains . In the 5-azacytidine-resistant strains restrictase activity was not detected.

Dtsch Tierarztl Wochenschr, 2001 Nov, 108(11), 454 - 8
Binding of E . coli isolates from pigs with postweaning diarrhea or edema disease to crude intestinal mucin of a weaned pig; Styriak I et al.; The presence of the fedA (gene coding F18 fimbriae) and genes coding STa and LTI enterotoxins and verotoxin Stx2v was determined in 30 E . coli strains isolated from weaned pigs with postweaning diarrhea (PWD) and edema disease (ED) . The fedA gene was detected in 22 strains (73.3%) . It was mostly associated with the presence of ST gene determinant (14 from 22 fedA positive strains, 63.6%) . Two strains possessed ST/Stx2v or LTI/Stx2v combination of genes for both toxins and two strains were negative for investigated toxin determinants . Among 8 fedA-negative strains, five strains without gene determinants for toxins were detected . All 30 E . coli strains were investigated for their binding to crude intestinal mucin of a weaned pig fixed in wells of microtitre plates . Positive mucin binding was observed in most of strains, however, great differences were shown between individual strains . Nineteen strains were classified as strongly adherent, 10 strains as weakly adherent, and only one nonadherent strain was found . Three E . coli strains, selected among the best mucin binders, bound to mucin in a concentration-dependent manner . A high mucin binding by E . coli strains was observed only after their cultivation on blood agar plates . Their cultivation in LB broth or on McConkey agar plates had negative effect on the mucin binding by these strains . The mucin binding is not restricted by the presence of fedA gene because the strains displaying very good binding are found either among fedA positive (1, 602/2, 4/3, 576/6) or fedA negative (DK 6, DK 8) E . coli strains . E . coli strains with the highest mucin binding ability belong to potential ST producents (strains 1, 602/2, 4/3, 6/2, 602/4) while the strains without genes coding toxin production displayed lower binding to mucin substratum with exception of the strains ZV5 and 13.

Int J Food Microbiol, 2001 Oct 22, 70(1-2), 143 - 54
Automated immunomagnetic separation and microarray detection of E . coli O157:H7 from poultry carcass rinse; Chandler DP et al.; We describe the development and application of an electromagnetic flow cell and fluidics system for automated immunomagnetic separation (IMS) of Escherichia coli O157:H7 directly from poultry carcass rinse . We further describe the biochemical coupling of automated sample preparation with nucleic acid microarrays . Both the cell concentration system and microarray detection method did not require cell growth or enrichment from the poultry carcass rinse prior to IMS . Highly porous Ni foam was used to enhance the magnetic field gradient within the flow path, providing a mechanism for immobilizing immunomagnetic particles throughout the fluid rather than the tubing wall . A maximum of 32% recovery efficiency of non-pathogenic E . coli was achieved within the automated system with 6 s cell contact times using commercially available antibodies targeted against the O and K antigens . A 15-min protocol (from sample injection though elution) provided a cell recovery efficiency that was statistically similar to > I h batch captures . O157:H7 cells were reproducibly isolated directly from poultry carcass rinse with 39% recovery efficiency at 10(3) CFU ml(-1) inoculum . Direct plating of washed beads showed positive recovery of O157:H7 directly from poultry carcass rinse at an inoculum of 10 CFU ml(-1) . Recovered beads were used for direct polymerase chain reaction (PCR) amplification and microarray detection, with a process-level detection limit (automated cell concentration though microarray detection) of < 10(3)CFU ml(-1) in poultry carcass rinse.

