Escherichia coli

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) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively . Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold . Similar to the GSA-AT of B . napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits . As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT . The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B . napus.

Protein Expr Purif, 2003 Jun, 29(2), 167 - 75
Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli; Ahmad S et al.; A major problem in assessing the vaccine and diagnostic potential of various proteins encoded by Mycobacterium tuberculosis genome is the inability to produce large quantities of these proteins, even when Escherichia coli or other heterologous systems are employed for recombinant protein production . To overcome these barriers, we have constructed a modified expression vector, using pGEX-4T-1 vector as the backbone . In addition to the features offered by the pGEX-4T vectors, the new vector allowed easy purification of recombinant proteins on the highly versatile Ni-NTA-agarose affinity matrix . The utility of the new vector was demonstrated by expressing and purifying, to near homogeneity, two M . tuberculosis proteins, i.e., Rv3872 (a member of the multi-gene PE subfamily) and Rv3873 (a member of the multi-gene PPE subfamily), which are encoded by the RD1 region of M . tuberculosis . The proteins encoded by rv3872 and rv3873 were expressed at high levels as fusion proteins with glutathione-S-transferase in E . coli . The recombinant Rv3872 and Rv3873 proteins were purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose and/or Ni-NTA-agarose affinity matrix and cleavage of the purified fusion proteins by thrombin protease . The recombinant Rv3872 protein was nearly homogeneous (more than 95% pure) while Rv3873 preparation was more than 90% pure . The recombinant Rv3872 and Rv3873 proteins were immunologically active and reacted with antibodies in sera from TB patients . Our results demonstrate the utility of the newly constructed expression vector with two affinity tags for efficient expression and purification of recombinant M . tuberculosis proteins expressed in E . coli, which could be used for further diagnostic and immunological studies.

Protein Expr Purif, 2003 Jun, 29(2), 148 - 55
Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase; Ayrapetov MK et al.; CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase) . They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities . However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer . To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli . The enzyme was highly expressed but unusually prone to proteolytic degradation during purification . Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein . The purified enzyme was able to phosphorylate and inactivate Src . Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain . The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.

DNA Repair (Amst), 2003 Jun 11, 2(6), 727 - 35
Participation of DNA polymerase II in the increased precise excision of Tn10; Nagel R et al.; In this work the involvement of polymerase II (Pol II) in the precise excision of Tn10 stimulated by a dnaB252 thermosensitive (Ts) mutant at the permissive temperature, by a uvrD mutant, or by mitomycin C (MMC) or ultraviolet (UV) light treatment, was investigated . A deltapolB::kan mutant showed a significant decrease in the excision of Tn10 induced by the dnaB mutation, or by MMC or UV treatment, indicating the participation of Pol II in this type of deletion process . However, no effect of Pol II was evidenced in the excision of Tn10 stimulated by the uvrD mutation . The effect of the polB mutation on Tn10 precise excision induced by all these treatments was compared to that of mutations in repair-recombination genes recF and recA . The results reveal that the degree of participation of these genes varies depending on the agent that stimulates the deletion event.

DNA Repair (Amst), 2003 Jun 11, 2(6), 695 - 705
The mutation frequency of 8-oxo-7,8-dihydroguanine (8-oxodG) situated in a multiply damaged site: comparison of a single and two closely opposed 8-oxodG in Escherichia coli; Malyarchuk S et al.; A multiply damaged site (MDS) is defined as > or =2 lesions within a distance of 10-15 base pairs (bp) . MDS generated by ionizing radiation contain oxidative base damage, and in vitro studies have indicated that if the base damage is <3bp apart, repair of one lesion is inhibited until repair of the lesion in the opposite strand is completed . Inhibition of repair could result in an increase in the mutation frequency of the base damage . We have designed an assay to determine whether a closely opposed lesion causes an increase in adenine insertion opposite an 8-oxodG in bacteria . We have positioned the MDS (an 8-oxodG in the transcribed strand and a second 8-oxodG immediately 5' to this lesion in the non-transcribed strand) within the firefly luciferase coding region . During two rounds of replication, insertion of adenine opposite the 8-oxodG in the transcribed (T) or non-transcribed (NT) strand results in a translation termination codon at position 444 or 445, respectively . The truncated luciferase protein is inactive . We have generated double-stranded oligonucleotides that contain no damage, each single 8-oxodG or the MDS . Each double-stranded molecule was ligated into the reporter vector and the ligation products transformed into wild-type or Mut Y-deficient bacteria . The plasmid DNA was isolated and sequenced from colonies that did not express luciferase activity . In wild-type bacteria, we detected a translation stop at a frequency of 0.15% (codon 444) and 0.09% (codon 445) with a single 8-oxodG in the T or NT strand, respectively . This was enhanced approximately 3-fold when single lesions were replicated in Mut Y-deficient bacteria . Positioning an 8-oxodG in the T strand within the MDS enhanced the mutation frequency by approximately 2-fold in wild-type bacteria and 8-fold in Mut Y-deficient bacteria, while the mutation frequency of the 8-oxodG in the NT strand increased by 6-fold in Mut Y-deficient bacteria . This enhancement of mutation frequency supports the in vitro MDS studies, which demonstrated the inability of base excision repair to completely repair closely opposed lesions.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jun 25, 790(1-2), 337 - 48
High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein; Heinz C et al.; MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis . Structural analysis was hampered by the scarce amount of pure protein . After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E . coli . The recombinant MspA (rMspA) monomer (M(r) 20000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture . This exceeded the yield of the native protein 10-fold . Circular dichroism revealed that rMspA is folded in a native-like structure . rMspA assembled partially to the channel-forming tetramer both during expression in E . coli and after purification in vitro . Thus, overexpression in E . coli and chromatographic purification are key steps towards a high resolution structure of MspA.

J Steroid Biochem Mol Biol, 2003 Apr, 84(5), 555 - 62
Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo; Haidar S et al.; Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo . Sa 40 {17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol}, its 3-acetyl derivate Sa 41 and BW 19 {3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene} are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis) . They have been compared with CB 7598 {abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol}, its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically . The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole . Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM) . No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment . In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active . The latter three compounds were equally active towards rat CYP 17 . Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally) . The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights . They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights . The only exception was BW 19 which did not change pituitary weights . Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.

Biochemistry, 2003 Jun 3, 42(21), 6588 - 95
Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity; Tang Y et al.; Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT) . This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit . In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups . Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units . Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT . This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step . To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT . In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly . In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module . In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates . Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units . Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs . Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs . Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.

Biochemistry, 2003 Jun 3, 42(21), 6582 - 7
Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic; Dioumaev AK et al.; At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively . It was reported, however {Friedrich, T . et al . (2002) J . Mol . Biol., 321, 821-838}, that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH . To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH . Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated . In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions . In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH . Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.

Biochemistry, 2003 Jun 3, 42(21), 6575 - 81
Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis; Karlsson J et al.; Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain . The latter harbors the proton channel . In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues . To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines . In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated . Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping . The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions . In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H) . Mutation of the remaining residues resulted in intermediate inhibitory effects . The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.

Biochemistry, 2003 Jun 3, 42(21), 6536 - 44
Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI; Rafie-Kolpin M et al.; In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation . Autophosphorylation sites in HRI were identified in order to investigate their functions . We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency . In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI . Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state . Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI . In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite . The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated . Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment . Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI . Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI . Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.

Biochemistry, 2003 Jun 3, 42(21), 6527 - 35
Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli; Liebl U et al.; In the heme-based oxygen sensor Dos from Escherichia coli, one of the axial ligands (Met 95) of a six-coordinate heme can be replaced by external ligands such as O(2), NO, and CO, which causes a switch in phosphodiesterase activity . To gain insight into the bidirectional switching mechanism, we have studied the interaction of ligands with the sensor domain DosH by flash photolysis experiments with femtosecond time resolution . The internal ligand can be photodissociated from the ferrous heme and recombines with time constants of 7 and 35 ps . This is somewhat slower than recombination of the external ligands NO, with which picosecond rebinding occurs with unprecedented efficiency (>99%) with a predominant phase of approximately 5 ps, and O(2) (97% in 5 ps, Liebl, U., Bouzhir-Sima, L., Negrerie, M., Martin, J.-L., and Vos, M . H . (2002) Proc . Natl . Acad . Sci . U.S.A . 99, 12771-12776) . Dissociated CO displays geminate rebinding in 1.5 ns with a very high yield (60%) . Together these results indicate that the heme environment provides a very tight pocket for external ligands, presumably preventing frequent switching events . Additional CO dissociation and rebinding experiments on a longer time scale reveal that (a) Met 95 binding, in 100 micros, occurs in competition with bimolecular CO binding, and (b) subsequent replacement of Met 95 by CO on the millisecond time scale occurs faster than in rapid-mixing experiments, suggesting a slow further relaxation . A minimal ligand binding model is proposed that suggests that Met 95 displacement from the heme is facilitated by the presence of an external ligand in the heme environment . Furthermore, the orders of magnitude difference between Met 95 binding after dissociation of internal and external ligands, as well as the spectral characteristics of photodissociation intermediates, indicate substantial rearrangement of the heme environment associated with ligand sensing . Further remarkable observations include evidence for stable (>4 ns) photooxidation of six-coordinate ferrous heme, with a quantum yield of 4-8%.

Biochemistry, 2003 Jun 3, 42(21), 6484 - 92
The role of the protein core in the inhibitory power of the classic serine protease inhibitor, chymotrypsin inhibitor 2; Radisky ES et al.; A synthetic cyclic peptide, reported to be a tight-binding inhibitor of serine proteases, is instead found to be a good substrate, as is the linear peptide of the same sequence . Both of the peptides, designed to mimic the binding loop of chymotrypsin inhibitor 2 (CI2), were cleaved by subtilisin primarily at the CI2 reactive-site Met-59-Glu-60 bond, revealing that the sequence, in the absence of the structural context of the inhibitor, provides sufficient specificity for hydrolysis of this bond . Insights from the crystal structure of the CI2/subtilisin complex, together with biochemical analysis of a CI2 Gly-83 deletion mutant, have allowed us to identify key features that make CI2 an effective inhibitor, while the cyclic and linear peptides are substrates.

Biochemistry, 2003 Jun 3, 42(21), 6460 - 6
Testing of the additivity-based protein sequence to reactivity algorithm; Qasim MA et al.; The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes . The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm . The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes . As the contact regions of these inhibitors are highly positive, nonadditivity was expected . Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive . For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive . These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains . The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign . It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.

Biochemistry, 2003 Jun 3, 42(21), 6453 - 9
Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Eschericia coli; Fenton AW et al.; The side chains of Escherichia coli phosphofructokinase (EcPFK) that interact with bound substrate, fructose 6-phosphate (Fru-6-P), are examined for their potential roles in allosteric regulation . Mutations that severely decrease Fru-6-P affinity and/or k(cat)/K(m) were created at each contact residue, with the exception of the catalytic base, D127 . Even though Fru-6-P affinity was greatly decreased for R162E, M169A, E222A/H223A, and R243E, the mutated proteins retained the ability to be activated by MgADP and inhibited by phosphoenolpyruvate (PEP) . R252E did not show an allosteric response to either MgADP or PEP . The H249E mutation retained MgADP activation but did not respond to PEP . R72E, T125A, and R171E maintained allosteric inhibition by PEP . Both R72E and T125A displayed a MgADP-dependent decrease in k(cat) but no MgADP-dependent K-type effects . R171E maintained MgADP-dependent K-type activation but also displayed a MgADP-dependent decrease in k(cat) . Localization of mutations that alter MgADP activation near the transferred phosphate group indicates the importance of the 1-methoxy region of Fru-6-P in allosteric regulation by MgADP . A region near the 6'-phosphate may be similarly important for PEP inhibition . R252 is uniquely positioned between the 1'- and 6'-phosphates of bound Fru-1,6-BP, and the mutation at this position may alter both allosterically responsive regions . The differential functions of specific regions in the Fru-6-P contact residues support different mechanisms for allosteric activation and inhibition . In addition, the lack of correlation between mutations that decrease Fru-6-P affinity and those that abolish allosteric communications supports the independence of affinity and allosteric coupling.

Biochemistry, 2003 Jun 3, 42(21), 6436 - 45
Characterization of two independent amino acid substitutions that disrupt the DNA repair functions of the yeast Apn1; Jilani A et al.; The members of the Endo IV family of DNA repair enzymes, including Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV, possess the capacity to cleave abasic sites and to remove 3'-blocking groups at single-strand breaks via apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities, respectively . In addition, Endo IV family members are able to recognize and incise oxidative base damages on the 5'-side of such lesions . We previously identified eight amino acid substitutions that prevent E . coli endonuclease IV from repairing damaged DNA in vivo . Two of these substitutions were glycine replacements of Glu145 and Asp179 . Both Glu145 and Asp179 are among nine amino acid residues within the active site pocket of endonuclease IV that coordinate the position of a trinuclear Zn cluster required for efficient phosphodiester bond cleavage . We now report the first structure-function analysis of the eukaryotic counterpart of endonuclease IV, yeast Apn1 . We show that glycine substitutions at the corresponding conserved amino acid residues of yeast Apn1, i.e., Glu158 and Asp192, abolish the biological function of this enzyme . However, these Apn1 variants do not exhibit the same characteristics as the corresponding E . coli mutants . Indeed, the Apn1 Glu158Gly mutant, but not the E . coli endonuclease IV Glu145Gly mutant, is able to bind DNA . Moreover, Apn1 Asp192Gly completely lacks enzymatic activity, while the activity of the E . coli counterpart Asp179Gly is reduced by approximately 40-fold . The data suggest that although yeast Apn1 and E . coli endonuclease IV exhibit a high degree of structural and functional similarity, differences exist within the active site pockets of these two enzymes.

