Mol Biol Evol, 2003 Aug, 20(8), 1188 - 94 Epub 2003 May 30. Loss of DNA recombinational repair enzymes in the initial stages of genome degeneration; Dale C et al.; Many obligate intracellular pathogens and symbionts undergo genome degeneration during long-term association with eukaryotic hosts; however, very little is known about genome changes that occur in the initial stages of such intracellular associations . By focusing on a clade of bacteria that have recently established symbiotic associations with insect hosts, we have identified events that may contribute to the reduction and degeneration of symbiont genomes . Unlike virtually all other bacteria, the obligate symbionts of maize and rice weevils each display substantial sequence divergence between multiple copies of their rDNA genes, resulting from a reduction in the efficacy of recombinational gene conversion, coincident with the inactivation of the recombinational repair gene recF in the common ancestor of both symbionts . The maize weevil endosymbiont also lacks a functional recA, resulting in further reduction in the efficacy of gene conversion between paralogous rDNAs and in a novel IS-mediated deletion in a 23S rDNA gene . Similar events may be pervasive during the evolution of symbiosis because symbiont genomes typically lack recombinational repair genes and have reduced numbers of ribosomal operons. J Biol Chem, 2003 Aug 8, 278(32), 30115 - 24 Epub 2003 May 30. A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure; Seigneuret M et al.; The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated . Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein . Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template . The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport . In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA . These differences can be related to those of the transported substrates . Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein . These may be important for size selection and proper positioning of drugs for transport . The relevance of the modeled conformation to P-gp function is discussed. J Biol Chem, 2003 Aug 15, 278(33), 31290 - 6 Epub 2003 May 29. An electrical potential in the access channel of catalases enhances catalysis; Chelikani P et al.; Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30-50 A in length . The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases . Changes in Val169, 8 A from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity . Changes in Asp181, 12 A from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues . Only the D181E variant retains wild type activity . Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169 . It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 A distant . The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron . Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues. J Biol Chem, 2003 Aug 15, 278(33), 30661 - 8 Epub 2003 May 30. CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis; Pojer F et al.; The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety . The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies . CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase . In the present study, the enzyme CloR was overexpressed in Escherichia coli, purified, and identified as a bifunctional non-heme iron oxygenase, which converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to 3DMA-4HB by two consecutive oxidative decarboxylation steps . In 18O2 labeling experiments we showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product 3DMA-4HB during the second reaction step . CloR does not show sequence similarity to known oxygenases . It apparently presents a novel member of the diverse family of the non-heme iron (II) and alpha-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an alpha-keto acid and as a hydroxylation substrate . The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature. Vet Res Commun, 2003 Apr, 27(3), 243 - 56 Construction, expression, purification, refold and activity assay of a specific scFv fragment against foot and mouth disease virus; ShengFeng C et al.; An active form of a single-chain antibody (scFv) from the murine monoclonal antibody (mAb) 1C7, which is specific for type O foot and mouth disease virus (FMDV), was produced in Escherichia coli . The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction . VH-(Gly4Ser)3-VL genes were screened by phage display technology . The sequencing results showed that the VH gene of scFv was composed of germline VH76-1BG-DFL16.1-JH4 and the VL gene of scFv consisted of germline bw20-JK2 . The resultant scFv gene was cloned to the pPRoEX HTc vector and expressed in E . coli as inclusion bodies . After extraction from the E . coli cells, the inclusion bodies were solubilized and denatured in the presence of 8 mol/L urea . The expressed scFv fusion proteins were purified by nickel-nitrilotriacetic acid and finally renatured by dialysis . The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay . The result revealed that the 1C7 scFv conserved the same characteristics of specific recognition and binding to type O FMDV as the parental 1C7 mAb. Plant Mol Biol, 2003 Apr, 51(6), 877 - 84 The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no alpha-L-fucosidase activity; Tarrago T et al.; An alpha-L-fucosidase purified from pea (Pisum sativum L . cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal alpha-L-fucosidic linkages from oligosaccharide fragments of xyloglucan . cDNA and genomic copies were further isolated and sequenced . The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines . This was the first alpha-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated . Here, our biochemical and immuno analyses suggest that fuc1 does not encode an alpha-L-fucosidase . Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without alpha-L-fucosidase activity . Pea plants had endogenous alpha-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E . coli . In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive . By chromatographic analysis of pea protein extracts, we separated alpha-L-fucosidase-active fractions from the 20 kDa protein fractions . We conclude that the alpha-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein . A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed. Am J Kidney Dis, 2003 Jun, 41(6), E13 - 7 Renal parenchymal malacoplakia: a rare cause of ARF with a review of recent literature; Tam VK et al.; Renal parenchymal malacoplakia is a rare cause of acute renal failure . Traditionally, it was associated with a high mortality rate and commonly resulted in renal failure requiring renal replacement therapy . The authors report on a 70-year-old woman who presented with acute renal failure caused by renal parenchymal malacoplakia . Her renal function recovered after levofloxacin treatment . All cases reported in the English-language literature since 1990, when fluoroquinolone was first used to treat malacoplakia, were reviewed . Although some patients still had renal failure, with renal biopsy and fluoroquinolone treatment, the patient mortality rate from renal parenchymal malacoplakia is remarkably low. Nat Rev Genet, 2003 Jun, 4(6), 419 - 31 The art and design of genetic screens: Escherichia coli; Shuman HA et al.; This article summarizes the general principles of selections and screens in Escherichia coli . The focus is on the lac operon, owing to its inherent simplicity and versatility . Examples of different strategies for mutagenesis and mutant discovery are described . In particular, the usefulness and effectiveness of simple colour-based screens are illustrated . The power of lac genetics can be applied to almost any bacterial system with gene fusions that hook any gene of interest to lacZ, which is the structural gene that encodes beta-galactosidase . The diversity of biological processes that can be studied with lac genetics is remarkable and includes DNA metabolism, gene regulation and signal transduction, protein localization and folding, and even electron transport. J Biol Chem, 2003 Aug 8, 278(32), 29979 - 86 Epub 2003 May 29. Specific binding of dehydroepiandrosterone to the N terminus of the microtubule-associated protein MAP2; Laurine E et al.; The effect of neurosteroids is mediated through their membrane or nuclear receptors . However, no dehydroepiandrosterone (DHEA)-specific receptors have been evidenced so far in the brain . In this paper, we showed by isothermal titration calorimetry that the DHEA specifically binds to the dendritic brain microtubule-associated protein MAP2C with an association constant of 2.7 x 10(7) m-1 and at a molar ratio of 1:1 . By partial tryptic digestions and mass spectrometry analysis, we found that the binding involved the N-terminal region of MAP2C . Interestingly, MAP2C displays homologies with 17 beta-hydroxysteroid dehydrogenase 1, an enzyme required for estrogen synthesis . Based on these sequence homologies and on the x-ray structure of the DHEA-binding pocket of 17 beta-hydroxysteroid dehydrogenase 1, we modeled the complex of DHEA with MAP2C . The binding of DHEA to MAP2C involved specific hydrogen bonds that orient the steroid into the pocket . This work suggests that DHEA can directly influence brain plasticity via MAP2C binding . It opens interesting ways for understanding the role of DHEA in the brain. J Bacteriol, 2003 Jun, 185(12), 3583 - 95 Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency; Blinkova A et al.; Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA . These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts) . One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro . Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III . The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive . The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication . We present five lines of evidence consistent with the initiation deficiency model . First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C . These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation . Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations . Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible . However, suppression was possible on glycerol medium at 38 degrees C . Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect . Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity. J Bacteriol, 2003 Jun, 185(12), 3538 - 46 Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids; Becker EC et al.; The primary DNA processing protein for conjugative mobilization of the plasmid R1162 is the transesterase MobA, which acts at a unique site on the plasmid, the origin of transfer (oriT) . Both MobA and oriT are members of a large family of related elements that are widely distributed among bacteria . Each oriT consists of a highly conserved core and an adjacent region that is required for binding by its cognate MobA . The sequence of the adjacent region is important in determining the specificity of the interaction between the Mob protein and the oriT DNA . However, the R1162 MobA is active on the oriT of pSC101, another naturally occurring plasmid . We show here that MobA can recognize oriTs having different sequences in the adjacent region and, with varying frequencies, can cleave these oriTs at the correct position within the core . Along with the structure of the oriTs themselves, these characteristics suggest a model for the evolution of this group of transfer systems. J Bacteriol, 2003 Jun, 185(12), 3524 - 6 Protein synthesis in Escherichia coli with mischarged tRNA; Min B et al.; Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli) . On the basis of the E . coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu . While large amounts of Asp-tRNA(Asn) are detrimental to E . coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase. J Bacteriol, 2003 Jun, 185(12), 3485 - 90 Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase; Kobayashi T et al.; An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2(Reu) . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers . Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1(Reu)) . The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers . However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time . PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R . eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies . When R . eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca . 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca . 1% of dry cell weight) . In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca . 5% in the stationary phase . A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca . 20%) and also an elevated PHB level (ca . 8%) in the stationary phase . These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R . eutropha along with PhaZ1. Int J Radiat Biol, 2003 Apr, 79(4), 281 - 6 Strong static magnetic field and the induction of mutations through elevated production of reactive oxygen species in Escherichia coli soxR; Zhang QM et al.; PURPOSE: Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently . Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF . MATERIALS AND METHODS: An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF . Various mutant strains of E . coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined . The expression of the soxS::lacZ fusion gene was assessed by measurement of beta-galactosidase activity . RESULTS: The results for survival or mutation were obtained with wild-type E . coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative . On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress . Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E . coli when exposed to 5 and 9 T, respectively . CONCLUSIONS: These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E . coli. J Biol . 2003;2(2):14 . Epub 2003 May 29. Environmental stresses can alleviate the average deleterious effect of mutations; Kishony R et al.; BACKGROUND: Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness . Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organism's ability to tolerate deleterious mutations . Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations . RESULTS: We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities . Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses . In contrast to the perception that stress always reduces the organism's ability to tolerate mutations, our measurements identified stresses that do the opposite - that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations . CONCLUSIONS: Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations . We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations . The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions. Biochem J, 2003 Sep 15, 374(Pt 3), 767 - 72 Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide; Roman E et al.; The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine) . The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH) . The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans . To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model . E . coli was transformed with the complete gene cluster for K5 polysaccharide production . Additional transformation with an extra copy of UDPGDH resulted in an approx . 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH . UDP-GlcA levels were increased 3-fold in overexpressing strains . However, metabolic labelling with {14C}glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide . No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains . Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production. Biochem J, 2003 Aug 15, 374(Pt 1), 193 - 8 Definition of structural elements in Plasmodium vivax and P . knowlesi Duffy-binding domains necessary for erythrocyte invasion; Singh SK et al.; Plasmodium vivax and P . knowlesi use the Duffy antigen as a receptor to invade human erythrocytes . Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion . Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains . In the present study, we report an analysis of the overall three-dimensional architecture of P . vivax and P . knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays . Our proteolysis experiments indicate that these domains are built of two distinct subdomains . The N-terminal region from Cys-1-4 (C1-C4) forms a stable non-functional subdomain . The region spanning C5-C12 forms another subdomain, which is capable of binding Duffy antigen . These subdomains are joined by a protease-sensitive linker . Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5-C8 of the Duffy-binding domains are sufficient for the binding receptor . Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition . Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module . These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors. Int J Surg Investig, 1999, 1(4), 277 - 83 Influence of C1-esterase inhibitor on tissue oxygenation of jejunal mucosa during endotoxemia; Schmidt W et al.; BACKGROUND: The complement system has been shown to play an important role in the pathogenesis of microcirculatory disturbances in trauma and sepsis . The intestinal mucosa is the most susceptible portion of the gut to impaired perfusion and oxygen delivery . The objective of this study was to investigate the effects of C1-esterase inhibitor (C1-INH) on arterial oxygenation (PaO2) and tissue oxygenation (PtiO2) of jejunal mucosa during endotoxemia . METHODS: Eighteen anesthetized and ventilated rats were laparotomized and a jejunal portion was exteriorized and fixed on a plexiglass stage . The jejunum was punctured and a Clark type microcatheter PO2 probe and a micro thermocouple were placed on the mucosa in order to measure PtiO2 . The animals were randomly assigned to receive one of the three treatments: infusion of Escherichia coli lipopolysaccharides (LPS) without C1-INH pretreatment (LPS group); or infusion of LPS with C1-INH pretreatment (C1-INH group); the control group (n = 6) without treatment of either C1-INH or LPS . The mean arterial pressure (MAP), heart rate (HR), PaO2 and PtiO2 were measured at baseline, 60 and 120 min after induction of endotoxemia . RESULTS: Hemodynamic parameters (MAP, HR) in all the three groups showed no significant changes during the study period . PaO2 significantly decreased in the LPS group . This decrease could be attenuated by pretreatment with C1-INH . The mucosal PtiO2 of the jejunum in the control group remained stable . It significantly decreased in the LPS and in the C1-INH groups without showing a significant difference after 120 min of endotoxemia . CONCLUSIONS: Pretreatment with C1-INH was able to diminish a decrease in PaO2 during endotoxemia, indicating that pulmonary injury was attenuated . Endotoxin-induced tissue hypoxia of the intestinal mucosa could not be prevented suggesting a minor involvement of complement activation in this pathophysiological process. Intensive Care Med, 2003 Jul, 29(7), 1164 - 8 Epub 2003 May 27. Risk factors for piperacillin/tazobactam-resistant Escherichia coli in ICU patients: a clinical study; Mohammedi I et al.; OBJECTIVE: To determine risk factors of infections with piperacillin/tazobactam-resistant Escherichia coli in critical care patients . DESIGN: Prospective, consecutive sample survey study . SETTING: Surgical intensive care unit (ICU) in a university hospital . PATIENTS: A consecutive series of 133 patients from whom culture results were positive for E . coli during their ICU stay . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: Multivariate logistic regression analysis identified the following significant independent factors associated with the emergence of a piperacillin/tazobactam-resistant Escherichia coli: prior use of amoxicillin (odds ratio, 4.15) and amoxicillin/clavulanate (odds ratio, 3.25) . CONCLUSIONS: Treatment with amoxicillin or amoxicillin/clavulanate is a major risk factor for the detection of piperacillin/tazobactam-resistant E . coli in ICU patients. Toxicol Sci, 2003 Aug, 74(2), 470 - 84 Epub 2003 May 28. Gene expression analysis of the acute phase response using a canine microarray; Higgins MA et al.; The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans . As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment . Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog . To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel . The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets . To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region . Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design) . Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts . To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS) . Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group) . Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge . Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose . In addition, the canine array identified several potential biomarkers of hepatic inflammation . Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP) . In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes. Exp Biol Med (Maywood), 2003 Jun, 228(6), 697 - 705 Regulation of vascular endothelial growth factor gene expression in murine macrophages by nitric oxide and hypoxia; Ramanathan M et al.; Vascular endothelial growth factor (VEGF) expression in murine peritoneal macrophages is strongly upregulated by hypoxia via transcriptional and posttranscriptional mechanisms . Interferon-gamma (IFN-gamma) with Escherichia coli lipopolysaccharide (LPS) also upregulates expression of VEGF, as well as of the inducible nitric oxide synthase (iNOS) . Hypoxia (1% O(2)) upregulates VEGF expression in macrophages from both wild-type and iNOS knockout mice, indicating that hypoxic upregulation of VEGF is independent of iNOS . However, the iNOS inhibitor aminoguanidine (AG) decreases the VEGF expression induced by LPS/IFN-gamma, indicating an important role for NO . NO-dependent induction of VEGF is strongly dependent on cell density . LPS/IFN-gamma treatment induces minimal VEGF protein expression in macrophages cultured at low cell densities (<0.25 x 10(6) cells/cm(2)); at higher cell densities (>0.25 x 10(6) cells/cm(2)) that lead to conditions of pericellular hypoxia, however, induction of VEGF expression was strong . Transient transfection of RAW 264.7 cells with luciferase reporter constructs of the murine VEGF promoter indicates that both hypoxia and LPS/IFN-gamma independently induce VEGF promoter activity, irrespective of cell density . Although LPS/IFN-gamma treatment induces transcriptional activation of the VEGF promoter, significant levels of VEGF protein are only expressed by cells at high density under conditions of pericellular hypoxia . This suggests an important regulatory role for hypoxia at the posttranscriptional level . Deletion analysis of the VEGF promoter shows that the hypoxia response element region and its immediate flanking sequences are essential for both hypoxia and LPS/IFN-gamma-induced VEGF promoter activation. Mol Cell Biol, 2003 Jun, 23(12), 4121 - 5 Effect of damage type on stimulation of human excision nuclease by SWI/SNF chromatin remodeling factor; Hara R et al.; To investigate the repair of different types of DNA lesions in chromatin, we prepared mononucleosomes containing an acetylaminofluorene-guanine adduct (AAF-G), a (6-4) photoproduct, or a cyclobutane pyrimidine dimer (CPD) and measured the repair of these lesions by reconstituted 6-factor human excision nuclease . We find that incorporation into nucleosomes inhibits the repair of CPD more severely than repair of the AAF-G adduct and the (6-4) photoproduct . Equally important, we find that SWI/SNF stimulates the removal of AAF-G and (6-4) photoproduct but not of CPD from nucleosomal DNA . These results shed new light on the low rate of repair of CPDs in human cells in vivo. J Biol Chem, 2003 Aug 15, 278(33), 30961 - 70 Epub 2003 May 28. Production, characterization, and immunogenicity of a soluble rat single chain T cell receptor specific for an encephalitogenic peptide; McMahan RH et al.; The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69-89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule . Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide . Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system . The recombinant molecule consists of the Valpha2 domain connected to the Vbeta8.2 domain via a 15-residue linker . Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure . C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69-89) peptide, as analyzed using surface plasmon resonance . Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness. J Biol Chem, 2003 Aug 15, 278(33), 31088 - 94 Epub 2003 May 28. Role of proline residues in the folding of serine hydroxymethyltransferase; Fu TF et al.; Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis . The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus . The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism . The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity . The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered . However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme . Mutation of both Pro258 and Pro264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity . The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability . Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme . The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5'-phosphate addition to the apo-enzyme. J Biol Chem, 2003 Aug 29, 278(35), 32631 - 7 Epub 2003 May 28. Structure-function analysis of recombinant substrate protein 22 kDa (SP-22) . A mitochondrial 2-CYS peroxiredoxin organized as a decameric toroid; Gourlay LJ et al.; Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases . It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168 . The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported . Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue . The enzyme forms a stable decameric toroid consisting of five basic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer . The disulfide bonds are not required for overall structural integrity . The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings . C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings . Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid. J Biol Chem, 2003 Aug 8, 278(32), 30311 - 6 Epub 2003 May 28. Crystal structure of the C-terminal 10-kDa subdomain of Hsc70; Chou CC et al.; The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions . They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain . The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain . Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 A . Its helical region adopted a helix-loop-helix fold . This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70 . Moreover, in the crystalline state, the 10-kDa subdomain formed dimers . The results of gel filtration chromatography further supported the view that this subdomain was self-associated . Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers . These findings suggest that the alpha-helical region of the 10-kDa subdomain dictates the chaperone self-association. J Biol Chem, 2003 Aug 15, 278(33), 30569 - 77 Epub 2003 May 27. Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover; Sundram V et al.; To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz . alphabeta and betagamma in Escherichia coli . After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively) . Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG . Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG . Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN . Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J . Biol . Chem . 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels. EMBO J, 2003 Jun 2, 22(11), 2614 - 22 A reducing system of the superoxide sensor SoxR in Escherichia coli; Koo MS et al.; The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide . When SoxR is activated by oxidation of its {2Fe-2S} cluster, it increases the synthesis of SoxS, which then activates its target gene expression . How the oxidized SoxR returns to and is maintained in its reduced state has been under question . To identity genes that constitute the SoxR-reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants . Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE-rseABC operon . In-frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype . The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR . Electron paramagnetic resonance analysis of SoxR and measurement of re-reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 731 - 5 Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation; Shoolingin-Jordan PM et al.; Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization . A mutation that affects a key catalytic residue, D99G, results in an inactive holo -protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo -protein and pre-uroporphyrinogen . The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo -protein . The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo -protein . The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly . In contrast, the mutant R167Q exists as a holo -enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme-substrate intermediates from the catalytic cycle. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 706 - 10 Genetic analysis of homologous recombination in Archaea: Haloferax volcanii as a model organism; Allers T et al.; Homologous recombination is a fundamental cellular process that rearranges genes both within and between chromosomes, promotes repair of damaged DNA and underpins replication . Much of our understanding of recombination stems from pioneering studies of bacterial and eukaryotic systems such as Escherichia coli and Saccharomyces cerevisiae . Since most archaeal species are extremophilic and difficult to cultivate, current knowledge of recombination in the Archaea is confined largely to comparative genomics and biochemistry . A clear view of what we can learn will not emerge until genetic and molecular systems have been established . We are developing such systems using Haloferax volcanii as a model organism, as it can be cultivated in the laboratory with ease and offers great potential for establishing tractable and informative genetic systems. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 664 - 8 Toll-like receptor 4 signalling: new perspectives on a complex signal-transduction problem; Vogel SN et al.; We previously reported that Toll-like receptor-2 (TLR2) agonists induce expression of a more limited repertoire of pro-inflammatory genes than TLR4 agonists . Murine macrophages stimulated with the TLR4 agonist, Escherichia coli lipopolysaccharide, induced signal transducer and activator of transcription 1 ('STAT1') tyrosine phosphorylation that was secondary to the autocrine/paracrine action of interferon (IFN)-beta, an immediate early gene . In contrast, TLR2 agonists failed to activate IFN-beta gene expression . TLR4-induced IFN-beta mRNA was found to be MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll/interleukin-1 receptor domain-containing adapter protein)/Mal (MyD88-adapter-like)-dependent . In the present paper, we outline the recent controversy over the role of TIRAP/Mal in TLR2 and TLR4 signalling in the context of the current molecular tools used for such studies . Collectively, our findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 563 - 71 Structural insights into the evolution of the pantothenate-biosynthesis pathway; Lobley CM et al.; Pantothenate is synthesized in bacteria, fungi and plants, and as vitamin B5 is a dietary requirement in animals . The three-dimensional structures of the four Escherichia coli enzymes involved in the production of pantothenate have been determined . We describe the use of comparative analyses of the sequences and structures to identify distant homologues of the four enzymes in an attempt to understand the evolution of the pathway . We conclude that it is likely to have evolved via a patchwork mechanism, whereby the individual enzymes were recruited separately. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 493 - 6 Oxygen activation in a copper-containing amine oxidase; Wilmot CM; The process by which molecular oxygen is activated to enable it to function as an electron acceptor in biology is poorly understood . The quinoprotein copper-containing amine oxidase (CuAO) catalyses the conversion of primary amines into aldehydes . As well as copper, the enzyme contains an organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ) . Following the formation of aldehyde, the enzyme is left as the two-electron reduced aminoquinol form . Reoxidation of the enzyme back to the resting state uses molecular oxygen, which is reduced to H(2)O(2) in the process, with the additional release of NH(3) . To understand the structural basis of oxygen activation in Escherichia coli CuAO (ECAO), catalytically competent crystals were used to trap catalytic intermediates by exposing then to amine substrate and then freeze-trapping under aerobic and anaerobic conditions . Single-crystal visible microspectrophotometry was used to probe the oxidation state of the quinone in the intermediates, as TPQ exhibits a rich palette of colour changes during catalytic turnover . This review will focus on one of these structures, that of the rate-determining species in the crystal under steady-state conditions . This structure has revealed many details regarding oxygen activation in ECAO, including the site of dioxygen binding, and the proton-transfer pathways involved in H(2)O(2) formation. Biochem J, 2003 Aug 15, 374(Pt 1), 207 - 17 A role for Sec1/Munc18 proteins in platelet exocytosis; Schraw TD et al.; A critical aspect of haemostasis is the release of clot-forming components from the three intra-platelet stores: dense-core granules, alpha granules and lysosomes . Exocytosis from these granules is mediated by soluble proteins {N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs)} and integral membrane proteins {vesicle and target SNAP receptors (v- and t-SNAREs)} . Three Sec1/Munc18 proteins (SM proteins) are present in platelets (Munc18a, Munc18b and Munc18c) and they bind to and potentially regulate specific syntaxin t-SNAREs . In resting platelets, these SM proteins associate with granules and open canalicular system membranes predominantly but not with the plasma membrane . Munc18a binds to syntaxin 2 alone and does not associate with other members of the core SNARE complex . Munc18b associates with a larger complex that contains synaptosome-associated protein of 23 kDa (SNAP-23) and cellubrevin/vesicle-associated membrane protein 3 . Munc18c associates with both syntaxins 2 and 4, with synaptosome-associated protein of 23 kDa (SNAP-23) and with a v-SNARE . On stimulation, most of the platelet SM proteins are still found in membrane fractions . Phosphorylation of each Munc18 increases in thrombin-treated cells and phosphorylated Munc18c remains associated with syntaxins 2 and 4, but its affinity for the SNAREs appears to be reduced . To determine the functional role of the platelet SM proteins, we examined the effects of Munc18-based peptides (Munc18a peptide 3 and Munc18c peptide 3) . Addition of the peptides to permeabilized platelets inhibits secretion from all three platelet granules . These peptides also inhibit agonist-induced aggregation in saponin-permeabilized platelets . These studies demonstrate a clear role for SM proteins in platelet exocytosis and aggregation and suggest a dominant role for Munc18c in all three granule-release events. Crit Care Med, 2003 May, 31(5), 1509 - 14 Species-specific modulation of the nitric oxide pathway after acute experimentally induced endotoxemia; Bachetti T et al.; OBJECTIVE: The derangement of the nitric oxide pathway is an important contributing factor to the pathogenesis of septic shock . The aim of this study was to investigate potential differences in modulation of such a pathway in two experimental models of endotoxemia . DESIGN: Prospective, randomized, placebo-controlled animal investigation . SETTING: Cardiovascular research laboratory . SUBJECTS: Male, anesthetized, and mechanically ventilated New-Zealand rabbits (n = 24) and Sprague-Dawley rats (n = 24) . INTERVENTIONS: After pretreatment with 1400W (1 mg kg(-1) subcutaneously), an inhibitor of inducible nitric oxide synthase, animals received an intravenous bolus of Escherichia Coli lipopolysaccharides (5 mg kg(-1)) . After 4 hrs, lungs, myocardial left ventricles, and aortas were collected . MEASUREMENTS AND MAIN RESULTS: Blood mean arterial pressure, pH, and nitrite/nitrate were monitored . Nitric oxide in the exhaled air was measured by chemiluminescence . Tissue activity of both constitutive nitric oxide synthase and inducible nitric oxide synthase was determined by measuring the conversion of {3H}L-arginine to {3H}L-citrulline . In lipopolysaccharide-treated animals, both mean arterial pressure (after 60 to 90 mins) and blood pH (after 4 hrs) decreased with respect to baseline values . 1400W prevented lipopolysaccharide-induced hypotension only in rats (p <.01) . Exhaled nitric oxide decreased in lipopolysaccharide-treated rabbits by 120 mins (from 12.6 +/- 0.6 to 8.4 +/- 0.6 ppb, p <.01) and remained low until the end of the experiment (p <.01 vs . baseline) . Conversely, exhaled nitric oxide increased in lipopolysaccharide-treated rats by 120 mins (from 0.4 +/- 0.1 to 5.3 +/- 1.7 ppb, p <.01) and reached a plateau by 210 mins (19.8 +/- 3.1 ppb, p <.01 vs . baseline) . 1400W prevented the lipopolysaccharide-induced increase in exhaled nitric oxide and blood nitrite/nitrate in rats (p <.05) . Inducible nitric oxide synthase activity increased in endotoxemic rabbit heart (0.19 +/- 0.05 vs . 0.07 +/- 0.02 pmol L-citrulline/min/mg protein in the control group, p <.05) and in all rat tissues, being more striking in the lungs (25.00 +/- 0.01 vs . 0.19 +/- 0.04 pmol L-citrulline/min/mg protein in the control group, p <.001) . CONCLUSIONS: The nitric oxide pathway is differently modulated between endotoxemic rabbits and rats. J Gen Virol, 2003 Jun, 84(Pt 6), 1607 - 12 Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host; Stevenson RA et al.; Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV) . As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies . In FMDV VP1, such antibodies commonly recognize linear epitopes present in the betaG-betaH loop region . To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins . These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice . Regions at the N- and C-termini as well as the betaE-betaF and the betaG-betaH loop regions contained B cell epitopes that elicited antibodies in the natural host . GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing . It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes. J Gen Virol, 2003 Jun, 84(Pt 6), 1577 - 82 Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli; Lu MW et al.; Dragon grouper (Epinephelus lanceolatus) nervous necrosis virus (DGNNV) comprises 180 copies of capsid protein that encapsulate a bipartite genome of single-stranded (+)-RNAs . This study reports that virus-like particles (VLPs) are formed in Escherichia coli expressing the full-length ORF encoding the DGNNV capsid protein . Two sizes of VLPs are observed . The heavier particles resemble the native piscine nodavirus in size and stain permeability, while the lighter ones are approximately two-thirds of the full size . The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus. Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7207 - 12 Epub 2003 May 27. Recombineering with overlapping single-stranded DNA oligonucleotides: testing a recombination intermediate; Yu D et al.; A phage lambda-based recombination system, Red, can be used for high-efficiency mutagenesis, repair, and engineering of chromosomal or episomal DNA in vivo in Escherichia coli . When long linear double-stranded DNA with short flanking homologies to their targets are used for the recombination, the lambda Exo, Beta, and Gam proteins are required . The current model is: (i) Gam inhibits the host RecBCD activity, thereby protecting the DNA substrate for recombination; (ii) Exo degrades from each DNA end in a 5' --> 3' direction, creating double-stranded DNA with 3' single-stranded DNA tails; and (iii) Beta binds these 3' overhangs to protect and anneal them to complementary sequences . We have tested this model for Red recombination by using electroporation to introduce overlapping, complementary oligonucleotides that when annealed in vivo approximate the recombination intermediate that Exo should create . Using this technique we found Exo-independent recombination . Surprisingly, a similarly constructed substrate with 5' overhangs recombined more efficiently . This 5' overhang recombination required both Exo and Beta for high levels of recombination and the two oligonucleotides need to overlap by only 6 bp on their 3' ends . Results indicate that Exo may load Beta onto the 3' overhang it produces . In addition, multiple overlapping oligonucleotides were successfully used to generate recombinants in vivo, a technique that could prove useful for many genetic engineering procedures. Nucleic Acids Res, 2003 Jun 1, 31(11), 2865 - 72 Tertiary structure base pairs between D- and TpsiC-loops of Escherichia coli tRNA(Leu) play important roles in both aminoacylation and editing; Du X et al.; To ensure the fidelity of protein biosynthesis, aminoacyl-tRNA synthetases (aaRSs) must recognize the tRNA identity elements of their cognate tRNAs and discriminate their cognate amino acids from structurally similar ones through a proofreading (editing) reaction . For a better understanding of these processes, we investigated the role of tRNA(Leu) tertiary structure in the aminoacylation and editing reactions catalyzed by leucyl-tRNA synthetase (LeuRS) . We constructed a series of Escherichia coli tRNA(Leu) mutated transcripts with alterations of the nucleotides involved in tertiary interactions . Our results revealed that any disturbance of the tertiary interaction between the tRNA(Leu) D- and TpsiC-loops affected both its aminoacylation ability and its ability to stimulate the editing reaction . Moreover, we found that the various tertiary interactions between the D- and TpsiC-loops (G18:U55, G19:C56 and U54:A58) functioned differently within the aminoacylation and editing