J Biotechnol, 2002 Feb 28, 93(3), 189 - 202
Structured model to predict intracellular amino acid shortages during recombinant protein overexpression in E . coli; Harcum SW; Recombinant protein overexpression and the classical stringent response have been shown to induce the same proteases . Since the stringent response was the result of an intracellular amino acid shortage, it was hypothesized that the overexpression of the recombinant protein also caused an intracellular amino acid shortage . A structured non-segregated kinetic mathematical model of recombinant Escherichia coli was developed to predict intracellular amino acid shortages during recombinant protein overexpression, and thus the induction of the stringent response . Two model recombinant proteins were examined, chloramphenicol acetyl-transferase (CAT) and an 'average protein' . The model predicted an aromatic amino acid shortage during CAT overexpression, as predicted based on the CAT's amino acid content . The model also predicted a shortage of the intracellular alanine family amino acid pool during CAT overexpression . This was unexpected due to the relatively low content of alanine family amino acids in CAT compared to the average E . coli protein . The model predicted alanine, glutarate, and aspartate family amino acid shortages during recombinant 'average protein' overexpression . Additionally, the model predicted a decrease in the ribosome pool at induction for both recombinant proteins, which agrees with published experimental results . Thus, the structured kinetic model was able to predict amino acid shortages, that could potentially cause a stringent response and elevated protease activity.

Insect Biochem Mol Biol, 2002 Feb, 32(2), 167 - 74
Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E . coli and purification in a functional state; Grebe M et al.; Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E . coli and purified by affinity chromatography . The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding . Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells . Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent . USP binds preferentially to DR5 . Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly . Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics . Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified receptor protein was able to bind ligand . The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C . tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A.

Genes Cells, 2001 Nov, 6(11), 987 - 1001
Mammalian homologue of E . coli Ras-like GTPase (ERA) is a possible apoptosis regulator with RNA binding activity; Akiyama T et al.; BACKGROUND: ERA (Escherichia coli Ras-like protein) is an E . coli GTP binding protein that is essential for proliferation . A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus . However, the physiological function of eukaryotic ERA-like proteins is not known . RESULTS: We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA . The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain . We performed transfection experiments with wild-type H-ERA or various H-ERA mutants . H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression . Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA . We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins . CONCLUSION: Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.

Structure (Camb), 2001 Dec, 9(12), 1153 - 64
The 1.6 A crystal structure of E . coli argininosuccinate synthetase suggests a conformational change during catalysis; Lemke CT et al.; BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles . In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock . RESULTS: The crystal structure of E . coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing . The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP) . Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain . The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding . CONCLUSIONS: The first three-dimensional structures of AS are reported . The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases . The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle . Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.

Biotechnol Prog, 2001 Nov-Dec, 17(6), 1169 - 79
Engineering the microstructure and permeability of thin multilayer latex biocatalytic coatings containing E . coli; Lyngberg OK et al.; The microstructure and permeability of rehydrated 20-100 microm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 microm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E . coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO(3) as the diffussant . The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles . Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM) . The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO(3) to its diffusivity in water (D(eff)/D) . Polymer particle coalescence was arrested by two methods to increase coating permeability . The first used glycerol with coating drying at 4 degrees C, near the glass transition temperature (T(g)) . The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away . D(eff)/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time . D(eff)/D varied from 3 x 10(-4) for unmodified SF091 coatings to 6.8 x 10(-2) for coatings containing sucrose . D(eff)/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC) . When corrected for the flattened particle layer, D(eff)/D of HEC-free coatings was as high as 0.20, which agreed with the value predicted from analysis of cryo-SEM images of the coat surface . D(eff)/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films . D(eff)/D of SF091 latex trilayer coatings containing viable E . coli HB101 cells decreased as cell loading was increased from 2.2 x 10(-2) for 64 g dry cell weight per liter of coat volume to 5 x 10(-3) for 151 g DCW/L of coat volume . The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for D(eff)/D in periodic composites.

Biochemistry, 2001 Dec 4, 40(48), 14706 - 14
Protein folding pathways of adenylate kinase from E . coli: hydrostatic pressure and stopped-flow studies; Ruan Q et al.; Adenylate kinase (AKe) from E . coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue . We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions . In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied . The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa . This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change . The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions . At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis . The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions . The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe . Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity . Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site . Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W . The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.