Biochemistry, 2003 Jun 3, 42(21), 6333 - 40
Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers; Valentine KG et al.; Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells . A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch . Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra . On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer . Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal . The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84 . This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase . Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution . These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.

Biopolymers, 2003 Jun, 69(2), 176 - 88
Acid-induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies; Yamasaki K et al.; Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies . The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state) . In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other . Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states . The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state . A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state . The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate .

Cytometry A, 2003 Jun, 53(2), 79 - 87
New flow cytometric method to quantify the inhibition of enteropathogenic Escherichia coli adhesion by anti-adhesin antibodies; Boullier S et al.; BACKGROUND: Pathogenesis of enteropathogenic Escherichia coli (EPEC) infections can be divided in three stages . The first one is the intestinal colonization mediated by bacterial adhesins . The second and third stages are characterized by an intimate attachment of bacteria to the enterocytes . Little information is available on the specific immune response against EPEC . Here, we describe and validate a new approach to quantify the function of anti-EPEC adhesin antibodies (Abs) . METHODS: We developed a new method to quantify the function of anti-adhesin Abs by flow cytometry . We used pEGFP-E22 (a rabbit EPEC E22 strain expressing the GFP protein) and HeLa cells . The adhesion of E22 bacteria to HeLa cells is mediated by AF/R2, the specific E22 adhesin . We performed short-time interaction (30 min) between pEGFP-E22 and HeLa cells . After extensive washes, 10,000 HeLa cells were acquired by flow cytometry and bacterial adhesion was quantified . Different sera were used to inhibit bacterial adhesion and recombinant MPB-Afr2G (Afr2G is the main AF/R2 subunit) was also tested in this system . RESULTS: We first verified that GFP expression by E22 did not modify bacterial adhesion . We then showed that this flow cytometry approach allowed easy quantification of bacterial adhesion and inhibition mediated by a specific anti-AF/R2 serum . Moreover, recombinant AF/R2 protein reversed the effect of the anti-AF/R2 serum . Finally, we validated our method using sera from E22 orally infected rabbits . We detected and quantified with this method functional specific anti-AF/R2 Abs in their sera . In addition, we correlated our results with an anti-AF/R2 enzyme-linked immunosorbent assay . CONCLUSIONS: We have developed a new method to detect and quantify specific anti-EPEC adhesin Abs by flow cytometry . This method is easy to use and highly reproducible . Its development could be extended to the search of specific anti-adhesin Abs in human EPEC infections .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 May, 35(5), 483 - 7
{Expression and purification of the N-domain of human canstatin and its bioactivity}; He GA et al.; Total RNA was extracted from placenta umbilical tissue . The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain . The amplified 1-89 aa N-domain was then cloned into pET-3c . The resulted plasmid pET-CN was transformed into E . coli BL21(DE3) . The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE . The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body . The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6% . CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 May, 35(5), 409 - 15
Identification of important amino acid residues for human IL-18 function by mutant construction; Fu Y et al.; To study the structure-function relationship of IL-18, two IL-18 mutants, N- and C-terminal mutant (Delta NC) and IL-1 signature-like sequence mutant S(154)A/Y(156)F/E(157)P/C(163)T (S), were constructed by PCR . The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, purified by Sephadex G-75 chromatography and renatured by stepwise dilution . The purity of the recombinant proteins was over 95% . The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma (IFN-gamma) production and NF-kappa B activation from human peripheral blood mononuclear cells (PBMC) . Our results showed that the two mutants induced significantly less amount of IFN-gamma from PBMC (13%, 48% of wild type rhIL-18 for Delta NC, S, respectively), and the activation of NF-kappa B also lower than wild type rhIL-18(69.7%, 89.8% of wild type rhIL-18 respectively), indicating that the deleted or mutated amino acids might be important for IL-18 function.

Nat Cell Biol, 2003 Jun, 5(6), 572 - 7
Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre; Lisby M et al.; DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms . To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells . Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize . Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process . In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest . Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.

J Biomol NMR, 2003 Jul, 26(3), 203 - 13
FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data; Cole R et al.; Herein we describe the program FAST-Modelfree for the fully automated, high throughput analysis of NMR spin-relaxation data . This program interfaces with the program Modelfree 4.1 and provides an intuitive graphical user interface for configuration as well as complete standalone operation during the model selection and rotational diffusion parameter optimization processes . FAST-Modelfree is also capable of iteratively assigning models to each spin and optimizing the parameters that describe the diffusion tensor . Tests with the protein Ribonuclease A indicate that using this iterative approach even poor initial estimates of the diffusion tensor parameters will converge to the optimal value within a few iterations . In addition to improving the quality of the final fit, this represents a substantial timesaving compared to manual data analysis and minimizes the chance of human error . It is anticipated that this program will be particularly useful for the analysis and comparison of data collected under different conditions such as multiple temperatures and the presence and absence of ligands . Further, this program is intended to establish a more uniform protocol for NMR spin-relaxation data analysis, facilitating the comparison of results both between and within research laboratories . Results obtained with FAST-Modelfree are compared with previous literature results for the proteins Ribonuclease H, E . coli glutaredoxin-1 and the Ca(2+)-binding protein S100B . These proteins represent data sets collected at both single and multiple static magnetic fields and which required analysis with both isotropic and axially symmetric rotational diffusion tensors . In all cases results obtained with FAST-Modelfree compared favorably with the original literature results.

J Biomol NMR, 2003 Apr, 25(4), 335 - 48
A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells; Bruggert M et al.; Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature . Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively (15)N-labeled proteins in insect cells . The quantities of (15)N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression . For the most studied amino acids essential for insect cells the (15)N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E . coli . Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species . Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system . Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E . coli and insect cells focused on nitrogen . For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.

J Mol Microbiol Biotechnol, 2003, 5(3), 154 - 60
Non-Shine-Dalgarno initiators of translation selected from combinatorial DNA libraries; Kolev V et al.; In a search for non-Shine-Dalgarno (non-SD) translational initiators, two combinatorial expression libraries (denoted R(1) and R(2)) were constructed containing randomized decanucleotide regions placed at either 6 (R(1)) or 11 (R(2)) nucleotides upstream of a modified chloramphenicol acetyltransferase (CAT) gene . To prevent sporadic formation of SD-like sequences the content of G in the randomized region was restricted to 3% only . The two libraries were transformed in Escherichia coli cells and screened for chloramphenicol (Cm) resistance . More than 50 clones capable of tolerating Cm concentrations from 50 micro g/ml to more than 800 micro g/ml were selected . With few exceptions only, the non-SD sequences found in the Cm-resistant clones did not show any significant homology with other known non-SD initiators or enhancers of translation . Statistical (chi(2)) analysis of the distribution of nucleotides in the new non-SD translational initiators showed a different pattern from that of the conventional SD sequences . In few of the clones the yield of CAT exceeded that of the referent (SD-containing) construct . The most productive clones carried the decanucleotides ATTTACCTCC, CCAATCTAC, TTCAATATTT, and TATTCCCCCA, and the corresponding yield of CAT obtained with them was 2.70, 2.06, 2.12 and 1.32 times, respectively, higher than that of the SD-bearing construct .

J Mol Microbiol Biotechnol, 2003, 5(3), 133 - 49
Reversing transmembrane electron flow: the DsbD and DsbB protein families; Kimball RA et al.; DsbD and DsbB are two proteins that in Escherichia coli catalyze transmembrane electron flow in opposite directions, thereby allowing reversible oxidoreduction of periplasmic dithiol/disulfide-containing proteins . We have identified all recognizable homologues of these two proteins in the databases and have conducted structural and phylogenetic analyses of the two families . The larger DsbD family is more diverse in sequence, topology, function and organismal distribution than the smaller DsbB family . DsbB homologues are rarely found outside of the proteobacteria, although DsbD homologues are found in many bacterial kingdoms as well as archaea and plant chloroplasts . Few organisms with a fully sequenced genome and a DsbB homologue lack a DsbD homologue, and most of these DsbD homologues fall within two clusters in the DsbD tree, exhibiting phylogenetic relationships that are the same as those observed for the DsbB proteins . These observations suggest that a subset of the DsbD homologues evolved in parallel with the DsbB family to perform a single unified function involving reversible extracytoplasmic protein dithiol-disulfide bond interchange . DsbD family proteins are shown to have arisen by an internal gene duplication event, and this observation leads to prediction of the pathway taken for the evolutionary appearance of the different protein topological types found within this family .

J Biol Chem, 2003 Aug 1, 278(31), 28588 - 92 Epub 2003 May 23.
Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C); Sapunaric FM et al.; The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C . A., and Levy, S . B . (2000) J . Biol . Chem . 275, 6101-6106) . Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis . Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene . The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed . Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity . These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation . The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.

J Biol Chem, 2003 Aug 8, 278(32), 29587 - 92 Epub 2003 May 24.
Adenine release is fast in MutY-catalyzed hydrolysis of G:A and 8-Oxo-G:A DNA mismatches; McCann JA et al.; MutY, a DNA repair enzyme, is unusual in that it binds exceedingly tightly to its products after the chemical steps of catalysis . Until now it was not known whether the product being released in the rate-limiting step was DNA, adenine, or both . MutY hydrolyzes adenine from 8-oxo-G:A (OG:A) base pair mismatches as the first step in the base excision repair pathway, as well as from G:A mismatches . The products are adenine and DNA containing an apurinic (AP) site . Tight product binding may have a physiological role in preventing further damage at the OG:AP site . We developed a rate assay using {8-14C}adenine in OG:A or G:A mismatches that distinguishes between adenine hydrolysis and adenine release . {8-14C}Adenine was released quickly from the MutY.AP-DNA.{8-14C}adenine complex, with a rate constant greater than 5 min-1 . This was much faster than the rate-limiting step, at 0.006-0.015 min-1 . Gel retardation experiments showed that AP-DNA release was very slow, consistent with it being the rate-limiting step . Thus, the kinetic mechanism involves fast adenine release after hydrolysis followed by rate-limiting AP-DNA release . Adenine appears to be buried deep in the protein.DNA interface, but there is enough flexibility or open space for it to dissociate from the MutY.APDNA.adenine complex . These results have implications for the catalytic mechanism of MutY.

J Biol Chem, 2003 Jul 18, 278(29), 26315 - 8 Epub 2003 May 24.
Split dnaE genes encoding multiple novel inteins in Trichodesmium erythraeum; Liu XQ et al.; Three inteins were found when analyzing a pair of split dnaE genes encoding the catalytic subunit of DNA polymerase III in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum . The three inteins (DnaE-1, DnaE-2, and DnaE-3) were clustered in a 70-amino acid (aa) region of the predicted DnaE protein . The DnaE-1 intein is 1258 aa long and three times as large as a typical intein, due to the presence of large tandem repeats in which a 57-aa sequence is repeated 17 times . The DnaE-2 intein has a more typical size of 428 aa with putative protein splicing and endonuclease domains . The DnaE-3 intein is a split intein consisting of a 102-aa N-terminal part and a 36-aa C-terminal part encoded on the first and second split dnaE genes, respectively . Synthesis of a mature DnaE protein is predicted to involve expression of two split dnaE genes followed by two protein cis-splicing reactions and one protein trans-splicing reaction . Tandem repeats in the DnaE-1 intein inhibited the protein splicing activity of this intein when tested in Escherichia coli cells and may potentially regulate DnaE synthesis in vivo.

Invest Ophthalmol Vis Sci, 2003 Jun, 44(6), 2683 - 8
Strong in vivo activation of NF-kappaB in mouse lenses by classic stressors; Alexander G et al.; PURPOSE: To examine the in vivo activation of nuclear factor (NF)-kappaB in mouse lens epithelia by using bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and UV-B radiation . METHODS: Transgenic mice containing the NF-kappaB-luciferase reporter were injected with LPS, TNF-alpha or, exposed to UV-B . After various exposure times, the mice were killed, and ocular, liver, lung, kidney, spleen, and skin tissue were obtained . Tissue homogenates were examined for luciferase activity with a luminometer . Groups of mice were also imaged in vivo through a light-intensified camera system to assess NF-kappaB activity . RESULTS: LPS- and TNF-alpha injected NF-kappaB-luciferase transgenic mice yielded 20- to 40-fold increases in lens NF-kappaB activity, similar to other LPS- and TNF-alpha-responsive organs . Peak NF-kappaB activity occurred 6 hours after injection of TNF-alpha and 12 hours after injection of LPS . Peak activities were, respectively, 3 and 6 hours later than that in other tissues . Mice exposed to 360 J/m(2) of UV-B exhibited a 16-fold increase in NF-kappaB activity 6 hours after exposure, which are characteristics similar to TNF-alpha-exposed mice . In vivo imaging of transgenic mice exposed to LPS, TNF-alpha, and UV-B radiation demonstrated a similarity between in vitro and in vivo measurements of NF-kappaB activity . CONCLUSIONS: In NF-kappaB-luciferase transgenic mice, NF-kappaB activity occurs in lens epithelial tissue and is activated when the intact mouse is exposed to bacterial LPS, TNF-alpha, or UV-B . Lens epithelial NF-kappaB kinetics were comparable to those of other tissues, indicating that NF-kappaB may play a role in progression or arrest of lens disorders.

