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-lengt