BMC Biochem . 2001;2(1):13 . Epub 2001 Nov 14.
The intein of the Thermoplasma A-ATPase A subunit: structure, evolution and expression in E . coli; Senejani AG et al.; BACKGROUND: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond . In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation . RESULTS: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T . acidophilum genome that has been identified to contain an intein . This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P . furiosus and P . horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae . In contrast to the other inteins in catalytic ATPase subunits, the T . acidophilum intein does not contain an endonuclease domain.T . acidophilum has different codon usage frequencies as compared to Escherichia coli . Initially, the low abundance of rare tRNAs prevented expression of the T . acidophilum A-ATPase A subunit in E . coli . Using a strain of E . coli that expresses additional tRNAs for rare codons, the T . acidophilum A-ATPase A subunit was successfully expressed in E . coli . CONCLUSIONS: Despite differences in pH and temperature between the E . coli and the T . acidophilum cytoplasms, the T . acidophilum intein retains efficient self-splicing activity when expressed in E . coli . The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase . Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

Bioorg Med Chem Lett, 2001 Dec 17, 11(24), 3107 - 10
Synthesis of P(1)-Citronellyl-P(2)-alpha-D-pyranosyl pyrophosphates as potential substrates for the E . coli undecaprenyl-pyrophosphoryl-N-acetylglucoseaminyl transferase MurG; Cudic P et al.; P(1)-Citronellyl-P(2)-alpha-D-pyranosyl pyrophosphates containing alpha-D-N-acetylglucoseaminyl, alpha-D-glucosyl, and alpha-D-N-acetylmuramyl carbohydrates were synthesized and used in substrate specificity studies of the Escherichia coli MurG enzyme . Oxalyl chloride activation of citronellyl phosphate for coupling to alpha-D-pyranose-1-phosphates resulted in markedly improved yields over traditional Khorana-Moffatt and diphenyl chlorophosphate activation strategies.

J Bacteriol, 2001 Dec, 183(24), 7273 - 84
Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E . coli intimin EaeA; Wentzel A et al.; Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors . The EaeA intimin from enterohemorrhagic E . coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope . We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins . We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E . coli K-12 via fusion to truncated intimin . Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene . Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E . coli K-12 to human platelets . Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds . These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E . coli and their exposure on the cell surface . Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.

Protein Sci, 2001 Dec, 10(12), 2618 - 22
Competing protein:protein interactions are proposed to control the biological switch of the E coli biotin repressor; Weaver LH et al.; A model is suggested for the complex between the biotin repressor of Escherichia coli, BirA, and BCCP, the biotin carboxyl carrier protein to which BirA transfers biotin . The model is consistent with prior physical and biochemical studies . Measurement of transfer rates for variants of BirA with single-site mutations in the proposed BirA:BCCP interface region also provides support . The unique feature of the proposed interaction between BirA and BCCP is that it uses the same beta-sheet region on the surface of BirA that the protein uses for homodimerization into a form competent to bind DNA . The resulting mutually exclusive protein:protein interfaces explain the novel feature of the BirA regulatory system, namely, that transcription of the genes involved in biotin synthesis is not determined by the level of biotin, per se, but by the level of unmodified BCCP . The model also provides a role for the C-terminal domain of BirA that is structurally similar to an SH3 domain.

Protein Sci, 2001 Dec, 10(12), 2608 - 17
The C-terminal domain of biotin protein ligase from E . coli is required for catalytic activity; Chapman-Smith A et al.; Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase . BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction . We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA . Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP . None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates . However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA . No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site . Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP . Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.

Ann N Y Acad Sci, 2001 Sep, 945, 116 - 8
Detection of circulating CEA molecules in human sera and leukopheresis of peripheral blood stem cells with E . coli expressed bispecific CEAScFv-streptavidin fusion protein-based immuno-PCR technique; Ren J et al.; In this study, a method called bispecific CEAScFv-streptavidin fusion protein-based immuno-PCR technique will be tested experimentally . The application of the bispecific fusion protein-based immuno-PCR technique has the significant advantage that it can be readily applied in the clinical setting, as well as tested as a potential screening tool in high-risk populations of certain types of cancer.