Diabetes, 2003 Jun, 52(6), 1355 - 63
Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes; Wu X et al.; Adiponectin is an abundant adipocyte-derived plasma protein with anti-atherosclerotic and insulin-sensitizing properties that suppresses hepatic glucose production and enhances glucose uptake into skeletal muscle . To characterize the potential effects of adiponectin on glucose uptake into adipose cells, we incubated isolated epididymal rat adipocytes with the globular domain of recombinant adiponectin purified from an E . coli expression system . Globular adiponectin increased glucose uptake in adipocytes without stimulating tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1, and without enhancing phosphorylation of Akt on Ser-473 . Globular adiponectin further enhanced insulin-stimulated glucose uptake at submaximal insulin concentrations and reversed the inhibitory effect of tumor necrosis factor-alpha on insulin-stimulated glucose uptake . Cellular treatment with globular adiponectin increased the Thr-172 phosphorylation and catalytic activity of AMP-activated protein kinase and enhanced the Ser-79 phosphorylation of acetyl CoA carboxylase, an enzyme downstream of AMP kinase in adipose cells . Inhibition of AMP kinase activation using two pharmacological inhibitors (adenine 9-beta-D-arabinofuranoside and compound C) completely abrogated the increase in glucose uptake stimulated by globular adiponectin, indicating that AMP kinase is integrally involved in the adiponectin signal transduction pathway . Coupled with recent evidence that the effects of adiponectin are mediated via AMP kinase activation in liver and skeletal muscle, the findings reported here provide an important mechanistic link in the signaling effects of adiponectin in diverse metabolically responsive tissues.

Biochim Biophys Acta, 2003 Jun 5, 1604(2), 55 - 9
Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme; Pedersen A et al.; Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel . Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme . Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium . Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH . In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H) . Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site . The corresponding reaction in the intact enzyme is not associated with proton pumping . It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer . It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E . Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.

Autoimmunity, 2002 Dec, 35(8), 501 - 13
A major CYP2D6 autoepitope in autoimmune hepatitis type 2 and chronic hepatitis C is a three-dimensional structure homologous to other cytochrome P450 autoantigens; Sugimura T et al.; Liver-kidney microsomal antibodies type 1 (LKM) are a diagnostic marker for autoimmune hepatitis type 2 (AIH-2), however, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C . The major target of LKM antibodies as evidenced by indirect immunofluorescence is cytochrome P4502D6 (CYP2D6) . Anti-CYP2D6 titers of 62 LKM positive sera, 196 sera of patients with hepatic and rheumatic diseases and 33 sera of healthy blood donors (BD) were determined by an in vitro transcription/in vitro translation assay (ITT) . Twenty five out of 26 AIH-2 sera and 33/36 LKM positive hepatitis C virus (HCV) sera were anti-CYP2D6 positive by ITT and antibody titers were similar in both patient groups . Epitope mapping experiments were performed by a series of truncated CYP2D6 proteins and by single epitopes of 257-269, 321-351, 373-389 and 410-419 amino acid (aa) expressed as DHFR-fusion proteins in Escherichia coli . The major linear epitope consists of 257-269 aa . This epitope is recognized with a significantly higher prevalence (64%) in AIH-2 than in LKM sera from patients with chronic hepatitis C (24%) (p < 0.001) . None of the other autoepitopes showed significant differences in the prevalence of recognition by sera from both patient groups . Minor binding sites consisted of 321-351 aa, which was recognized by less than 20% of LKM sera and in the C-terminal region of 350-494 aa, which was recognized by less than 5% of LKM sera Our study revealed an epitope of 321-379 an on CYP2D6, which was shown to be conformation dependent . It was recognized by the vast majority of LKM sera, specifically by 76% of sera from HCV positive LKM patients and also by 76% of sera from patients with AIH-2 . This epitope is homologous to three-dimensional epitopes detected by autoantibodies directed against hepatic cytochromes P450s in drug induced hepatitis and to an autoepitope on CYP21B associated with adrenal failure.

Mol Genet Genomics, 2003 Jul, 269(4), 508 - 16 Epub 2003 May 22.
A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification; Hirouchi T et al.; Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine {6-4} pyrimidone photoproduct (6-4 product) . Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage . These photolyases are specific for either CPDs or 6-4 products . Two classes of photolyases (class I and class II) repair CPDs . A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii . We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification . The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50% . This gene encodes a protein with CPD photolyase activity when expressed in E . coli . The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5 . One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues . The two RNAs occur in about equal amounts in O . sativa cells.

Appl Microbiol Biotechnol, 2003 Jun, 61(5-6), 495 - 501 Epub 2003 Feb 20.
Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB; Mergulhao FJ et al.; The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described . Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium . These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E . coli . It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

Science, 2003 May 23, 300(5623), 1300 - 3
Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo; Pages V et al.; Numerous agents attack DNA, forming lesions that impair normal replication . Specialized DNA polymerases transiently replace the replicative polymerase and copy past lesions, thus generating mutations, the major initiating cause of cancer . We monitored, in Escherichia coli, the kinetics of replication of both strands of DNA molecules containing a single replication block in either the leading or lagging strand . Despite a block in the leading strand, lagging-strand synthesis proceeded further, implying transient uncoupling of concurrent strand synthesis . Replication through the lesion requires specialized DNA polymerases and is achieved with similar kinetics and efficiencies in both strands.

J Biol Chem, 2003 Aug 1, 278(31), 28572 - 9 Epub 2003 May 22.
Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1; Jean L et al.; Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors . Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion . Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31 . To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes . CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins . Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1 . Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49-60-fold higher than that for PfSUB-1 . Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition . None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1 . These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain.

Behav Processes, 2003 May 28, 63(1), 9 - 19
Characterization of NFH-LacZ transgenic mice with the SHIRPA primary screening battery and tests of motor coordination, exploratory activity, and spatial learning; Lalonde R et al.; NFH-LacZ transgenic mice express a fusion protein between a truncated form of the endogenous neurofilament of heavy molecular weight and the complete E . coli beta-galactosidase . NFH-LacZ transgenic mice could be distinguished from controls in the SHIRPA neurological battery by the appearance of action tremor and hindlimb clasping and a lower body weight . Despite normal exploratory activity and spatial learning, NFH-LacZ transgenic mice were deficient in stationary beam, coat-hanger, and rotorod tests of motor coordination . These results are concordant with neuropathological findings in spinal motoneurons and the cerebellum and indicate that despite the absence of paralysis, these transgenic mice may serve as an experimental model of the early stage of amyotrophic lateral sclerosis.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 586 - 91
Fusion-type lycopene beta-cyclase from a thermoacidophilic archaeon Sulfolobus solfataricus; Hemmi H et al.; Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a thermoacidophilic archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene beta-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes . The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of beta-carotene in the cells, which verifies the function of the gene . Homologues of the archaeal lycopene beta-cyclase from various organisms such as bacteria, archaea, and fungi have been reported . Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 534 - 40
On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells; Umehara S et al.; We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes . This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers . After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles . When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h . The results indicate that the cell's growth and division are directly related to their nutrient condition . The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 476 - 83
Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli; Neugebauer R et al.; Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor . The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit . Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture . After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist {3H}MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively . In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained . These results redefine the S1S2 domains required for high-affinity glycine binding . Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.

In Silico Biol, 2003, 3(1-2), 33 - 47 Epub 2003 Apr 14.
An algorithm for identification of regulatory signals in unaligned DNA sequences, its testing and parallel implementation; Danilova LV et al.; We describe an algorithm (IRSA) for identification of common regulatory signals in samples of unaligned DNA sequences . The algorithm was tested on randomly generated sequences of fixed length with implanted signal of length 15 with 4 mutations, and on natural upstream regions of bacterial genes regulated by PurR, ArgR and CRP . Then it was applied to upstream regions of orthologous genes from Escherichia coli and related genomes . Some new palindromic binding and direct repeats signals were identified . Finally we present a parallel version suitable for computers supporting the MPI protocol . This implementation is not strictly bounded by the number of available processors . The computation speed linearly depends on the number of processors.

Protein Sci, 2003 Jun, 12(6), 1216 - 21
A wheat germ cell-free system is a novel way to screen protein folding and function; Morita EH et al.; For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system . Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited . A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms . This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins . To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them . The structural analysis of these proteins has already progressed with an E . coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained . Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity . This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.

Protein Sci, 2003 Jun, 12(6), 1205 - 15
Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli; Mehl AF et al.; The GrpE heat shock protein from Escherichia coli has a homodimeric structure . The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement . We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation . Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region . Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface . These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer . Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure . A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.

J Biochem (Tokyo), 2003 Apr, 133(4), 485 - 91
A novel screening system for self-mRNA targeting proteins; Ying BW et al.; Here we describe the application of an in vitro translation system for genetic screening, to identify RNA-binding proteins that bind to their own mRNAs . It is a relatively novel system designed using an advanced cell-free translation system reconstructed with purified translational components . Due to the absence of nucleases and proteases, the complex of mRNA and nascent polypeptide synthesized in this system is expected to exhibit high stability ensuring the following efficient selection toward the protein . Escherichia coli ribosomal protein S15, which is known to bind to its own mRNA, was employed as a model molecule to evaluate the system . Wild-type S15 mRNA specifically isolated from a mutant mRNA lacking the secondary structure responsible for binding the S15 protein accumulated markedly after several rounds of selection-amplification . The success of this selection demonstrates the potentiality of the systematic screening of self-mRNA targeting proteins through direct and functional selection . This strategy as a method to identify peptides or proteins that bind to their own mRNAs, is of general interest and has different potential applications, such as, the identification of new regulatory proteins or peptide motifs for RNA recognition, the study of self-mRNA-protein interactions, etc.

J Biol Chem, 2003 Aug 8, 278(32), 29995 - 30004 Epub 2003 May 21.
The Pleckstrin homology domains of phospholipases C-beta and -delta confer activation through a common site; Guo Y et al.; Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators . PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) . For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation . Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2 . The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1 . Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma . Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1 . A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera . Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.

J Biol Chem, 2003 Aug 1, 278(31), 28912 - 20 Epub 2003 May 21.
Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member; Alanen HI et al.; Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins . Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete . Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase . We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold . From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site . Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation . Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues . This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation . A putative physiological role for Erp18 in native disulfide bond formation is discussed.

J Biochem (Tokyo), 2003 Jan, 133(1), 33 - 42
D-arginase of Arthrobacter sp . KUJ 8602: characterization and its identity with Zn(2+)-guanidinobutyrase; Arakawa N et al.; D-Arginase activity was found in the cells of an isolate, Arthrobacter sp . KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized . Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer . The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine . The V(max)/K(m) values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 micro mol/min/mg/mM, respectively . Accordingly, the enzyme is regarded as a kind of guanidinobutyrase {EC 3.5.3.7} . The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively . The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors . The enzyme contained about 1 g-atom of firmly bound Zn(2+) per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity . The inactivated enzyme was reactivated markedly by incubation with either Zn(2+) or Co(2+), and slightly by incubation with Mn(2+) . The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M(r): 37,933) . The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn(2+)-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

J Biochem (Tokyo), 2003 Feb, 133(2), 219 - 24
Cloning, expression, and characterization of the first archaeal ATP-dependent glucokinase from aerobic hyperthermophilic archaeon Aeropyrum pernix; Sakuraba H et al.; The gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli . The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum . The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa . The enzyme retained its full activity on heating at 70 degrees C for 10 min and retained 65% of the activity after 10-min incubation at 100 degrees C . The enzyme exclusively catalyzed the phosphorylation of D-glucose using ATP as a phosphoryl donor . ITP was accepted in addition to ATP . The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent K(m) values of 0.054 and 0.50 mM, respectively . The enzyme activity required divalent cations; Mg(2+), which was most effective, could partially be replaced by Mn(2+) or Ca(2+) . Phylogenetic analysis revealed that the glucokinase from A . pernix does not belong to the clusters of enzymes found in bacteria and eukarya . This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon.

Infect Immun, 2003 Jun, 71(6), 3619 - 22
The FimH A27V mutation is pathoadaptive for urovirulence in Escherichia coli B2 phylogenetic group isolates; Hommais F et al.; Correlations between FimH mutations and virulence were established by studying a collection of human commensal and extraintestinal pathogenic Escherichia coli natural isolates . Pathoadaptive (A27V and, to a lesser extent, A119V) and "commensal-adaptive" (A202V) mutations were evidenced in B2 phylogenetic group strains . fimH phylogenetic analysis indicates that these pathoadaptive mutations occurred several times.

Infect Immun, 2003 Jun, 71(6), 3551 - 62
Catalases of Aspergillus fumigatus; Paris S et al.; Upon infection of a host, the pathogenic fungus Aspergillus fumigatus is attacked by the reactive oxygen species produced by phagocytic cells . Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response . A . fumigatus produces three active catalases; one is produced by conidia, and two are produced by mycelia . The mycelial catalase Cat1p was studied previously . Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants . CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent . This enzyme is a dimeric protein with 84.5-kDa subunits . The 749-amino-acid polypeptide exhibits high levels of similarity to the Aspergillus nidulans CatA catalase and to bacterial catalase HPII of Escherichia coli . In spite of increased sensitivity to H(2)O(2), killing of DeltacatA conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type . In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent . This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria . Surprisingly, mycelium of the double Deltacat1Deltacat2 mutant with no catalase activity exhibited only slightly increased sensitivity to H(2)O(2) and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain . However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain . These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.

Infect Immun, 2003 Jun, 71(6), 3310 - 9
Secretin of the enteropathogenic Escherichia coli type III secretion system requires components of the type III apparatus for assembly and localization; Gauthier A et al.; At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined . A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin . Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction . Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel . Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location . Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components . Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm . EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation . These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.

Infect Immun, 2003 Jun, 71(6), 3302 - 9
The immunoglobulin D-binding protein MID from Moraxella catarrhalis is also an adhesin; Forsgren A et al.; The Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD . MID is found in the majority of M . catarrhalis strains . In the present paper, we show that MID-expressing M . catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells . In contrast, M . catarrhalis isolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity . To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule . The truncated MID proteins were expressed in Escherichia coli, purified, and used for raising polyclonal antibodies in rabbits . Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID(764-913) . In addition, antibodies against full-length MID, MID(764-913), or a 30-amino-acid consensus sequence (MID(775-804)) inhibited adhesion to alveolar epithelial cells . Antibodies against UspA1, an outer membrane protein expressed in essentially all M . catarrhalis strains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells . Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine against M . catarrhalis.