reactions . In these two reactions, the role of base pair 19:56 was closely correlated and dependent on the hydrogen bond number . In contrast, U54:A58 was more important in aminoacylation than in editing . Taken together, our results suggest that the elbow region of tRNA formed by the tertiary interactions between the D- and TpsiC-loops affects the interactions between tRNA and aaRS effectively both in aminoacylation and in editing. Nucleic Acids Res, 2003 Jun 1, 31(11), 2803 - 10 Purification and characterisation of a novel DNA methyltransferase, M.AhdI; Marks P et al.; We have cloned the M and S genes of the restriction-modification (R-M) system AhdI and have purified the resulting methyltransferase to homogeneity . M.AhdI is found to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M2S2 (where the M and S subunits are responsible for methylation and DNA sequence specificity, respectively) . Sedimentation equilibrium experiments show that the tetrameric enzyme dissociates to form a heterodimer at low concentration, with K(d) approximately 2 microM . The intact (tetrameric) enzyme binds specifically to a 30 bp DNA duplex containing the AhdI recognition sequence GACN5GTC with high affinity (K(d) approximately 50 nM), but at low enzyme concentration the DNA binding activity is governed by the dissociation of the tetramer into dimers, leading to a sigmoidal DNA binding curve . In contrast, only non-specific binding is observed if the duplex lacks the recognition sequence . Methylation activity of the purified enzyme was assessed by its ability to prevent restriction by the cognate endonuclease . The subunit structure of the M.AhdI methyltransferase resembles that of type I MTases, in contrast to the R.AhdI endonuclease which is typical of type II systems . AhdI appears to be a novel R-M system with properties intermediate between simple type II systems and more complex type I systems, and may represent an intermediate in the evolution of R-M systems. Nucleic Acids Res, 2003 Jun 1, 31(11), 2778 - 85 Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family; Bernstein DA et al.; RecQ DNA helicases function in DNA replication, recombination and repair . Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer . In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (approximately 59 kDa) N-terminal and a small (approximately 9 kDa) C-terminal domain . A short N-terminal segment (7 or 21 residues) is also shown to be sensitive to proteases . The effects of removing these regions of RecQ are tested in vitro . Removing 21 N-terminal residues from RecQ severely diminishes its DNA-dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays . In contrast, removing the approximately 9 kDa C-terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA . These results establish the biochemical roles of an N-terminal sequence motif in RecQ catalytic function and for the C-terminal RecQ domain in stable DNA binding. J Biol Chem, 2003 Aug 8, 278(32), 29581 - 6 Epub 2003 May 27. Direct demonstration of ATP-dependent release of SecA from a translocating preprotein by surface plasmon resonance; de Keyzer J et al.; Translocase mediates the transport of preproteins across the inner membrane of Escherichia coli . SecA binds with high affinity to the membrane-embedded protein-conducting SecYEG complex and serves as both a receptor for secretory proteins and as an ATP-driven molecular motor . Cycles of ATP binding and hydrolysis by SecA drive the progressive movement of the preprotein across the membrane . Surface plasmon resonance allows an online monitoring of protein interactions . Here we report on the kinetic analysis of the interaction between SecA and the membrane-embedded SecYEG complex . Immobilization of membrane vesicles containing overproduced SecYEG on the Biacore Pioneer L1 chip allows the detection of high affinity SecA binding to the SecYEG complex and online monitoring of the translocation of the secretory protein proOmpA . SecA binds tightly to the SecYEG.proOmpA complex and is released only upon ATP hydrolysis . The results provide direct evidence for a model in which SecA cycles at the SecYEG complex during translocation. Carcinogenesis, 2003 May, 24(5), 911 - 7 Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells; Yasui M et al.; Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT) . Equilin and equilenin are the major components of the widely prescribed drug used for ERT . These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages . To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF, was exposed to 4-OHEQ and transfected into human fibroblast . Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing . The proportion of plasmids with the mutated supF gene was increased dose-dependently . The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions . Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions . The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site . C:G --> G:C and C:G --> A:T mutations were detected preferentially with lesser numbers of C:G --> T:A transitions . Sixty-two percent of base substitutions were observed particularly at C:G pairs in (5')-TC/AG-(5') sequences . Using (32)P-post-labeling/gel electrophoresis analysis, 4-OHEN-dC was a major adduct, followed by lesser amounts of 4-OHEN-dA adduct . Mutations observed at C:G pairs may result from 4-OHEN-dC adduct . These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in (5')-TC/AG-(5') sequences. Br J Pharmacol, 2003 May, 139(2), 289 - 98 Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum; Hsu MJ et al.; 1 The polysaccharide from Ganoderma lucidum (PS-G) has been reported to enhance immune responses and to elicit antitumor effects . In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis when cultured in vitro . Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS-G on these two cell functions, and the molecular events leading to these actions . 2 Using latex beads and heat-inactive Escherichia coli serving as particles for neutrophil engulfment, we found that PS-G is able to enhance phagocytic activity of human primary neutrophils and neutrophilic-phenotype cells differentiated from all trans retinoic acid-treated HL-60 cells . 3 Chemotactic assay using Boyden chamber also revealed the ability of PS-G to increase neutrophil migration . 4 Exposure of neutrophils to PS-G time dependently caused increases in protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), Hck, and Lyn activities . 5 Results with specific kinase inhibitors indicate that phagocytic action of PS-G was reduced by the presence of wortmannin (Phosphatidylinositol 3-kinase, PI3K inhibitor), pyrazolpyrimidine 2 (Src-family tyrosine kinase inhibitor), Ro318220 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), but not by PD98059 (mitogen-activated protein/ERK kinase inhibitor) . Moreover, chemotactic action of PS-G requires the activities of PI3K, p38 MAPK, Src tyrosine kinases and PKC . 6 All these results demonstrate the abilities of PS-G to enhance neutrophil function in phagocytosis and chemotaxis, and further provide evidence to strengthen the beneficial remedy of G . lucidum in human to enhance defense system. Br J Pharmacol, 2003 May, 139(2), 263 - 70 Low endotoxemia prevents the reduction of gastric blood flow induced by NSAIDs: role of nitric oxide; Calatayud S et al.; 1 The role of nitric oxide (NO) in the effects of low endotoxemia on gastric damage and blood flow has been evaluated in indomethacin-treated rats . 2 Pretreatment (-1 h) with endotoxin (40 micro g kg(-1)) reduced gastric damage induced by indomethacin (20 mg kg(-1)) in conscious rats . 3 Endotoxin prevented the reduction in gastric blood flow (laser Doppler flowmetry) induced by indomethacin in pentobarbital-anaesthetised rats . 4 Pretreatment with an NO-synthase (NOS) inhibitor (L-NAME, 1 mg kg(-1)) reversed the protective effect of endotoxin on gastric blood perfusion . 5 Endotoxin did not modify the expression of mRNA for endothelial NOS or inducible NOS in the gastric corpus when evaluated 1 h postinjection . However, a 3.8-fold increase in inducible NOS mRNA and a 61% reduction in endothelial NOS mRNA were observed in the gastric corpus 4 h after endotoxin administration . 6 Evaluation of both total and Ca(2+)-dependent NOS activity by analysing the rate of conversion of L-arginine to L-citrulline in gastric corpus homogenates showed no differences between animals treated with endotoxin and those treated with saline 1 or 4 h beforehand . Ca(2+)-independent NOS activity was almost non-apparent in control as well as in endotoxin-treated rats at all the time points analysed . 7 Low endotoxemia preserves blood perfusion and protects the gastric mucosa against the deleterious effects of indomethacin through the endogenous NO release . NO synthesis in response to endotoxin does not involve the inducible NOS, but probably depends on the post-translational/biochemical regulation in vivo of a Ca(2+)-dependent NOS, most probably endothelial NOS. Biophys J, 2003 Jun, 84(6), 3564 - 82 Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E; Reblova K et al.; Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs . The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs . The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence . The deep groove of Loop E motifs provides unique sites for cation binding . Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width . In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove . The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus . Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times . The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters . The ordered hydration is intimately connected with RNA local conformational variations. Chem Biol, 2003 May, 10(5), 453 - 62 Biosynthesis of structurally novel carotenoids in Escherichia coli; Lee PC et al.; Previously, we utilized in vitro evolution to alter the catalytic functions of several carotenoid enzymes and produce the novel carotenoids tetradehydrolycopene and torulene in Escherichia coli . Here we report on the successful extension of these pathways and the C(30) carotenoid diaponeurosporene pathway with additional carotenoid genes . Extension of the known acyclic C(30) pathway with C(40) carotenoid enzymes-spheroidene monooxygenase and lycopene cyclase-yielded new oxygenated acylic products and the unnatural cyclic C(30) diapotorulene, respectively . Extension of acyclic C(40) pathways with spheroidene monooxygenase generated novel oxygenated carotenoids including the violet phillipsiaxanthin . Extension of the torulene biosynthetic pathway with carotene hydroxylase, desaturase, glucosylase, and ketolase yielded new torulene derivatives . These results demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds. FEMS Immunol Med Microbiol, 2003 Jun 10, 37(1), 59 - 67 Serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid; Lukasiewicz J et al.; The covalent conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid was prepared using reaction of reductive amination . The neoglycoconjugate was a good immunogen in rabbits yielding a high level of anti-lipopolysaccharide (LPS) antibodies of the IgG class . It was found that antiserum was able to react with the smooth LPS molecules of identical (R4) or related (R1) core type . The reactions were shown in the enzyme-linked immunosorbent assay and the immunoblotting test . Flow cytometry showed that anti-core antibodies reacted with LPS present on intact, live, smooth bacteria labelling more than 90% of cells . The anti-OS R4-TT serum used for in vitro studies showed high endotoxin neutralization activity . The serum inhibited endotoxin-induced tumor necrosis factor alpha and nitric oxide synthesis by the J-774A.1 cell line and attenuated pulmonary retention of YAC-1 cells. FEMS Microbiol Lett, 2003 May 28, 222(2), 237 - 42 Expression and biochemical characterization of the 1-HO-carotenoid methylase CrtF from Rhodobacter capsulatus; Badenhop F et al.; In purple bacteria, acyclic 1-methoxy carotenoids like spheroidene or spirilloxanthin are essential components of the photosynthetic apparatus . One of the last steps of their biosynthesis involves O-methylation of the 1-hydroxy group . The 1-HO-carotenoid methylase CrtF from Rhodobacter capsulatus catalyzing this type of reaction was expressed in Escherichia coli in an active form . It was then purified by affinity chromatography and biochemically characterized . The enzymatic reaction depends on S-adenosylmethionine as the only cofactor . By complementation in E . coli, the substrate specificity of the enzyme was determined . It could be shown that the enzyme converts not only all possible 1-hydroxy carotenoids in the spheroidene/1'-HO-spheroidene biosynthetic pathway of R . capsulatus but also carotenoid intermediates leading to the formation of spirilloxanthin in a pathway which is absent in R . capsulatus but present in related species. FEMS Microbiol Lett, 2003 May 28, 222(2), 199 - 203 Multilocus sequence typing (MLST) of Escherichia coli O78 strains; Adiri RS et al.; Strains of Escherichia coli serotype O78 are associated with many diseases, including invasive infections, in humans and farm animals . The clonal relationship between strains from different hosts is therefore important for assessing the risk of zoonotic infections . Here we propose a multilocus sequence typing scheme for E . coli, based on six housekeeping genes . Preliminary, but significant, results indicate that clonal division in E . coli O78 strains is host independent, and closely related clones reside in different hosts . There was a positive correlation between virulence and clonal origin. J Insect Physiol, 2000 Jan, 46(1), 1 - 11 Parasitization of Lacanobia oleracea (Lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, suppresses haemocyte-mediated recognition of non-self and phagocytosis; Richards EH et al.; Although many endoparasitic wasps suppress the haemocyte-mediated immune defences of their insect hosts, the effects of ectoparasitoids are virtually unknown . In view of this, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and its host, the tomato moth, Lacanobia oleracea . For unparasitized insects, in vitro assays indicated that less than 3.0% of L . oleracea haemocytes on a monolayer formed rosettes with yeast cells or fresh rabbit erythrocytes (rbc), and virtually no phagocytosis of these particles occurred . In addition, although fixed rbc formed rosettes with 51.21% of haemocytes, only about 3.0% of the haemocytes ingested one or more of these particles . In contrast to this, B . cereus and E . coli were readily phagocytosed by 14.75% and 53.70% of haemocytes, respectively . These results indicate that L . oleracea haemocytes can recognise different types of non-self particles and demonstrate that ingestion does not necessarily follow attachment . When rosetting and phagocytosis assays were performed with fixed rbc and FITC-labelled E . coli, and haemocytes from starved L . oleracea, PBS injected L . oleracea, and experimentally envenomated insects on day five of treatment, there was no significant difference in the percentage of rosetting or phagocytosis occurring . When haemocytes from parasitized insects on day five of treatment were utilised, however, rosetting and phagocytosis were reduced by 31.41% and 34.94%, respectively . Thus, the effects of parasitization and experimental envenomation are not the same . In addition, suppression of host haemocyte-mediated recognition and phagocytosis was not a secondary effect of nutritional deprivation and was not due to ectoparasitoid venom components, rather it was a direct result of parasitization of L . oleracea by E . pennicornis . The putative nature and source of the immunosuppressive factor(s) involved is discussed with reference to those produced by endoparasitic wasps. J Insect Physiol, 2002 Sep, 48(9), 845 - 855 Larvae of the ectoparasitic wasp, Eulophus pennicornis, release factors which adversely affect haemocytes of their host, Lacanobia oleracea; Richards EH et al.; When larvae of the ectoparasitic wasp Eulophus pennicornis were incubated for 4 h on balls of cotton wool soaked in tissue culture medium (TC-100), they released a variety of factors . Subsequent incubation of these larval wasp secretions with monolayers of haemocytes from their host, Lacanobia oleracea, demonstrated that they adversely affect haemocyte morphology, behaviour and viability . For instance, when monolayers of haemocytes were incubated for 18 h in TC-100, approximately 73% of the cells present, attached firmly to and spread over the tissue culture surface by extending pseudopods . By contrast, when incubated in TC-100 containing larval wasp secretions, only about 27% of the haemocytes present remained attached to the tissue culture surface after washing . The majority of these had a rounded configuration and neither spread nor extended pseudopods . Furthermore, viability assays indicated that approximately 36% of the attached haemocytes were dead, as opposed to 11-12% in the controls . The E . pennicornis secretions also significantly reduced the ability of L . oleracea haemocytes to move across the surface of the slide and form clumps (p</=0.0005) and to phagocytose FITC-labelled Escherichia coli in vitro (p</=0.0005) . These results indicate that secretions from E . pennicornis larvae contain an anti-haemocyte factor(s) that can kill and/or alter the behaviour of host haemocytes . As a result, the ability of the haemocytes to execute important immune responses is compromised . Preliminary data suggest that the active molecules are proteins, and that their mechanism of action may involve inhibition of polymerization and/or disorganization of the haemocyte cytoskeleton. Mol Cell, 2003 May, 11(5), 1337 - 47 RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair; Morimatsu K et al.; Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three proteins is unknown . Here, we show that the concerted action of the RecFOR complex directs the loading of RecA protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) protein, thereby accelerating DNA strand exchange . The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5' terminus at the junction . Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions . This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair . We propose that the behavior of the RecFOR proteins is mimicked by functional counterparts that exist in all organisms. Mini Rev Med Chem, 2003 Aug, 3(5), 375 - 85 Human insulin genome sequence map, biochemical structure of insulin for recombinant DNA insulin; Chakraborty C et al.; Insulin is a essential molecule for type I diabetes that is marketed by very few companies . It is the first molecule, which was made by recombinant technology; but the commercialization process is very difficult . Knowledge about biochemical structure of insulin and human insulin genome sequence map is pivotal to large scale manufacturing of recombinant DNA Insulin . This paper reviews human insulin genome sequence map, the amino acid sequence of porcine insulin, crystal structure of porcine insulin, insulin monomer, aggregation surfaces of insulin, conformational variation in the insulin monomer, insulin X-ray structures for recombinant DNA technology in the synthesis of human insulin in Escherichia coli. Comb Chem High Throughput Screen, 2003 Jun, 6(4), 381 - 7 Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening; Eglen RM et al.; In this review, the applications of beta galactosidase complementation are described . alpha Complementation is a naturally occurring process in bacteria and in engineered cells, and can also occur in eukaryotic cells . Two forms of alpha complementation have been used in high throughput screening (HTS), in which interacting fragments complement with either low or high affinity . Low affinity complementation is used to monitor protein protein interactions, such as those occurring in homodimerization of the epidermal growth factor receptor (EGFR), and provides a robust screen for detection of EGFR inhibitors . High affinity complementation provides the basis for several HTS assays, in which analytes, such as cAMP or IP(3), are detected in crude cell lysates . A development of the latter approach is protein labeling, providing for measurement of cell protein expression and trafficking . Collectively, the use of beta galactosidase complementation provides a novel and flexible technology for highly sensitive, homogeneous HTS assay development. Biotechnol Bioeng, 2003 Jul 20, 83(2), 149 - 57 Biomass/adsorbent electrostatic interactions in expanded bed adsorption: a zeta potential study; Lin DQ et al.; Expanded bed adsorption is an integrated technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed . The biomass particles contained in the feedstock have to be treated as an integral part of the process and potential interactions between suspended biomass and the adsorbent must be excluded during process design . Because the electrostatic forces dominate the interactions between the biomass and adsorbent, the zeta potential has been studied as a tool to characterize biomass/adsorbent electrostatic interactions . The zeta potentials of four types of biomass (yeast intact cells, yeast homogenate, Escherichia coli intact cells, and E . coli homogenate) and two types of ion exchanger were measured systematically at varying process conditions . Using the cell transmission index from biomass pulse-response experiments as a parameter, the relations between zeta potential and the biomass/adsorbent interaction were evaluated . Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)), and biomass size (d), parameter (-zeta(a)zeta(b)d) was found to be an appropriate indicator of the biomass/adsorbent interactions in expanded beds under various liquid-phase conditions for different types of biomass . The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV(2) microm for cell transmission of >90%, which means that systems with (-zeta(a)zeta(b)d) < 120 may have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions . Extremophiles, 2003 Jun, 7(3), 249 - 57 Epub 2003 Apr 09. Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii; Lingl A et al.; Archaeal A(1)A(O) ATP synthase/ATPase operons are highly conserved among species and comprise at least nine genes encoding structural proteins . However, all A(1)A(O) ATPase preparations reported to date contained only three to six subunits and, therefore, the study of this unique class of secondary energy converters is still in its infancy . To improve the quality of A(1)A(O) ATPase preparations, we chose the hyperthermophilic, methanogenic archaeon Methanococcus jannaschii as a model organism . Individual subunits of the A(1)A(O) ATPase from M . jannaschii were produced in E . coli, purified, and antibodies were raised . The antibodies enabled the development of a protocol ensuring purification of the entire nine-subunit A(1)A(O) ATPase . The ATPase was solubilized from membranes of M . jannaschii by Triton X-100 and purified to apparent homogeneity by sucrose density gradient centrifugation, ion exchange chromatography, and gel filtration . Electron micrographs revealed the A(1) and A(O) domains and the central stalk, but also additional masses which could represent a second stalk . Inhibitor studies were used to demonstrate that the A(1) and A(O) domains are functionally coupled . This is the first description of an A(1)A(O) ATPase preparation in which the two domains (A(1) and A(O)) are fully conserved and functionally coupled. Extremophiles, 2003 Jun, 7(3), 229 - 33 Epub 2003 Mar 08. Distribution of 16S rRNA introns among the family Thermoproteaceae and their evolutionary implications; Itoh T et al.; Novel 16S rRNA introns were detected in four new strains within the family Thermoproteaceae . Pyrobaculum oguniense TE7(T) and Thermoproteus sp . IC-062 housed introns of 32 and 665-668 bp after positions 1205 and 1213 ( Escherichia coli numbering system), respectively . Caldivirga maquilingensis IC-167(T) had two introns of 37 and 140 bp after positions 901 and 908, respectively . Vulcanisaeta distributa IC-065 had a 691-bp intron after position 1391 . All the introns larger than 650 bp encoded the LAGLI-DADG type proteins . The intron-encoded proteins of P . oguniense TE7(T) and Thermoproteus sp . IC-062 are cognate with the proteins encoded by introns inserted at the same position in other Pyrobaculum/ Thermoproteus strains and phylotypes . The intron-encoded protein of V . distributa IC-065 is partially related to that of a Pyrobaculum phylotype . A large-scale deletion in the second intron of Caldivirga maquilingensis IC-167(T) is suspected . Based on these newly found introns and hitherto known 16S rRNA introns, the evolutionary movements of the 16S rRNA introns and the encoded LAGLI-DADG type proteins are discussed. Protoplasma, 2003 May, 221(1-2), 19 - 30 A major integral protein of the plant plasma membrane binds flavin; Lorenz A et al.; Abundant flavin binding sites have been found in membranes of plants and fungi . With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL . hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family . Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related . The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs . When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein . Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min . Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor . Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. Radiat Environ Biophys, 2003 Jul, 42(2), 113 - 8 Epub 2003 May 27. Extremely low frequency magnetic fields affect transposition activity in Escherichia coli; Del Re B et al.; The aim of this study was to verify whether extremely low frequency (ELF) magnetic fields (MF) could affect transposition activity like some environmental stress factors such as heat shock or UV irradiation . Using an Escherichia coli Lac Z(-) strain transformed with a plasmid containing a Tn 10 derivative element expressing beta-galactosidase only after transposition, it was possible to determine the events of transposition evaluating the rate at which the colonies developed dark coloured papillae (Lac Z(+)) . We found that those bacteria that had been exposed for a long time (58 h) to a 50 Hz low intensity MF (0.1-1 mT) gave colonies with significantly lower transposition activity compared to sham-exposed bacteria . Such reduction in transposition activity was positively correlated to the intensity of the MF, in a dose-effect manner . This phenomenon was not affected by bacterial cell proliferation, since no significant differences were observed in number, diameter and perimeter between sham-exposed and MF-exposed colonies. J Virol, 2003 Jun, 77(12), 7150 - 5 Trans activity of the norovirus Camberwell proteinase and cleavage of the N-terminal protein encoded by ORF1; Seah EL et al.; The virus-encoded proteinase of Camberwell virus, a genogroup 2 norovirus, was synthesized in Escherichia coli . The purified proteinase had correct N and C termini and showed trans activity in cell-free assays . trans activity was also demonstrated in COS cells transfected with constructs encoding either the proteinase or a proteinase-polymerase fusion . The N-terminal protein of ORF1 was cleaved in COS cells, possibly at the site E(194)/S. J Virol, 2003 Jun, 77(12), 7143 - 9 Flavivirus capsid is a dimeric alpha-helical protein; Jones CT et al.; The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance . The oligomeric properties of the capsid protein in solution were investigated . In the absence of nucleic acid, both proteins were predominantly dimeric in solution . Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical . The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments . The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices . The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character . In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein. J Virol, 2003 Jun, 77(12), 6692 - 9 Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice; Richards CM et al.; The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model . Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant . The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a . Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions . Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals . This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection . After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice . Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate . Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced . Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease . The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed. J Biol Chem, 2003 Aug 22, 278(34), 32246 - 50 Epub 2003 May 26. The closed structure of the MscS mechanosensitive channel . Cross-linking of single cysteine mutants; Miller S et al.; Mechanosensitive channels must make a large conformational change during the transition from the closed to the open state . The crystal structure of the open form of the Escherichia coli MscS channel was recently solved and depicts a homoheptamer (1) . In this study, cross-linking of site-specific cysteine substitutions demonstrates that residues up to 10-33 A apart in the crystal structure readily form disulfide bridges in the closed form and can also be cross-linked by a 10-A linker . Cross-linking between adjacent subunits stabilizes the heptameric form of the channel providing biochemical evidence to support the crystal structure . The data are consistent with the published model (1) in that the membrane domain is highly flexible and that the closed to open transition may involve a significant displacement of transmembrane helices 1 and 2, possibly by as much as 30 A . The data are also consistent with significant flexibility of the cytoplasmic domain. Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 1073 - 7 Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides; Cai JP et al.; MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors . During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module . The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified . mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM . Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E . coli mutT(-) cells . Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals . MTH2 could act as an MTH1 redundancy factor. Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 1057 - 60 Role of Shiga toxin 2 (Stx2)-binding protein, human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15; Kimura T et al.; Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections . Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP) . Here, we report the role of HuSAP in STEC infections . HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice . By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP . These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP. J Mol Biol, 2003 Jun 6, 329(3), 611 - 22 Prudent modeling of core polar residues in computational protein design; Bolon DN et al.; Hydrogen bond interactions were surveyed in a set of protein structures . Compared to surface positions, polar side-chains at core positions form a greater number of intra-molecular hydrogen bonds . Furthermore, the majority of polar side-chains at core positions form at least one hydrogen bond to main-chain atoms that are not involved in hydrogen bonds to other main-chain atoms . Based on this structural survey, hydrogen bond rules were generated for each polar amino acid for use in protein core design . In the context of protein core design, these prudent polar rules were used to eliminate from consideration polar amino acid rotamers that do not form a minimum number of hydrogen bonds . As an initial test, the core of Escherichia coli thioredoxin was selected as a design target . For this target, the prudent polar strategy resulted in a minor increase in computational complexity compared to a strategy that did not allow polar residues . Dead-end elimination was used to identify global minimum energy conformations for the prudent polar and no polar strategies . The prudent polar strategy identified a protein sequence that was thermodynamically stabilized by 2.5 kcal/mol relative to wild-type thioredoxin and 2.2 kcal/mol relative to a thioredoxin variant whose core was designed without polar residues. J Mol Biol, 2003 Jun 6, 329(3), 599 - 610 The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd; Martin A et al.; The gene-3 protein (G3P) of filamentous phages is essential for the infection of Escherichia coli . The carboxy-terminal domain anchors this protein in the phage coat, whereas the two amino-terminal domains N1 and N2 protrude from the phage surface . We analyzed the folding mechanism of the two-domain fragment N1-N2 of G3P (G3P(*)) and the interplay between folding and domain assembly . For this analysis, a variant of G3P(*) was used that contained four stabilizing mutations (IIHY-G3P(*)) . The observed refolding kinetics extend from 10 ms to several hours . Domain N1 refolds very rapidly (with a time constant of 9.4 ms at 0.5 M guanidinium chloride, 25 degrees C) both as a part of IIHY-G3P(*) and as an isolated protein fragment . The refolding of domain N2 is slower and involves two reactions with time constants of seven seconds and 42 seconds . These folding reactions of the individual domains are followed by a very slow, spectroscopically silent docking process, which shows a time constant of 6200 seconds . This reaction was detected by a kinetic unfolding assay for native molecules . Before docking, N1 and N2 unfold fast and independently, after docking they unfold slowly in a correlated fashion . A high energy barrier is thus created by domain docking, which protects G3P kinetically against unfolding . The slow domain docking is possibly important for the infection of E.coli by the phage . Upon binding to the F pilus, the N2 domain separates from N1 and the binding site for TolA on domain N1 is exposed . Since domain reassembly is so slow, this binding site remains accessible until pilus retraction has brought N1 close to TolA on the bacterial surface. J Mol Biol, 2003 Jun 6, 329(3), 495 - 503 Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ); Cai SJ et al.; The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes . Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution . By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc . By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed . The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein . The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction . By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit . Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer. J Mol Biol, 2003 Jun 6, 329(3), 441 - 65 Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein . Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex; Galletto R et al.; Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and fluorescence energy transfer techniques . The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding . Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB-DnaC complex formation . The DnaC binding to the unmodified helicase has been characterized in competition experiments . The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer . The positive cooperative interactions are limited to the two neighboring DnaC molecules . Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(+/-0.5)x10(5)M(-1) and cooperativity parameter sigma=21+/-5 . These data suggest strongly that the DnaC-DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution . The DnaC nucleotide-binding site is not involved in the stabilization of the complex . Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex . The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding . However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC-DnaB-ssDNA, formed in the presence of AMP-PNP as compared to ADP . The topology of the ternary complex DnaC-DnaB-ssDNA has been determined using the fluorescence energy transfer method . In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase . The significance of the results in the functioning of the DnaB helicase-DnaC protein complex is discussed. Protein Expr Purif, 2003 Jun, 29(2), 311 - 20 A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles; Millard CS et al.; Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies . Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming . We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles . The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures . The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved . Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures . The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions . After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures . The approach appears to be suitable for high-throughput production of proteins for structural or functional studies. Protein Expr Purif, 2003 Jun, 29(2), 291 - 303 Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies; Kessler N et al.; The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively . The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy . The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates . This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus . The V3 loop is an immunodominant neutralizing epitope of HIV-1 . To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed . A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm . For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon . A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers . The forward primers for the V(L) and V(H) were based on constant region sequences . The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli . High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment . The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N) . Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible. Protein Expr Purif, 2003 Jun, 29(2), 265 - 71 Recombinant expression of Mus musculus myoglobin; Bianchi M et al.; The cDNA encoding for Mus musculus myoglobin (Mb) was amplified using standard RT-PCR techniques and cloned in an appropriate bacterial expression vector . For the first time, mouse Mb was recombinantly expressed in Escherichia coli cells, BL21(DE3), and purified in sufficient amounts to carry out a preliminary characterization . As shown by mass spectrometry, the protein is found in complex with glutathione, which binds the Cys residue in the topological position E9, in the proximity of the heme group . In recombinant murine Mb, azide affinities are only slightly dependent on the Cys(E9) oxidation state . This suggests that Cys(E9) does not provide a relevant contribution for the stabilization of ligands bound to the heme iron atom . Recombinant expression of M . musculus Mb might have an important role in order to investigate the eventual involvement of Cys(E9) in the new physiological roles proposed for Mb. Protein Expr Purif, 2003 Jun, 29(2), 252 - 8 Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies; Dutta A et al.; MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system . We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies . Under the various conditions that we have standardized for the affinity purification of His(6) MutH, the yield of the purified MutH has been estimated to be 35-40 mg of protein from 1liter of M9 minimal media . The yield, thus, obtained by this method is significantly higher than those of previously reported methods . Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis revealed that the protein was pure and existed essentially in a monomeric form . Uniformly 15N-labeled protein, thus, produced has been characterized by recording a sensitivity enhanced 2D {15N}-{1H} HSQC spectrum . The dispersion seen in 15N-1H cross-peaks indicates the presence of a well-ordered structure for MutH and proper folding of the purified protein . The spectrum confirms further the existence of MutH as a monomer. Protein Expr Purif, 2003 Jun, 29(2), 209 - 16 Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography; Smith VR et al.; A major obstacle to investigating the structure of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein . The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been cloned as a fusion protein containing a C-terminal hexa-histidine tag . This fusion protein has been expressed at an abundant level in a mutant strain of Escherichia coli BL21 (DE3) called C41 (DE3) . The protein accumulated as inclusion bodies and none was detected in the bacterial inner membrane . The denatured protein was refolded to reconstitute functional properties similar to the native protein . Solubilized inclusion body protein was immobilized using nickel-chelating affinity chromatography, and purified and refolded in a single step . The protein eluted as a monomer which was stable in mild detergent, at a yield equivalent to 15 mg active protein/liter bacterial culture . The reconstituted fusion protein displayed the same transport characteristics as the wild-type, demonstrating that the tag does not perturb the structure of the protein . The oxoglutarate carrier is one member of an extensive family of mitochondrial transport proteins . These proteins transport many different metabolites across the inner mitochondrial membrane and share a common mechanism of action . Therefore, it is likely that this folding protocol can be applied successfully to other mitochondrial transport proteins, thus providing sufficient protein for extensive crystallization trials with a wide range of family members. Protein Expr Purif, 2003 Jun, 29(2), 193 - 201 Expression of a Brassic napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein; Tsang EW et al.; Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis . ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles . To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation . A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector . The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column . The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg . Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity . However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag . The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP) . Addition of glutamate and pyridoxal 5' phosphate (PLP)