J Mol Biol, 2001 Nov 9, 313(5), 1195 - 206
A structural census of metabolic networks for E . coli; Saqi MA et al.; A structural survey of the Escherichia coli proteins occurring in metabolic networks in the KEGG database (release 19 of LIGAND) has been carried out . A measure of structural coverage of a network is defined and calculated for each network . Twenty-four networks have 50 % or more of the enzyme steps assigned in E . coli and of these 21 have a structural coverage of 50 % or more . For those proteins that have a region matching a SCOP domain 50 % fall on or below the 30 % sequence identity threshold and represent non-trivial comparative modelling targets highlighting the need for experimental structure determination studies . The survey reveals the predominance of alpha/beta and alpha+beta folds for enzymes involved in metabolic pathways and that this general trend is maintained at the level of each pathway . The most popular superfamilies are coenzyme binding domains and are involved in the supply of energy to reactions . Although a few superfamilies are found in many pathways, in general there is a specificity of a particular superfamily for a particular pathway .

Receptors Channels, 2001, 7(4), 303 - 18
Reconstitution of human 5-hydroxytryptamine5A receptor--G protein coupling in E . coli and Sf9 cell membranes with membranes from Sf9 cells expressing mammalian G proteins; Francken BJ et al.; The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E . coli) to verify its pharmacological profile in the absence of G proteins . In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach . Agonists displayed lower affinities for h5-ht5A-E . coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E . coli . Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor . To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E . coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins . The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of {35S}GTP gamma S binding . The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists . Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the {35S}GTP gamma S binding assay.

Bioorg Khim, 2000 Nov, 26(11), 844 - 51
{Cleavage of RNA in hybrid duplexes by E . coli ribonuclease H . II . Substrate properties of nucleotides containing non-nucleotide linkers}; Vorob'ev PE et al.; The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers . Hybrid complexes of the bridged oligonucleotides proved to be substrates for the E . coli ribonuclease H . The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold . It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers . RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucleotide . The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.

Vopr Med Khim, 2001 Jul-Aug, 47(4), 382 - 92
{Heterologous expression of eukaryotic CYP450 . 1 . Heterologous expression of cytochrome P450 2B4 using groups with various affinity in E . coli}; Zhgun AA et al.; The expression levels of cytochrome P450 2B4 variants with N- and C-terminal modifications were compared and some of the enzymatic characteristics of recombinant proteins studied . Following C-terminal hybrids for CYP2B4 gene were constructed: 1) with intein-chitin binding domain cassette 2) with hexahistidine tag . These modifications were combined with P450 2B4 glutathione-S-transferase N-terminal fusions {Pernecky S.J., et . al., (1995) Arch . Biochem . Biophys., 318, 446-456} . The obtained constructs provided for the synthesis of full-length protein products in E . coli cells with holoenzyme yield at the levels of 200-1000 nmoles/l of the bacterial culture . Partial in vivo proteolysis was observed for C-terminal fusions with intein moiety despite the presence of glycine aminoacid residue at the junction of two proteins . The principle inapplicability of standard purification scheme for isolation of P450 2B4-intein fusions is demonstrated, since the P450 domain is inactivated at 40 mM DTT concentrations . The recombinant full-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control of T7 promoter and purified using immobilized metal-ion chelating chromatography . The C-terminal hexahistidine tag does not affect the catalytic properties of recombinant enzyme in 7-pentoxyresorufin O-dealkylation reaction.

Biosens Bioelectron, 2001 Dec, 16(9-12), 745 - 55
A MEMS based amperometric detector for E . coli bacteria using self-assembled monolayers; Gau JJ et al.; We developed a system for amperometric detection of Escherichia coli (E . coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification . Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable . Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E . coli . Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au . The biotinylated thiol approach yielded the best results . High specificity for E . coli was achieved using ssDNA-rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme . The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min . The detection system was capable of detecting 1000 E . coli cells without polymerase chain reaction with high specificity for E . coli vs . the bacteria Bordetella bronchiseptica.