Infect Immun, 2003 Jun, 71(6), 3020 - 7
Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells; Kim S et al.; Brucella spp . are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans . The mechanism and factors of virulence are not fully understood . To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant . Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth . From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth . Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes . These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages . These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

Bioinformatics, 2003 May 22, 19(8), 1027 - 34
Hierarchical analysis of dependency in metabolic networks; Gagneur J et al.; MOTIVATION: Elucidation of metabolic networks for an increasing number of organisms reveals that even small networks can contain thousands of reactions and chemical species . The intimate connectivity between components complicates their decomposition into biologically meaningful sub-networks . Moreover, traditional higher-order representations of metabolic networks as metabolic pathways, suffers from the lack of rigorous definition, yielding pathways of disparate content and size . RESULTS: We introduce a hierarchical representation that emphasizes the gross organization of metabolic networks in largely independent pathways and sub-systems at several levels of independence . The approach highlights the coupling of different pathways and the shared compounds responsible for those couplings . By assessing our results on Escherichia coli (E.coli metabolic reactions, Genetic Circuits Research Group, University of California, San Diego, 'model v 1.01 . reactions') against accepted biochemical annotations, we provide the first systematic synopsis of an organism's metabolism . Comparison with operons of E.coli shows that low-level clusters are reflected in genome organization and gene regulation . AVAILABILITY: Source code, data sets and supplementary information are available at http://www.mas.ecp.fr/labo/equipe/gagneur/hierarchy/hierarchy.html

Bioinformatics, 2003 May 22, 19(8), 987 - 98
Codon pairs in the genome of Escherichia coli; Boycheva S et al.; MOTIVATION: The effect of two neighboring codons (codon pairs) on gene expression is mediated via the interaction of their cognate tRNAs occupying the two functional ribosomal sites during the translation elongation step . For steric reasons it is reasonable to assume that not all combinations of codons and therefore of tRNAs are equally favorable when situated on the ribosome surface . Aiming of identifying preferential and rare codon pairs, we have determined the frequency of occurrence of all possible combinations of codon pairs in the entire genome of Escherichia coli (E.coli) . RESULTS: The frequency of occurrence of the 3904 codon pairs comprising both sense:sense and sense:stop codon pairs in the full set of E.coli 4289 ORFs was found to vary from zero to 4913 times . For most of the pairs we have observed a significant difference between the real and statistically predicted frequency of occurrence . The analysis of 334 highly expressed and 303 poorly expressed E.coli genes showed that codon pair usage is different for the two gene categories . Using an especially defined criterion (Delta(REG)), the codon pairs are classified as 'hypothetically attenuating' (HAP) and 'hypothetically non-attenuating' (HNAP) and their possible effect on translation is discussed . AVAILABILITY: The program used in this study is available at http://www.bio21.bas.bg/codonpairs/

J Parasitol, 2003 Apr, 89(2), 209 - 14
A comparative study of biochemical and immunological properties of triosephosphate isomerase from Taenia solium and Sus scrofa; Jimenez L et al.; We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa . Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a Km value of 0.758, and S . scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a Km value of 0.51 . The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes . Anti-T . solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S . scrofa, human, or protozoan TPIs . These antibodies inhibited T . solium TPI activity but did not affect S . scrofa enzymatic activity . Immunizations with 1 microg of the T . solium TPI reduced 52% of cysticerci in a mouse-Taenia crassiceps model 1 mo after challenge . Our findings show that T . solium and S . scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti-T . solium TPI antibodies did not recognize S . scrofa TPI . Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.

J Biol Chem, 2003 Jul 25, 278(30), 27712 - 20 Epub 2003 May 19.
The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity; Legate KR et al.; The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane . We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase . We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP . SRbeta possesses no detectable GTPase activity . Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains . A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase . Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.

J Biol Chem, 2003 Aug 8, 278(32), 29435 - 41 Epub 2003 May 20.
Two functional heads are required for full activation of smooth muscle myosin; Li XD et al.; The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity . To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e . S236T or G457A) . The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively . If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value . The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head . Consistently, the actin-gliding velocity of the asymmetric HMM (i.e . S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM . The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.

Anal Biochem, 2003 Jun 15, 317(2), 255 - 8
Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins; Kery V et al.; High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics . A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host . Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process . Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA) . An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane . Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP . The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay . Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis . It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.

Anal Biochem, 2003 Jun 15, 317(2), 226 - 32
Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein-protein interactions; Scheibner KA et al.; Determination of protein oligomerization state can be technically challenging . We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states . We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins . The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET . Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization . These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.

Anal Biochem, 2003 Jun 15, 317(2), 156 - 65
A high-throughput solid-phase extraction assay capable of measuring diverse polyprenyl phosphate: sugar-1-phosphate transferases as exemplified by the WecA, MraY, and MurG proteins; Hyland SA et al.; The bacterial proteins WecA and MraY are members of the polyprenyl phosphate:N-acetylhexosamine-1-phosphate transferase family, each of which catalyzes the transfer of a specific hexosamine 1-P from a soluble UDP-hexosamine substrate to a bactoprenyl phosphate carrier at the membrane surface . Currently, assays designed to quantitate the activity of these enzymes rely on paper chromatography or liquid-liquid extractions or are specialized to a few members of the family . We describe a generalizable, high-throughput, one-pot assay for these activities that uses a solid-liquid bead-based separation system to selectively adsorb the highly hydrophobic products of reaction . By judicious choice of radiolabeled UDP-hexosamine precursor, the same format can be used to quantitate not only diverse members of this transferase family, but also enzymes that catalyze the further modification of these transferase products . This possibility is exemplified by the MurG protein of bacterial cell wall synthesis, which catalyzes the addition of an N-acetylglucosamine residue to the product of the MraY reaction . Thus, the use of this flexible assay tool will allow a critical biochemical and enzymologic analysis of many such membrane-bound transferases in a similar setting.

Biochim Biophys Acta, 2003 May 30, 1648(1-2), 99 - 104
Creation of soybean beta-conglycinin beta with strong phagocytosis-stimulating activity; Maruyama N et al.; beta-Conglycinin is composed of three kinds of subunit: alpha, alpha' and beta . A phagocytosis-stimulating peptide sequence (MITLAIPVNKPGR), soymetide, exists in the alpha' subunit of beta-conglycinin . Met at N terminus of the soymetide is essential for the activity . When Thr at the third residue from N terminus of the soymetide is replaced by Phe or Trp, the phagocytosis-stimulating activity greatly increases (Thr<Phe<Trp) . The beta subunit does not exhibit the phagocytosis-stimulating activity because the residues corresponding to the first and third residues in the soymetide are Ile and Lys, respectively . In this study, we introduced the phagocytosis-stimulating peptide sequence (Ile-->Met, Lys-->Thr, Phe, or Trp) into the beta subunit after confirmation of the effects of residue replacements by molecular modeling, suggesting that the introduced mutations might not prevent the correct folding . The studies of circular dichroism (CD), gel filtration and differential scanning calorimetry (DSC) of the mutants (I122M/K124T, I122M/K124F, I122M/K124W) expressed in E . coli demonstrated that they folded and self-assembled similarly to the wild type . This was confirmed by X-ray analysis of I122M/K124W crystal where the biggest residue tryptophane was introduced . The three mutants exhibited phagocytosis activities after digestion by trypsin, and the order was the wild type<I122M/K124T<I122M/K124F<I122M/K124W as expected.

Biochim Biophys Acta, 2003 May 30, 1648(1-2), 84 - 9
Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA; Chant A et al.; The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes . We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein . A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E . coli . Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (approximately 4 M), exhibiting a sharp transition . Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence . The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.

Biochim Biophys Acta, 2003 May 30, 1648(1-2), 17 - 23
Expression of recombinant human L-glutaminase in Escherichia coli: polyclonal antibodies production and immunological analysis of mouse tissues; Campos JA et al.; The first complete sequence of human L-glutaminase was deduced from breast cancer glutaminase cDNA cloned in our laboratory . This cDNA clone has now been engineered to synthesize both precursor and mature forms of the protein in Escherichia coli . Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-3c) was found to be the most efficient for glutaminase overproduction . Upon induction, precursor glutaminase accounts for about 25% of total E . coli protein, whereas a lower amount (12%) was achieved for the putative mature protein . The optimal length of the translational spacer on the ribosome binding site was shown to be eight nucleotides . However, using this length of spacer, we were unable to obtain expression in the pQE vector, tagged with a 6x His sequence at the NH(2)-terminus, stressing the importance of the 5'-coding sequence in the expression efficiency . Although the precursor and mature recombinant forms of glutaminase were devoid of catalytic activity, the purified protein allowed us to obtain highly specific polyclonal antibodies, as shown by immunoblot analysis of mouse tissues . Furthermore, the antibodies were able to immunoprecipitate the in vitro translated enzyme using a reticulocyte lysate system; these antibodies might be a valuable tool for studies on L-glutaminase expression in mammalian tissues.

J Mol Biol, 2003 May 30, 329(2), 363 - 70
Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA; Yamazaki J et al.; RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction . We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein . RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains . The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea . Thus, the C-terminal domain of RecA is unfolded after the central part unfolds . The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains . This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA . ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change . ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it . Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.

J Mol Biol, 2003 May 30, 329(2), 253 - 69
Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI; Chevalier B et al.; Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame . These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites . Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences . Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers . We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site . This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts . The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.

FEMS Microbiol Lett, 2003 May 16, 222(1), 115 - 21
60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage; Yamamoto T et al.; Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher . However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC . Stx production was observed in parallel to the phage induction . Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing . Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage . The findings suggest that (1) . although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2) . irradiation differentially inactivates some activities of Stx.

J Virol Methods, 2003 Jun 9, 110(1), 91 - 7
Production of recombinant Potato mop-top virus coat protein in Escherichia coli and generation of antisera recognising native virus protein; Helias V et al.; Potato mop-top virus (PMTV, Pomovirus) is difficult to detect because it is unevenly distributed and present at low concentration in infected tissues . The production of PMTV-free seed relies on sensitive and specific detection methods of virus detection, including serological methods . The possibility of using a PMTV recombinant coat protein (CP) as an antigen for antiserum production was investigated . The region encoding the PMTV CP was inserted into pET3A, expressed in Escherichia coli, and the recombinant PMTV CP produced was used to raise antibodies in rabbits . Three antisera were produced . All recognised efficiently the recombinant CP in Western blot analysis and the most sensitive antiserum (H5003) detected native CP on Western blots and in ELISA . Thus, recombinant CP can be used as an alternative to purified virus for the production of specific antibodies against PMTV.

Free Radic Biol Med, 2003 Jun 1, 34(11), 1447 - 57
Expression of human MutT homologue (hMTH1) protein in primary non-small-cell lung carcinomas and histologically normal surrounding tissue; Kennedy CH et al.; In situ, oxidation of deoxyguanosine yields 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), which is mutation prone and results in a G:C --> T:A transversion following DNA replication . Another pathway to the formation of DNA containing 8-oxo-dG is by the misincorporation of 8-oxo-dGTP via DNA polymerase . Human MutT homologue (hMTH1), an 8-oxo-dGTPase, prevents misincorporation of this oxidized nucleotide by hydrolyzing 8-oxo-dGTP to 8-oxo-dGMP . Previous studies have shown that hMTH1 mRNA is overexpressed in human renal cell carcinomas and breast tumors . Elevated levels of hMTH1 protein have also been detected in brain tumors . In the current study, we determined whether hMTH1 protein is overexpressed in primary non-small-cell lung carcinomas as compared to adjacent histologically normal lung tissue . Twenty matched human lung tumor/normal pairs were examined by Western analysis for expression of hMTH1 protein . Overexpression in the tumors was detected in 4/8 (50%) adenocarcinomas, 4/4 (100%) adenocarcinomas with bronchioalveolar (BAC) features, 2/2 (100%) BACs, and 3/6 (50%) squamous cell carcinomas . The data from Western analysis were validated by immunohistochemical staining for hMTH1 protein . The results of this study indicate that hMTH1 protein may be a potential marker for the detection of persistent oxidative stress in lung cancer.

Cell, 2003 May 16, 113(4), 469 - 82
The pathogen-inducible nitric oxide synthase (iNOS) in plants is a variant of the P protein of the glycine decarboxylase complex; Chandok MR et al.; A growing body of evidence indicates that nitric oxide (NO) plays important signaling roles in plants . However, the enzyme(s) responsible for its synthesis after infection was unknown . Here, we demonstrate that the pathogen-induced, NO-synthesizing enzyme is a variant form of the P protein of glycine decarboxylase (GDC) . Inhibitors of the P protein of GDC block its NO synthase (NOS)-like activity, and variant P produced in E . coli or insect cells displays NOS activity . The plant enzyme shares many biochemical and kinetic properties with animal NOSs . However, only a few of the critical motifs associated with NO production in animals can be recognized in the variant P sequence, suggesting that it uses very different chemistry for NO synthesis . Since nitrate reductase is likely responsible for NO production in uninfected or nonelicited plants, our results suggest that plants, like animals, use multiple enzymes for the synthesis of this critical hormone.

J Coll Physicians Surg Pak, 2003 May, 13(5), 297 - 301
A review of rotavirus diarrhea in Pakistan: how much do we know?
Ali NK, Bhutta ZA.
INTRODUCTION: Rotavirus diarrhea has a worldwide distribution, infecting almost all children by the age of 3-5 years . EPIDEMIOLOGY: A comparable etiological 2-year survey carried out by the W.H.O Diarrheal Disease control (CDD) Program in 1991, in a multicenter study in 5 developing countries including Pakistan revealed that Rotavirus was found to be the most frequently detected pathogen in diarrheal episodes, during the first year of life, with the highest incidence (20%) occurring among 6-11 months old . Two other studies done in Pakistan, in under five children done in Lahore (between 1985 and 1991) and Rawalpindi (between May 1983 and April 1984) showed that Rotavirus was the second most common Diarrhea causing enteric pathogen following E.Coli TRANSMISSION: Rotaviruses are shed in high concentrations 2 days before and as many as 10 days after onset of symptoms in immunocompetent hosts, thus being an important source of viral transmission . CLINICAL COURSE: A multicenter study in 5 developing countries including Pakistan conducted by WHO CDD program revealed that only 1.8 % of cases presented with severe dehydration and these were mostly due to Rotavirus, V.Cholerae and ETEC13 . DIAGNOSTIC TESTS: A study conducted in local hospitals in Pakistan during the period of October 1985-April 1986 compared the different diagnostic modalities for the detection of rotavirus in the faeces of children with acute diarrhea . The study all methods detected Rotavirus to varying degrees but ELISA was found to be the most sensitive method with 72.4% stools being positive . PREVENTIVE STRATEGIES: A study was conducted in Lahore (Pakistan) among 72 infants 6 weeks old in 1991 to assess safety and efficacy of RRV vaccine . It was found that of all infants given RRV with OPV, 50% had a two to four-fold rise in neutralization titers against rotavirus . RRV was found to be safe and not associated with adverse reactions in the 6 weeks old infants . CONCLUSION: With regards to Pakistan, there is a great need for defining rotavirus associated disease burden and strain prevalence . We also need to conduct Rotavirus vaccine trials to assess its efficacy and safety in our setting.

Bioconjug Chem, 2003 May-Jun, 14(3), 679 - 83
PNA-based RNA-triggered drug-releasing system; Ma Z et al.; A three-component sequence-specific RNA-triggered drug-releasing system is described that consists of an 8-mer PNA linked to a coumarin ester (the prodrug component) and a 14-mer PNA linked to histidine (the catalytic component) that are complementary to the C loop of E . coli 5S rRNA (the triggering component) . Binding of the catalytic component to the RNA creates a prodrug-metabolizing enzyme that catalyzes a 60,000-fold acceleration in the rate of coumarin release from the prodrug compared to the rate of coumarin release from the ester subunit catalyzed by imidazole alone . RNA-triggered release of hydroxycoumarin is only slightly less efficient than that triggered by a short unfolded DNA sequence corresponding to the PNA binding sites . The lower efficiency results from a decrease in k(cat) and an increase in K(M), presumably due to the bent nature of the RNA . The efficiency of DNA-triggered hydroxycoumarin release was found to depend on the distance between the catalytic and prodrug components.

Adv Exp Med Biol, 2003, 529, 101 - 4
Mapping of possible laminin binding sites of Y . pestis plasminogen activator (Pla) via phage display; Benedek O et al.; We tried to determine amino acid motifs of Y . pestis plasminogen activator (Pla) involved in laminin binding . We selected heptamer peptides using a random phage library which was tested against immobilised laminin . Two sequences seemed to inhibit Pla mediated laminin binding of E . coli and exhibited a strong laminin binding capacity revealing a competition with Pla for the same laminin binding site . The motifs are also involved in plasminogen activation because they caused fibrinolysis on fibrin films . The patterns were localised outside the putative surface-exposed loops (Kukkonen et al., 2001).

Arch Virol, 2003 Jun, 148(6), 1211 - 8
Molecular evidence that aphid-transmitted Alpinia mosaic virus is a tentative member of the genus Macluravirus; Liou RF et al.; Alpinia mosaic virus (AlpMV), once assigned to the genus Potyvirus, infects primarily plants of the ginger family . To seek molecular evidence for correct classification of this virus, a cDNA clone corresponding to the 3' portion of the AlpMV genome was obtained by reverse transcriptase-PCR and TA cloning . The authenticity of the cDNA clone was confirmed by expression of the coat protein (CP) in E . coli followed by immunoblot analysis . Sequence analysis indicated that, in contrast to its low identity with all the other genera of the family Potyviridae, the deduced amino acid sequence of AlpMV CP was 42.9 - 61.9% identical to members of the genus Macluravirus . Phylogenetic analysis also demonstrated that the AlpMV CP clustered with those of Cardamom mosaic virus and Chinese yam necrotic mosaic virus . These results indicate that AlpMV should be classified as a tentative species within the genus Macluravirus rather than Potyvirus as proposed previously.

Arch Virol, 2003 Jun, 148(6), 1185 - 93
Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India; Hema M et al.; Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae . The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV-AP) was cloned and expressed in Escherichia coli . The recombinant coat protein was used to raise high quality antiserum . The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India . The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz . Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV-AP . The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level . This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV . In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Mol Genet Genomics, 2003 May, 269(2), 215 - 26 Epub 2003 Mar 12.
Construction of a mitotic linkage map of Fusarium oxysporum based on Foxy-AFLPs; Teunissen HA et al.; Construction of the first mitotic linkage map of the asexual fungus Fusarium oxysporum, based on a population of 32 parasexual fusion products, is reported . Molecular markers were developed using a modified AFLP technique which combines a Foxy-specific primer with standard adapter primers . The retroposon Foxy is abundantly present and highly variable in location in F . oxysporum isolates: 43% of the Foxy-AFLP markers tested appeared to be polymorphic between the strains Fol004 and Fol029 . Of the 102 Foxy markers obtained, 83 segregated in a 1:1 ratio . The remaining fragments showed a skewed segregation pattern in which the Fol004 derived Foxy fragments were overrepresented . Foxy markers were observed to be clustered, suggesting that active Foxy elements may not transpose very far from their initial insertion sites, or that hotspots for insertion may exist . Linkage analysis revealed 23 linkage groups . Physical linkage between segregating markers predicted to be 20 cM apart was confirmed, indicating that the mitotic linkage map is reliable.

Mol Genet Genomics, 2003 May, 269(2), 180 - 7 Epub 2003 Feb 11.
Importance of transmembrane segments in Escherichia coli SecY; Shimokawa N et al.; To assess the functional importance of the transmembrane regions of SecY, we constructed a series of SecY variants, in which the six central residues of each transmembrane segment were replaced by amino acid residues from either transmembrane segment 3 or 4 of LacY . The SecY function, as assessed by the ability to complement cold-sensitive secYmutants with respect to their growth and translocase defects, was eliminated by the alterations in transmembrane segments 2, 3, 4, 7, 9 and 10 . Among them, those in segments 3 and 4 had especially severe effects . In contrast, transmembrane segments 1, 5, 6, and 8 were more tolerant to the sequence alterations . The purified protein with an altered transmembrane segment 6 retained, in large measure, the ability to support SecA-dependent preprotein translocation in vitro . These results will help us to further understand how the SecYEG protein translocation channel functions.

RNA, 2003 Jun, 9(6), 734 - 45
Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P; Zahler NH et al.; The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit . It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence . However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined . Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site . Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1) . Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation . Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes . These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.

RNA, 2003 Jun, 9(6), 722 - 33
Effect of transcription on folding of the Tetrahymena ribozyme; Heilman-Miller SL et al.; Sequential formation of RNA interactions during transcription can bias the folding pathway and ultimately determine the functional state of a transcript . The kinetics of cotranscriptional folding of the Tetrahymena L-21 ribozyme was compared with refolding of full-length transcripts under the same conditions . Sequential folding after transcription by phage T7 or Escherichia coli polymerase is only twice as fast as refolding, and the yield of native RNA is the same . By contrast, a greater fraction of circularly permuted variants folded correctly at early times during transcription than during refolding . Hybridization of complementary oligonucleotides suggests that cotranscriptional folding enables a permuted RNA beginning at G303 to escape non-native interactions in P3 and P9 . We propose that base pairing of upstream sequences during transcription elongation favors branched secondary structures that increase the probability of forming the native ribozyme structure.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 6980 - 5 Epub 2003 May 19.
Correlated motion and the effect of distal mutations in dihydrofolate reductase; Rod TH et al.; Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate . The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme {Rajagopalan, P . T . R, Lutz, S., and Benkovic, S . J . (2002) Biochemistry 41, 12618-12628} . On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed . The parameters we study are relative populations of different conformations sampled and hydrogen bonds . We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme . Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations . We finally examine the role a structural perturbation will have on the hydride transfer step . On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

J Biol Chem, 2003 Aug 15, 278(33), 31078 - 87 Epub 2003 May 19.
Identification, purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi; Armengaud J et al.; Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented . Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria . In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway . We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944) . Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide . The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction . The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry . Potassium glutamate was found to stabilize the protein at 400 mm . The enzyme behaves as a monomeric protein . Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT . The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.

J Biol Chem, 2003 Aug 1, 278(31), 29298 - 307 Epub 2003 May 19.
Catalytic mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase in complex with sulfate, magnesium, and triton; Chang SY et al.; Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP) . UPPs from Escherichia coli is a dimer, and each subunit consists of 253 amino acid residues . The chain length of the product is modulated by a hydrophobic active site tunnel . In this paper, the crystal structure of E . coli UPPs was refined to 1.73 A resolution, which showed bound sulfate and magnesium ions as well as Triton X-100 molecules . The amino acid residues 72-82, which encompass an essential catalytic loop not seen in the previous apoenzyme structure (Ko, T.-P., Chen, Y . K., Robinson, H., Tsai, P . C., Gao, Y.-G., Chen, A . P.-C., Wang, A . H.-J., and Liang, P.-H . (2001) J . Biol . Chem . 276, 47474-47482), also became visible in one subunit . The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site . The Mg2+ is chelated by His-199 and Glu-213 from different subunits and possibly plays a structural rather than catalytic role . However, the metal ion is near the IPP-binding site, and double mutation of His-199 and Glu-213 to alanines showed a remarkable increase of Km value for IPP . Inside the tunnel, one Triton surrounds the top portion of the tunnel, and the other occupies the bottom part . These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site . Kinetic analysis indicated that a high concentration (>1%) of Triton inhibits the enzyme activity.

J Cell Biol, 2003 May 26, 161(4), 679 - 84 Epub 2003 May 19.
Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome; Ullers RS et al.; As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell . In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome . The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs) . Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP . Both TF and SRP were found to interact with the SA with partially overlapping binding specificity . In addition, extensive contacts with L23 and L29 were detected . Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP . The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

Drug Metab Dispos, 2003 Jun, 31(6), 697 - 700
Enantioselectivity of human hydroxysteroid sulfotransferase ST2A3 with naphthyl-1-ethanols; Sheng JJ et al.; Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of several endogenous steroids and many hydrophobic xenobiotic alcohols . The substrate stereoselectivities of sulfotransferases may be critically important in determining their overall roles in metabolism of drugs, carcinogens, and other xenobiotics . In the present work, stereoselectivity of the human hydroxysteroid sulfotransferase ST2A3 (also variously named as SULT2A1 or human DHEA-ST) was examined through analysis of its catalytic activities with the enantiomers of 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol . The kcat/Km value for sulfation of the R-(+)-enantiomer of 1-naphthyl-1-ethanol catalyzed by ST2A3 was 3.3 min-1mM-1, whereas the S-(-)-enantiomer was not a substrate for the enzyme . S-(-)-1-naphthyl-1-ethanol did however interact with ST2A3 as an inhibitor of the sulfation of dehydroepiandrosterone . This substrate stereospecificity was not present with the enantiomers of 2-naphthyl-1-ethanol, since both were substrates for the enzyme . Such differences between the sulfation of 1- and 2-naphthyl-1-ethanol are consistent with the importance of steric interactions between the ethanol group and a hydrogen atom at the peri-position (C8) on the naphthyl ring in 1-naphthyl-1-ethanol that combine with the topology of the enzyme's active site to determine stereospecificity.

J Vet Pharmacol Ther, 2003 Jun, 26(3), 225 - 31
Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs; Bozic F et al.; The present study tested the hypothesis that levamisole exerts its immunopotentiating activity in weaned pigs vaccinated against colibacillosis by priming the lymphocytes and macrophages in the mesenteric lymph node (MLN) . Ten weaned piglets were used and allocated into two equal groups . The experimental group was intramuscularly primed with levamisole at an immunostimulatory dose of 2.5 mg/kg given daily, in three consecutive days, and controls received saline according to the same schedule . Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0 and challenged with the virulent E . coli strain 7 days later . All pigs were killed on postchallenge day 6 . Upon virulent challenge the health status of the two groups was evaluated by clinical observations, and expression of CD25, SWC7 and SWC9 activation antigens by MLN and spleen T and B cells and macrophages, respectively, was tested using flow cytometry . Priming by levamisole significantly contributed to the effectiveness of a live attenuated oral vaccine against porcine postweaning colibacillosis, as evidenced by a good health status of primed vaccinated vs . un-primed vaccinated pigs . The CD3+, CD25+ and SWC9+ MLN but not spleen T cells and macrophages increased in experimental vs . control pigs, implying that levamisole exerts its potentiating activity in the MLN by augmenting both recruitment and activation of cells that participate in cell-mediated immunity.