J Ind Microbiol Biotechnol, 2001 Aug, 27(2), 129 - 34
Comparison of an immunochromatographic method and the TaqMan E . coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching; Fratamico PM et al.; An immunochromatographic-based assay (Quixtrade mark E . coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E . coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds . Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E . coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting . The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested . E . coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process . However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E . coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds . The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid . At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E . coli O157:H7 . All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E . coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h . In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts . In conclusion, E . coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays . The data also revealed that blanching may not be effective to completely inactivate all the E . coli O157:H7 that may be present in sprouts.

Protein Sci, 2001 Nov, 10(11), 2272 - 9
E . coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide; Boschi-Muller S et al.; The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide . The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site . The core is wrapped by two long N- and C-terminal extended chains . The catalytic mechanism of the E . coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation . In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities . All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol . However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner . The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant . Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM . Thus, thioredoxin is likely the reductant in vivo . Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO) . Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.

Protein Sci, 2001 Nov, 10(11), 2186 - 94
Influence of a sulfhydryl cross-link across the allosteric-site interface of E . coli phosphofructokinase; Johnson JL et al.; To assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in E . coli phosphofructokinase by changing N288 to cysteine . N288 is located in close proximity to the equivalent residue on an adjacent subunit . Although SDS-PAGE of oxidized N288C indicates monomeric protein, blocking the six native cysteine residues with N-ethyl maleimide (NEM) reveals dimers of N288C on non-native gels . Subsequent addition of dithiothreitol (DTT) to NEM-labeled N288C regenerates the monomer on SDS-PAGE, reflecting the reversibility of intersubunit disulfide bond formation . KSCN-induced hybrid formation between N288C and the charged-tagged mutant E195,199K exhibits full monomer-monomer exchange only upon DTT addition, providing a novel assessment of disulfide bond formation without NEM treatment . N288C also exhibits a diminished tendency toward nonspecific aggregation under denaturing conditions, a phenomenon associated with monomer formation in PFK . Pressure-induced dissociation and urea denaturation studies further indicate that oxidized N288C exhibits increased quaternary stability along both interfaces of the tetramer, suggesting a synergistic relationship between active site and allosteric site formation . Although the apparent binding affinities of substrates and effectors change somewhat upon disulfide formation in N288C, little difference is evident between the maximally inhibited and activated forms of the enzyme in oxidizing versus reducing conditions . Allosteric influence, therefore, is not correlated to subunit-subunit affinity, and does not involve substantial interfacial rearrangement.

Zhonghua Yi Xue Za Zhi, 1999 Mar, 79(3), 221 - 3
{High-level expression of 6B11 ovarian carcinoma anti-idiotypic antibody scFv genes in E . Coli}; Liu Y et al.; OBJECTIVE: To express high-level 6B11 ovarian carcinoma anti-idiotypic antibody single chain Fv (scFv) genes in E . Coli . METHODS: Using PCR cloning technique, We cloned 6B11scFv genes into bacterial expression vector pKPL-3a . Recombinant plasmid vector pL-6B11scFv was transformed into E . Coli pop2136 with temperature control to produce proteins . Renaturated proteins were purified on DEAE-Sepharose Fast Flow ion exchange column with salt gradient elution . Immunoactivity was determined with ELISA and inhibition ELISA tests respectively . RESULTS: 6B11scFv genes were expressed as inclusion bodies in the cytoplasm . The purity of 6B11scFv turned out by SDS-PAGE was more than 95% . The expressed proteins after purification specifically reacted with anti-ovarian carcinoma monoclonal antibody COC166-9 and efficiently inhibited the reaction between primary ovarian carcinoma antigen OC166-9 and COC166-9 . CONCLUSION: We successfully expressed high-level 6B11scFv genes in E . Coli . Expressed proteins showed pretty good immunoactivity.