J Vet Pharmacol Ther, 2003 Jun, 26(3), 159 - 64
Influence of endotoxin on the disposition kinetics and dosage regimens of oxytetracycline in calves; Kumar R et al.; The influence of endotoxin on the disposition kinetics of oxytetracycline (OTC) (10 mg/kg) was investigated in five healthy ruminating male crossbred calves . The serum concentration-time data of OTC before and after endotoxin challenge were best described by a two-compartment open model . Repeated administration of Escherichia coli endotoxin (1 microg/kg, i.v.) at an interval of 12 h up to 48 h produced a clear rise in the body temperature and an increase in the pulse and respiration rates . Endotoxin caused a significant reduction in mean transit time in tissue compartment (MTTT) (P < or = 0.05), mean residence time in the peripheral tissue compartment (MRTT) (P < or = 0.05), mean residence time in the body (MRTB) (P < or = 0.05), elimination half-life (t1/2lambda2) (P < or = 0.05) and distribution space in tissues (VT) (P < or = 0.01) and at steady-state (Vd(ss)) (P < or = 0.01) . Endotoxin had no effect on the distribution clearance (ClD), systemic clearance (Cl) and distribution half-life of OTC, while the values of first order rate constant of transfer of drug from tissue to central compartment (K21) and the zero time intercept at terminal phase (C2) were significantly high . The drug dosage regimens to maintain serum OTC concentrations of 0.5, 1, 2, 4, 6 and 8 microg/mL were also determined in febrile and clinically healthy animals.

Mol Ecol, 2003 Jun, 12(6), 1681 - 5
Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers; De Marchis F et al.; Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment . Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants . In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined . Plants of Lotus corniculatus L . transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor . Nontransgenic plants belonging to the species L . corniculatus L., L . tenuis Waldst . and Kit . ex Willd, and L . pedunculatus Cav., were utilized as recipients . Two experimental fields were established in two areas of central Italy . Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker . No transgene flow between L . corniculatus transformants and the nontransgenic L . tenuis and L . pedunculatus plants was detected . As regards nontransgenic L . corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot . In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

Biochemistry, 2003 May 27, 42(20), 6283 - 92
Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli; Copik AJ et al.; To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared . Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data . Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme . These data indicate that the metal binding site has not been affected by altering H178 . The effect of altering H178 on activity is, in general, due to a decrease in k(cat) . The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA . The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A . The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction . The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I . Therefore, H178A is catalytically important but is not required for catalysis . Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I . Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule . The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.

Biochemistry, 2003 May 27, 42(20), 6241 - 8
BmSPN2, a serpin secreted by the filarial nematode Brugia malayi, does not inhibit human neutrophil proteinases but plays a noninhibitory role; Stanley P et al.; The filarial nematode, Brugia malayi, is a causative agent of lymphatic filariasis . Bm-spn-2, one of two serpin genes identified in B . malayi, is expressed only in humans where the encoded protein, BmSPN2, is secreted by blood-dwelling microfilariae . Previous work reported that BmSPN2 could inhibit the activities of elastase and cathepsin G from human neutrophils, despite an atypical amino acid sequence . This did not fit with accepted theories as to the sequence requirements of serpins for proteinase inhibition . We have cloned and expressed Bm-spn-2 in Escherichia coli and characterized the structural and functional properties of recombinant BmSPN2 . Sequence alignment, circular dichroism spectroscopy, and susceptibility to cleavage by proteinases all suggest that BmSPN2 shares the tertiary structure typical of the serpin family including an accessible reactive center loop . However, we have found that BmSPN2 has no effect on the activity of neutrophil elastase or cathepsin G and does not form SDS-stable complexes with these proteinases . We provide evidence that BmSPN2 cannot undergo the characteristic stressed to relaxed transition required for proteinase inhibition by serpins . We conclude that BmSPN2 is not an atypical inhibitor but is a new noninhibitory serpin, in keeping with its sequence.

Biochemistry, 2003 May 27, 42(20), 6115 - 20
Binding of phlorizin to the isolated C-terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence; Xia X et al.; Phlorizin, a phloretin 2'-glucoside, is a potent inhibitor of the Na(+)/glucose cotransporter (SGLT1) . On the basis of transport studies in intact cells, a binding site for phlorizin was suggested in the region between amino acids 604-610 of the C-terminal loop 13 . To further investigate phlorizin binding titration experiments of the intrinsic Trp fluorescence of isolated wild-type loop 13 and two mutated loops (Y604K and G609K) were carried out . Phlorizin (135 microM) produced approximately 40% quenching of the fluorescence of wild-type loop 13; quenching could also be observed with the two mutated loops . The apparent K(d) was lowest for the wild-type loop 13 (K(d) approximately 23 microM), followed by mutant G609K (57 microM) and mutant Y604K (70 microM) . Binding of phlorizin was further confirmed by a decrease of the accessibility of loop 13 to the collisional quencher acrylamide . The interaction involves the aromatic moiety of the aglucone since phloretin (the aglucone of phlorizin) showed almost the same effects as phlorizin, while d-glucose did not . MALDI-TOF experiments revealed that loop 13 contained a disulfide bond between Cys 560 and Cys 608 that is very important for phlorizin-dependent fluorescence quenching . These studies provide direct evidence that loop 13 is a site (important amino acids including 604-609) for the molecular interaction between SGLT1 and phlorizin . They confirm that the aglucone part of the glucoside is responsible for this interaction.

Biochemistry, 2003 May 27, 42(20), 6099 - 105
Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1; Koteiche HA et al.; Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE . For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1) . This segment has been implicated in the catalytic mechanism of EmrE due to the presence of the highly conserved glutamate 14, a residue absolutely required for ligand binding . Sequence-specific variation in the accessibilities of the introduced nitroxides to molecular oxygen reveals a transmembrane helical conformation along TMS1 . One face of the helix is in contact with the hydrocarbon interior of the detergent micelle while the other face appears to be solvated by an aqueous environment, resulting in significant exposure of the nitroxides along this face to NiEDDA . TMS1 from two different subunits are in close proximity near a 2-fold axis of symmetry as revealed by the analysis of spin-spin interactions at sites 14 and 18 . The limited extent of spin-spin interactions is consistent with a scissor-like packing of the two TMS1 . This results in a V-shaped chamber which is in contact with the aqueous phase near the N-terminus . The spatial organization of TMS1, particularly the close proximity of E14, is consistent with a proposed mechanistic model of EmrE {Yerushalmi, H., and Schuldiner, S . (2000) Biochemistry 39, 14711-14719} where substrate extrusion is coupled to proton influx through electrostatic interactions and shifts of the glutamate 14 pK(a) during the cycle.

Biochemistry, 2003 May 27, 42(20), 6090 - 8
Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase; Bok D et al.; Lecithin retinol acyltransferase (LRAT) catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester, an essential reaction in the vertebrate visual cycle . Since all-trans-retinyl esters are the substrates for the isomerization reaction that generates 11-cis-retinoids, this esterification reaction is essential in the operation of the visual cycle . In addition, LRAT is the founder member of a series of proteins, which are of novel sequence and have unknown functions . Native LRAT is an integral membrane protein and has never been purified . To obtain a pure LRAT, the N- and C-transmembrane termini were deleted and replaced with a poly His tag for the purpose of purification . This truncated form of LRAT, referred to as tLRAT, has been expressed in bacteria and fully purified . tLRAT is catalytically active and processes all-trans-retinol at least 10-fold more efficiently than 11-cis-retinol, the precursor to the visual chromophore . While tLRAT can be robustly expressed in bacteria, it requires detergent for extraction, as the enzyme still contains hydrophobic domains, which may interact . Indeed, tLRAT can oligomerize and forms dimers . Native LRAT also forms functional homodimers . These studies pave the way for the preparation of large-scale amounts of pure tLRAT for further mechanistic and structural studies.

Biochemistry, 2003 May 27, 42(20), 6043 - 56
Rational design, synthesis, evaluation, and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid; Zhang Y et al.; Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades . Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined . Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials . Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase . Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation . The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate . The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site . Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

Antonie Van Leeuwenhoek, 2003, 83(1), 35 - 43
An Azospirillum brasilense Tn5 mutant with modified stress response and impaired in flocculation; Galindo Blaha CA et al.; The analysis of an A . brasilense Tn5 mutant shows significant phenotypic differences compared to the wild type isogenic strain . The transposon was located disrupting an open reading frame of 840 bp (ORF280) which exhibits similarity to the universal stress protein (USP) family . The USP family encompasses proteins that are expressed as a response to cell growth arrest . The mutant revealed a pleiotrophic phenotype with respect to different stress conditions . The ORF mutation results in an increased sensitivity of cells to carbon starvation and heat-shock treatment . However, the mutant strain displays a higher tolerance to oxidative stress agents . In contrast to the isogenic parent strain, colonies of the mutant are weakly stained by Congo red added to solid media and are impaired in flocculation . Scanning electron micrographs revealed that the mutant lacks part of the surface material present as a thick layer of exopolysaccharides on the surface of the wild type cells . The pleiotrophic phenotype revealed for this mutant and the similarity of the C-terminal region of ORF280 to UspA from E . coli indicates that the A . brasilense ORF280 may be a Usp-like protein.

C R Biol, 2003 Feb, 326(2), 215 - 7
Hardware (DNA) circuits; D'Ari R et al.; A scheme is presented whereby a new genetic control circuit can be introduced into an organism, permitting the experimenter to turn the expression of a given gene (or set of genes) on or off at will . The proposed scheme involves a positive feedback loop--here, a positive regulator, the CII protein of phage lambda, with its structural gene engineered so as to require CII for its expression . This feedback loop creates the possibility of two stable steady states, with gene cII ON or OFF . Genes added downstream of cII and lacking a promoter will follow the same expression as cII . Two additional circuits allow the experimenter to switch at will between the ON and OFF states.

Exp Mol Med, 2003 Apr 30, 35(2), 106 - 12
Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli; Lee YS et al.; 8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species . In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA . In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro . Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini . Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E . coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis . DNA polymerase and DNA ligase then completed the repair . These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.

J Lipid Res, 2003 Aug, 44(8), 1489 - 98 Epub 2003 May 16.
A single intravenous dose of endotoxin rapidly alters serum lipoproteins and lipid transfer proteins in normal volunteers; Hudgins LC et al.; Endotoxemia is associated with rapid and marked declines in serum levels of LDL and HDL by unknown mechanisms . Six normal volunteers received a single, small intravenous (iv) dose of endotoxin (Escherichia coli 0113, 2 ng/kg) or saline in a random order, cross-over design . After endotoxin treatment, volunteers had mild, transient flu-like symptoms and markedly increased serum levels of tumor necrosis factor and its soluble receptors, interleukin-6, cortisol, serum amyloid A, and C-reactive protein . Triglyceride (TG), VLDL-TG, and nonesterified fatty acid increased (peak at 3-4 h), then TG declined (nadir at 9 h), and then cholesterol, LDL cholesterol, apolipoprotein B (apoB), and phospholipid declined (nadirs at 12-24 h) . HDL cholesterol and apoA-I levels were not affected, but half of the decrease in phospholipid was HDL phospholipid . Lipopolysaccharide binding protein (LBP) rose 3-fold (peak at 12 h), with smaller and later decreases in the activities of phospholipid transfer protein and cholesteryl ester transfer protein . In conclusion, a decline in LDL was rapidly induced in normal volunteers with a single iv dose of endotoxin . The selective loss of phospholipid from HDL may have been mediated by LBP and, after more intense or prolonged inflammation, could result in increased HDL clearance and reduced HDL levels.

J Biol Chem, 2003 Jul 25, 278(30), 27630 - 5 Epub 2003 May 16.
Certain metals trigger fibrillation of methionine-oxidized alpha-synuclein; Yamin G et al.; The aggregation and fibrillation of alpha-synuclein has been implicated as a key step in the etiology of Parkinson's disease and several other neurodegenerative disorders . In addition, oxidative stress and certain environmental factors, including metals, are believed to play an important role in Parkinson's disease . Previously, we have shown that methionine-oxidized human alpha-synuclein does not fibrillate and also inhibits fibrillation of unmodified alpha-synuclein (Uversky, V . N., Yamin, G., Souillac, P . O., Goers, J., Glaser, C . B., and Fink, A . L . (2002) FEBS Lett . 517, 239-244) . Using dynamic light scattering, we show that the inhibition results from stabilization of the monomeric form of Met-oxidized alpha-synuclein . We have now examined the effect of several metals on the structural properties of methionine-oxidized human alpha-synuclein and its propensity to fibrillate . The presence of metals induced partial folding of both oxidized and non-oxidized alpha-synucleins, which are intrinsically unstructured under conditions of neutral pH . Although the fibrillation of alpha-synuclein was completely inhibited by methionine oxidation, the presence of certain metals (Ti3+, Zn2+, Al3+, and Pb2+) overcame this inhibition . These findings indicate that a combination of oxidative stress and environmental metal pollution could play an important role in triggering the fibrillation of alpha-synuclein and thus possibly Parkinson's disease.

J Bacteriol, 2003 Jun, 185(11), 3469 - 72
Error-prone polymerase, DNA polymerase IV, is responsible for transient hypermutation during adaptive mutation in Escherichia coli; Tompkins JD et al.; The frequencies of nonselected mutations among adaptive Lac(+) revertants of Escherichia coli strains with and without the error-prone DNA polymerase IV (Pol IV) were compared . This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain . Thus, the mutations that occur during hypermutation are due to Pol IV.

J Bacteriol, 2003 Jun, 185(11), 3436 - 45
Open reading frame sso2387 from the archaeon Sulfolobus solfataricus encodes a polypeptide with protein-serine kinase activity; Lower BH et al.; The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily . sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli . The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea . The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues . The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases . By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII . Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme . Autophosphorylation was detected only at temperatures >or=60 degrees C, whereas phosphorylation of exogenous proteins was detectable at 37 degrees C . Similarly, replacement of one of the potential sites of autophosphorylation, Ser(548), with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

J Bacteriol, 2003 Jun, 185(11), 3429 - 35
Cysteine-scanning analysis of the dimerization domain of EnvZ, an osmosensing histidine kinase; Qin L et al.; EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli . The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B . Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A . The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized . From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity . Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A . The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation . Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities . The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.

J Bacteriol, 2003 Jun, 185(11), 3410 - 5
Role of feedback regulation of pantothenate kinase (CoaA) in control of coenzyme A levels in Escherichia coli; Rock CO et al.; Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters . The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex . CoaA{R106A}, CoaA{H177Q}, and CoaA{F247V} were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA . CoaA{R106A} retained 50% of the catalytic activity of CoaA, whereas the CoaA{H177Q} and CoaA{F247V} mutants were less active . The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA{R106A} in strain ANS3 {coaA15(Ts) panD2} . Cells expressing CoaA{R106A} had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein . These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.

J Bacteriol, 2003 Jun, 185(11), 3344 - 51
Concentration and assembly of the division ring proteins FtsZ, FtsA, and ZipA during the Escherichia coli cell cycle; Rueda S et al.; The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique . Immunostaining of the three proteins revealed no organized structure in newly born cells . In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction . FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ . The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins . Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.

J Biol Chem, 2003 Aug 8, 278(32), 29837 - 55 Epub 2003 May 15.
Global gene expression profiling in Escherichia coli K12 . The effects of oxygen availability and FNR; Salmon K et al.; The work presented here is a first step toward a long term goal of systems biology, the complete elucidation of the gene regulatory networks of a living organism . To this end, we have employed DNA microarray technology to identify genes involved in the regulatory networks that facilitate the transition of Escherichia coli cells from an aerobic to an anaerobic growth state . We also report the identification of a subset of these genes that are regulated by a global regulatory protein for anaerobic metabolism, FNR . Analysis of these data demonstrated that the expression of over one-third of the genes expressed during growth under aerobic conditions are altered when E . coli cells transition to an anaerobic growth state, and that the expression of 712 (49%) of these genes are either directly or indirectly modulated by FNR . The results presented here also suggest interactions between the FNR and the leucine-responsive regulatory protein (Lrp) regulatory networks . Because computational methods to analyze and interpret high dimensional DNA microarray data are still at an early stage, and because basic issues of data analysis are still being sorted out, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence . In particular, we describe an approach for identifying gene expression patterns (clusters) obtained from multiple perturbation experiments based on a subset of genes that exhibit high probability for differential expression values.

J Biol Chem, 2003 Aug 8, 278(32), 29901 - 12 Epub 2003 May 16.
A bipartite mechanism for ERK2 recognition by its cognate regulators and substrates; Zhang J et al.; Mitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates . We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins . We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2 . Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation . MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site . Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation . In addition to the shared residues, there are also residues that are unique to each target recognition . There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive . Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process . In this model, one part of the ERK2-binding proteins (e.g . the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g . the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules.

J Biol Chem, 2003 Aug 8, 278(32), 29701 - 9 Epub 2003 May 15.
Thermoirreversible and thermoreversible promoter opening by two Escherichia coli RNA polymerase holoenzymes; Kamali-Moghaddam M et al.; Promoter opening, in which the complementary DNA strands separate around the transcriptional start site, is generally thermoreversible . An exceptional case of thermoirreversible opening of the T4 late promoter has been analyzed by KMnO4 footprinting and transcription . T4 late promoters, which consist of an 8-base pair (bp) TATA box "-10" element, are recognized by the small, phage-encoded, highly diverged sigma-family initiation subunit gp55 . The T4 late promoter only opens above 15-20 degrees C, but once it has been formed remains open and transcriptionally active for days at -0.5 degrees C . The low temperature-trapped open complex and its isothermally formed state are shown to be structurally distinctive . Two "extended -10" sigma 70 promoters, which, like the T4 late promoter, lack "-35" sites, have been subjected to a comparative analysis: the T4 middle promoter PrIIB2 opens and closes thermoreversibly under conditions of basal and MotA- and AsiA-activated transcription . The open galP1 promoter complex, whose transcription bubble is very AT-rich, also closes reversibly upon shift to -0.5 degrees C, but more slowly than does the rIIB2 promoter . Formation of a trapped-open low temperature state of the promoter complex appears to be a singular property of gp55-RNA polymerase holoenzyme.

Biochem Pharmacol, 2003 May 15, 65(10), 1685 - 90
Enzymatic characterization of recombinant mouse retinal dehydrogenase type 1; Gagnon I et al.; Retinal dehydrogenases (RALDHs) convert retinal into retinoic acids (RAs), which are important signaling molecules in embryogenesis and tissue differentiation . We expressed mouse RALDH type 1 (mRALDH1) in Escherichia coli and studied the kinetic properties of the recombinant enzyme for retinal substrates . Purified recombinant mRALDH1 catalyzed the oxidation of all-trans and 9-cis retinal but not 13-cis retinal, and exhibited two pH optimums, 7.8 and 9.4, for all-trans and 9-cis retinal substrates, respectively . The K(m) for all-trans retinal (11.6 micro M) was 3-fold higher than for 9-cis retinal (3.59 micro M) . However, the conversion efficiencies of either all-trans or 9-cis retinal to the respective RAs were similar . MgCl(2) inhibited the oxidation of both all-trans and 9-cis retinal . Chloral hydrate and acetaldehyde competitively suppressed all-trans retinal oxidation with inhibition constants (K(i)) of 4.99 and 49.4 micro M, respectively . Retinol, on the other hand, blocked the reaction uncompetitively . These data extend the kinetic characterization of mRALDH1, provide insight into the possible role of this enzyme in the biogenesis of RAs, and should give useful information on the determination of amino acid residues that play crucial roles in the catalysis of all-trans and 9-cis retinal.

FEBS Lett, 2003 May 22, 543(1-3), 164 - 9
Crystal structure of DsbDgamma reveals the mechanism of redox potential shift and substrate specificity(1); Kim JH et al.; The Escherichia coli transmembrane protein DsbD transfers electrons from the cytoplasm to the periplasm through a cascade of thiol-disulfide exchange reactions . In this process, the C-terminal periplasmic domain of DsbD (DsbDgamma) shuttles the reducing potential from the membrane domain (DsbDbeta) to the N-terminal periplasmic domain (DsbDalpha) . The crystal structure of DsbDgamma determined at 1.9 A resolution reveals that the domain has a thioredoxin fold with an extended N-terminal stretch . In comparison to thioredoxin, the DsbDgamma structure exhibits the stabilized active site conformation and the extended active site alpha2 helix that explain the domain's substrate specificity and the redox potential shift, respectively . The hypothetical model of the DsbDgamma:DsbDalpha complex based on the DsbDgamma structure and previous structural studies indicates that the conserved hydrophobic residue in the C-X-X-C motif of DsbDgamma may be important in the specific recognition of DsbDalpha.

J Autoimmun, 2003 May, 20(3), 255 - 63
Promiscuous T cells selected by Escherichia coli: OGDC-E2 in primary biliary cirrhosis; Tanimoto H et al.; The etiology of primary biliary cirrhosis (PBC) remains enigmatic . One theory that has attracted attention proposes that PBC is induced via molecular mimicry with Escherichia coli . If molecular mimicry is responsible for the immunogenic response in PBC, then T cell clones specific for E . coli antigens should stimulate and be cross-reactive with peptides specific for the human immunodominant autoepitopes . To address this issue, we developed T cell clones specific for E . coli OGDC-E2 peptide . Importantly, we demonstrate the presence of T cell clones specific for E . coli OGDC-E2 that react promiscuously with the human mitochondrial equivalents . Indeed, there was a significant increase in the liver derived T cell precursor frequency of such reactivity and such liver clones were only found in patients with PBC . In conclusion, these data suggest that PBC is a multi-hit disease involving a genetic predisposition, a mucosal response, and activation of promiscuous T cells; such activation may occur either directly from bacterial antigens, or indirectly through chemically-modified bacterial antigens . Dissection of the mechanisms involved will lead not only to understanding the immunogenetic basis of PBC, but likely its pathogenic etiology.

Vet Immunol Immunopathol, 2003 May 30, 93(1-2), 69 - 79
Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay; Funke C et al.; Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response . As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value . A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli . Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma . Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6 . Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6 . The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6 . A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml . The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin . The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure.

Pigment Cell Res, 2003 Jun, 16(3), 273 - 9
Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability; Farmer PJ et al.; Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes . We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within . Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species . The electron paramagnetic resonance (EPR)-active 5,5'-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals . A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress . Melanoma cultures grown under 95% O2/5% CO2 atmospheres show markedly reduced viability as compared with normal melanocytes . Cu- and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity . The antimelanoma effect of metal- and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes.

Mol Microbiol, 2003 May, 48(4), 1005 - 16
Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli; Inoue K et al.; Era is a small GTP-binding protein and essential for cell growth in Escherichia coli . It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains . It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro . Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells . The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain . The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA . Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells . The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits . RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain . The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells . Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era . The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era . It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells . These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.

Mol Microbiol, 2003 May, 48(4), 947 - 58
Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption; Canizalez-Roman A et al.; We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects . Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres . Pet effects require its internalization into epithelial cells . We have also shown that Pet degrades erythroid spectrin . Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin) . Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells . When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs . Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa . Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain . Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet . Pet also cleaves epithelial fodrin from cultured Pet-treated cells . A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin . This is the first report showing cleavage of alpha-fodrin by a bacterial protease . Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.

Mol Microbiol, 2003 May, 48(4), 863 - 74
Region 4 of sigma as a target for transcription regulation; Dove SL et al.; Bacterial sigma factors play a key role in promoter recognition, making direct contact with conserved promoter elements . Most sigma factors belong to the sigma70 family, named for the primary sigma factor in Escherichia coli . Members of the sigma70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation . We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of sigma.

Mol Microbiol, 2003 May, 48(4), 855 - 61
Small non-coding RNAs, co-ordinators of adaptation processes in Escherichia coli: the RpoS paradigm; Repoila F et al.; Adaptation to the changing environment requires both the integration of external signals and the co-ordination of internal responses . Around 50 non-coding small RNAs (sRNAs) have been described in Escherichia coli; the levels of many of these vary with changing environmental conditions . This suggests that they play a role in cell adaptation . In this review, we use the regulation of RpoS (sigma38) translation as a paradigm of sRNA-mediated response to environmental conditions; rpoS is currently the only known gene regulated post-transcriptionally by at least three sRNAs . DsrA and RprA stimulate RpoS translation in response to low temperature and cell surface stress, respectively, whereas OxyS represses RpoS translation in response to oxidative shock . However, in addition to regulating RpoS translation, DsrA represses the translation of HNS (a global regulator of gene expression), whereas OxyS represses the translation of FhlA (a transcriptional activator), allowing the cell to co-ordinate different pathways involved in cell adaptation . Environmental cues affect the synthesis and stability of specific sRNAs, resulting in specific sRNA-dependent translational control.

Eur J Biochem, 2003 May, 270(10), 2236 - 43
Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin; Lagarrigue E et al.; Gelsolin is an abundant calcium dependent actin filament severing and capping protein . In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites . It is generally held that a 'helical-latch' at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch . Here we provide evidence for a calcium dependent conformational change within G2 (Kd = approximately 15 micro m) . We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193 . It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch . We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6 . Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.

Eur J Biochem, 2003 May, 270(10), 2195 - 206
Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase . Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis; Simard D et al.; Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen . When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis . Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis . Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity . Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP) . The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP . K187A was able to form tetramers in the presence of UTP and ATP . Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain . Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage . The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation . For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered . Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold . This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.

Eur J Biochem, 2003 May, 270(10), 2186 - 94
An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase; Hemmi H et al.; (All-E) prenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphates with allylic prenyl diphosphates, producing products with various chain-lengths that are unique for each enzyme . Some short-chain (all-E) prenyl diphosphate synthases, i.e . farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product . However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III geranylgeranyl diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features . In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae . This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme . We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity . These results provide valuable information on the molecular evolution of (all-E) prenyl diphosphate synthases.

Allergy, 2003 May, 58(5), 412 - 9
Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11); Ramos JD et al.; BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously . This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms . METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS . Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli . Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test . The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies . RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD . The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11 . Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart . CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11 . This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.

APMIS, 2003 Mar, 111(3), 389 - 97
Recombinant HpaA purified from Escherichia coli has biological properties similar to those of native Helicobacter pylori HpaA; Lundstrom AM et al.; The aim of this study was to recombinantly produce and purify Helicobacter pylori adhesin A (HpaA) from Escherichia coli and compare it to purified native H . pylori HpaA, for potential use as a vaccine antigen . The hpaA gene was cloned from H . pylori, transferred to two different expression vectors, and transformed into E . coli . Expression of rHpaA was analysed by immunoblot, inhibition ELISA, and semi-quantitative dot-blot . Using affinity chromatography, rHpaA was purified from E . coli and native HpaA from H . pylori . The binding of both purified proteins to sialic acid was analysed and antibody titres to native and rHpaA were compared after intraperitoneal immunisation of C57/Bl mice . The rHpaA protein was highly expressed in E . coli from both vectors . Purified recombinant and native HpaA bound similarly to fetuin but also to the non-sialylated asialofetuin . Both native HpaA and rHpaA induced comparable amounts of specific antibodies in serum after immunisation and they were identical in double immunodiffusion . In conclusion, rHpaA was successfully produced in E . coli . Purified rHpaA showed biological properties similar to those of native HpaA isolated from H . pylori and may therefore be further used as an antigen in the development of a vaccine against H . pylori infection.

Br J Haematol, 2003 May, 121(4), 653 - 6
Endotoxaemia modulates Toll-like receptors on leucocytes in humans; Marsik C et al.; The modulation of Toll-like receptors (TLR) 1, 2 and 4 was studied during experimental human endotoxaemia . Healthy volunteers received 2 ng/kg of lipopolysaccharide (LPS) endotoxin (n = 10) . TLR1, 2 and 4 expression occurred on monocytes and neutrophils, with monocytes expressing higher baseline levels of TLR2 . LPS infusion downmodulated TLR4 expression on neutrophils, with maximal downregulation occurring at 24 h (-62% from baseline; P < 0.03 versus baseline) . Monocyte TLRs were upregulated in vivo (TLR1 and 2), and in vitro (TLR1, 2 and 4) 8 h after LPS bolus (P < 0.05 versus baseline) . Therefore, neutrophils and monocytes differentially express surface TLRs, and endotoxaemia differentially regulates TLR expression.