Infect Immun, 2001 Nov, 69(11), 7152 - 8
Inhibition of attaching and effacing lesion formation following enteropathogenic Escherichia coli and Shiga toxin-producing E . coli infection; Johnson-Henry K et al.; Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E . coli (STEC) induce cytoskeletal changes in infected epithelial cells . To further characterize host cytosolic responses to infection, a series of specific cell-signaling inhibitors were employed . Initial bacterial adhesion to HEp-2 epithelial cells was not reduced, whereas alpha-actinin accumulation in infected cells was blocked by a phosphoinositide-specific phospholipase C inhibitor (ET-18-OCH3), phosphoinositide 3-kinase inhibitors (wortmannin and LY294002), and a 5-lipoxygenase inhibitor, nordihydroguaretic acid . A cyclooxygenase-2 inhibitor (NS-398), however, did not block alpha-actinin reorganization in response to EPEC and STEC infections . Understanding signal transduction responses to enteric pathogens could provide the basis for the development of novel therapeutic strategies.

Mutat Res, 2001 Nov 1, 487(1-2), 51 - 8
Overexpression of Ogt reduces MNU and ENU induced transition, but not transversion, mutations in E . coli; Beenken K et al.; Studies of alkylation-induced mutations in Escherichia coli FX-11 revealed that both N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) produced tRNA suppressor mutations (G:C to A:T) but only ENU produced a significant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G) . Further, the ENU-induced transversions were absent in a UmuC-defective strain . This suggested that transition mutations could result from alkylation of guanine or thymine at the O(6)- and O(4)-positions, respectively, but that transversions might result from alkylation of thymine at the O(2)-position . To test this idea, the gene encoding O(6)-alkylguanine-DNA methyltransferase (ogt) was recombined into a plasmid to overexpress the cellular levels of this enzyme . Ogt protein can de-alkylate O(6)-alkylguanine and O(4)-alkylthymine, but not O(2)-alkylthymine . Cells harboring the plasmid (or a control plasmid lacking the ogt gene) were exposed to different concentrations of MNU or ENU and the resulting mutations were analyzed . With either MNU or ENU, the frequency of GlnV(o) suppressors was reduced about 70-fold in the Ogt-overexpressing cells, suggesting that Ogt eliminated O(6)-alkylguanine . Similarly, GlnU(o) suppressor frequencies were substantially reduced . In contrast, the reduction in frequency for the backmutations was slight, only about 2.5-fold with MNU and less than two-fold for ENU . However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by overexpression of Ogt, suggesting repair of O(4)-alkylthymine . The frequency of transversions, in comparison, was essentially unaltered . These results implicate O(2)-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.

Gene, 2001 Oct 3, 276(1-2), 89 - 99
Analysis of codon usage diversity of bacterial genes with a self-organizing map (SOM): characterization of horizontally transferred genes with emphasis on the E . coli O157 genome; Kanaya S et al.; With increases in the amounts of available DNA sequence data, it has become increasingly important to develop tools for comprehensive systematic analysis and comparison of species-specific characteristics of protein-coding sequences for a wide variety of genomes . In the present study, we used a novel neural-network algorithm, a self-organizing map (SOM), to efficiently and comprehensively analyze codon usage in approximately 60,000 genes from 29 bacterial species simultaneously . This SOM makes it possible to cluster and visualize genes of individual species separately at a much higher resolution than can be obtained with principal component analysis . The organization of the SOM can be explained by the genome G+C% and tRNA compositions of the individual species . We used SOM to examine codon usage heterogeneity in the E . coli O157 genome, which contains 'O157-unique segments' (O-islands), and showed that SOM is a powerful tool for characterization of horizontally transferred genes.

J Am Chem Soc, 2001 Oct 10, 123(40), 9860 - 6
Site-specific amide hydrogen/deuterium exchange in E . coli thioredoxins measured by electrospray ionization mass spectrometry; Kim MY et al.; Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach . In this study, site-specific amide deuterium content was measured in oxidized and reduced E . coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7) . The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants . The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data . In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels . Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences . The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms . The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32 . It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.

J Mol Biol, 2001 Oct 5, 312(5), 1121 - 34
Rough energy landscapes in protein folding: dimeric E . coli Trp repressor folds through three parallel channels; Gloss LM et al.; The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development . Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases . The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration . Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species . A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer . The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations . These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain . One, perhaps both, of these packing modes contains non-native contacts . Copyright 2001 Academic Press.

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