Biol Chem, 2003 Apr, 384(4), 673 - 9
Solvent isotope effect on the reaction catalysed by the pyruvate dehydrogenase complex from Escherichia coli; Liu X et al.; The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide . The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction . The pH profile and, accordingly, the delta pK1 and delta pK2 values were shifted by 0.6 units to higher pL values . A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0% . This is an indication for a single proton transfer . It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1' nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue . The proton of its carboxylic group exchanges very fast with deuterons of the solvent.

Biol Chem, 2003 Apr, 384(4), 653 - 6
Biosynthesis of trypanothione in Trypanosoma brucei brucei; Comini M et al.; Trypanothione {T(SH)2}, the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps . A full-length reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T . brucei as template and primers based on fragments of putative TryS genes . The recombinant protein produced by E . coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography . The enzyme catalyzed both reactions of T(SH)2 biosynthesis . Thus, T(SH)2 synthesis appears to be similar in African (T . brucei) and New World (T . cruzi) trypanosomes but distinct from that of Crithidia.

DNA Seq, 2003 Feb, 14(1), 71 - 4
Cloning and sequencing a HemK-family gene in Porphyromonas gingivalis; Kusaba A et al.; HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyltransferases (MTases) . In the present study, we sequenced a 5026 bp DNA fragment just downstream of the PgPepO gene reported previously . The DNA sequence analysis revealed three ORFs . The ORF2 gene encoded a protein of 294 amino acids with a calculated molecular weight of 32,160 Da . The deduced amino acid sequence of the ORF2 gene exhibited a significant similarity to sequence of HemK from E . coli (35% identical residues) . The ORF2 gene complemented an E . coli hemK mutant . Thus, ORF2 was named PgHemK . From the point of veiw of our recent finding, that E . coli HemK catalyses the methylation of polypeptide chain release factors such as RF1 and RF2, we postulated that PgHemK might function as a protein MTase containing the DNA MTase motif.

Science, 2003 May 16, 300(5622), 1159 - 62
Catalysis of ribosomal translocation by sparsomycin; Fredrick K et al.; During protein synthesis, transfer RNAs (tRNAs) are translocated from the aminoacyl to peptidyl to exit sites of the ribosome, coupled to the movement of messenger RNA (mRNA), in a reaction catalyzed by elongation factor G (EF-G) and guanosine triphosphate (GTP) . Here, we show that the peptidyl transferase inhibitor sparsomycin triggers accurate translocation in vitro in the absence of EF-G and GTP . Our results provide evidence that translocation is a function inherent to the ribosome and that the energy to drive this process is stored in the tRNA-mRNA-ribosome complex after peptide-bond formation . These findings directly implicate the peptidyl transferase center of the 50S subunit in the mechanism of translocation, a process involving large-scale movement of tRNA and mRNA in the 30S subunit, some 70 angstroms away.

J Biol Chem, 2003 Aug 1, 278(31), 28976 - 84 Epub 2003 May 14.
Structure and ubiquitin binding of the ubiquitin-interacting motif; Fisher RD et al.; Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps) . Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules . Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM) . We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin . Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues . NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin . The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix . Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

J Biol Chem, 2003 Aug 8, 278(32), 29728 - 43 Epub 2003 May 15.
Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element; Burrows PC et al.; The sigma 54 promoter specificity factor is distinct from sigma 70-type factors . The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes . The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70 . Here, sigma 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of sigma 54 is close to and may therefore interact with the consensus -24 promoter element . We show that the spatial relationship between the sigma 54-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase . In contrast, the spatial relationship between sigma 54-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one . We provide evidence that some -12 promoter region-sigma 54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP . We also show the beta-subunit flap domain contributes to different sets of sigma-promoter DNA interactions at sigma 54- and sigma 70-dependent promoters.

J Biol Chem, 2003 Aug 1, 278(31), 28635 - 43 Epub 2003 May 15.
Characterization of the final two genes of the gibberellin biosynthesis gene cluster of Gibberella fujikuroi: des and P450-3 encode GA4 desaturase and the 13-hydroxylase, respectively; Tudzynski B et al.; Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined . Here we describe the function of the sixth gene, P450-3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster . We have thereby defined the complete GA biosynthesis gene cluster in this fungus . The predicted amino acid sequence of orf3 revealed no close homology to known proteins . High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA1 and GA4, rather than GA3 and GA7, as the major C19-GA products, suggesting that orf3 encodes the GA4 1,2-desaturase . This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des . The transformants converted GA4 to GA7, and also metabolized GA9 (3-deoxyGA4) to GA120 (1,2-didehydroGA9), but the 2alpha-hydroxylated compound GA40 was the major product in this case . We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA7 to GA3, in the last step of the pathway . This enzyme also catalyzes the 13-hydroxylation of GA4 to GA1 . Disruption of the des gene in an UV-induced P450-3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA4 . The des and P450-3 genes differ in their regulation by nitrogen metabolite repression . In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen.

Plasmid, 2003 May, 49(3), 193 - 204
Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals; Mannik A et al.; The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals . Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system . We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector . The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid . This method resulted in successful production of transgenic animals . Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50% . The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.

Hepatogastroenterology, 2003 Mar-Apr, 50(50), 507 - 9
Infected hepatic cyst; Yoshida H et al.; We describe an unusual case involving an infected hepatic cyst . An 88-year-old woman presented with acute onset of right upper quadrant abdominal pain, mild left lower abdominal pain, diarrhea, and fever . On admission, computed tomography revealed multiple hepatic cysts including an 8-cm cyst located in the left medial segment of the liver, which demonstrated a thickened wall enhanced with contrast media . Ultrasonography showed an 8-cm hypoechoic lesion which differed in appearance from the other, anechoic hepatic cysts . The serum concentration of C-reactive protein was 29.8 mg/dL; white blood cell count, 12,800/microL; CA19-9, 96 U/mL; and CEA, 2.2 ng/mL . Diagnosis of infected hepatic cyst was made by percutaneous transhepatic drainage of the cyst . Milky fluid was obtained and the patient's right upper quadrant abdominal pain resolved after drainage . The cyst fluid CA19-9 concentration was 18,000 U/mL . Cytology of the cyst fluid was negative . Serum CA19-9 (41 U/mL) and CEA (1.8 ng/mL) concentrations were improved 1 week after drainage . Escherichia coli was cultured from the drainage fluid . The patient was discharged 27 days after admission . Percutaneous transhepatic drainage is effective in the treatment of infected hepatic cysts.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6849 - 54 Epub 2003 May 14.
Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana; Schluepmann H et al.; Genes for trehalose metabolism are widespread in higher plants . Insight into the physiological role of the trehalose pathway outside of resurrection plant species is lacking . To address this lack of insight, we express Escherichia coli genes for trehalose metabolism in Arabidopsis thaliana, which manipulates trehalose 6-phosphate (T6P) contents in the transgenic plants . Plants expressing otsA {encoding trehalose phosphate synthase (TPS)} accumulate T6P whereas those expressing either otsB {encoding trehalose phosphate phosphatase (TPP)} or treC {encoding trehalose phosphate hydrolase (TPH)} contain low levels of T6P . Expression of treF (encoding trehalase) yields plants with unaltered T6P content and a phenotype not distinguishable from wild type when grown on soil . The marked phenotype obtained of plants accumulating T6P is opposite to that of plants with low T6P levels obtained by expressing either TPP or TPH and consistent with a critical role for T6P in growth and development . Supplied sugar strongly inhibits growth of plants with reduced T6P content and leads to accumulation of respiratory intermediates . Remarkably, sugar improves growth of TPS expressors over wild type, a feat not previously accomplished by manipulation of metabolism . The data indicate that the T6P intermediate of the trehalose pathway controls carbohydrate utilization and thence growth via control of glycolysis in a manner analogous to that in yeast . Furthermore, embryolethal A . thaliana tps1 mutants are rescued by expression of E . coli TPS, but not by supply of trehalose, suggesting that T6P control over primary metabolism is indispensable for development.

J Med Microbiol, 2003 Jun, 52(Pt 6), 499 - 504
Genotypic and phenotypic characterization of attaching and effacing Escherichia coli (AEEC) isolated from children with and without diarrhoea in Londrina, Brazil; Nunes EB et al.; Attaching and effacing Escherichia coli (AEEC) have been implicated in diarrhoea in humans in several countries . A total of 919 E . coli strains, isolated from 125 children with diarrhoea and 98 without diarrhoea, was investigated by PCR for the presence of the EAF, bfp, eae and stx genes . Thirty-four of these isolates were found to carry the eae gene; they were isolated from 27 (79.4 %) children with diarrhoea and seven (20.6 %) controls, in the city of Londrina, Brazil . These strains were investigated for their genotypic and phenotypic characteristics . Different genetic profiles were observed; strains containing the eae gene alone were most common (47.1 %) . The characteristic genetic profile of typical enteropathogenic E . coli (EPEC), eae, bfp and EAF, was only found in isolates from children with diarrhoea . The stx gene was not detected in any of the 34 strains studied . Ten (29.4 %) strains were negative in the fluorescent actin-staining test . Localized adhesion (LA) was the most common pattern of adhesion (44.1 %), followed by the aggregative adhesion (AA) (23.5 %) and localized adhesion-like (LAL) (14.7 %) patterns . The results showed a strong association between strains presenting the LA pattern and diarrhoea . Forty-seven per cent of the strains studied belonged to classical O-serogroups of EPEC . The most common serotype found was O119 : H6; these isolates all showed the LA pattern, were positive for fluorescent actin-staining and were associated with diarrhoea . Intimin beta was detected in seven strains, four of which belonged to serotype O119 : H6 and three to serotype ONT : H7; all were associated with diarrhoea . On the other hand, intimin epsilon was detected in two strains of serotype O111 : H38 and one of serotype ONT : H19, isolated from children without diarrhoea . To our knowledge, this is the first report of the occurrence of intimin epsilon in strains of E . coli isolated from humans in Brazil.

J Med Microbiol, 2003 Jun, 52(Pt 6), 461 - 9
NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator; Cooksley C et al.; The Helicobacter pylori protein NapA has been identified as a homologue of the Escherichia coli protein Dps . It is shown in this study that, like Dps, NapA is produced maximally in stationary phase cells and contributes to the ability of H . pylori to survive under oxidative stress conditions . Moreover, NapA co-localizes with the nuclear material, suggesting that it can interact with DNA in vivo . Furthermore, it is demonstrated that repression of NapA production by iron starvation was not so pronounced in a H . pylori fur mutant, suggesting that the ferric uptake regulator (Fur) is involved in napA regulation, and a potential fur box by which this control could be mediated is identified . This finding is consistent with the regulation of iron-binding proteins by Fur and also the modulation of Fur during oxidative stress, thus allowing NapA levels to be increased in the environmental conditions under which its ability to protect DNA from attack by toxic free radicals is most beneficial to the cell.

J Biol Chem, 2003 Jul 25, 278(30), 27663 - 71 Epub 2003 May 14.
Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system; Gubbins MJ et al.; F-like plasmid transfer is mediated by the FinOP fertility inhibition system . Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO . FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer . FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails . Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro . In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number . Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation . Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.

J Biol Chem, 2003 Jul 25, 278(30), 27575 - 85 Epub 2003 May 13.
Regulation of the mhp cluster responsible for 3-(3-hydroxyphenyl)propionic acid degradation in Escherichia coli; Torres B et al.; The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP) . In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches . The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5'-ends . By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent . DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position -58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter . Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium . Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter . Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter.

J Biol Chem, 2003 Jul 25, 278(30), 28284 - 93 Epub 2003 May 14.
Characterization of the DNA damage-inducible helicase DinG from Escherichia coli; Voloshin ON et al.; The dinG promoter was first isolated in a genetic screen scoring for damage-inducible loci in Escherichia coli (Lewis, L . K., Jenkins, M . E., and Mount, D . W . (1992) J . Bacteriol . 174, 3377-3385) . Sequence analysis suggests that the dinG gene encodes a putative helicase related to a group of eukaryotic helicases that includes mammalian XPD (Koonin, E . V . (1993) Nucleic Acids Res . 21, 1497), an enzyme involved in transcription-coupled nucleotide excision repair and basal transcription . We have characterized the dinG gene product from E . coli using genetic and biochemical approaches . Deletion of dinG has no severe phenotype, indicating that it is non-essential for cell viability . Both dinG deletion and over-expression of the DinG protein from a multicopy plasmid result in a slight reduction of UV resistance . DinG, purified as a fusion protein from E . coli cells, behaves as a monomer in solution, as judged from gel filtration experiments . DinG is an ATP-hydrolyzing enzyme; single-stranded (ss) DNA stimulates the ATPase activity 15-fold . Kinetic data yield a Hill coefficient of 1, consistent with one ATP-hydrolyzing site per DinG molecule . DinG possesses a DNA helicase activity; it translocates along ssDNA in a 5' --> 3' direction, as revealed in experiments with substrates containing non-natural 5'-5' and 3'-3' linkages . The ATP-dependent DNA helicase activity of DinG requires divalent cations (Mg2+, Ca2+, and Mn2+) but is not observed in the presence of Zn2+ . The DinG helicase does not discriminate between ribonucleotide and deoxyribonucleotide triphosphates, and it unwinds duplex DNA with similar efficiency in the presence of ATP or dATP . We discuss the possible involvement of the DinG helicase in DNA replication and repair processes.

J Biol Chem, 2003 Aug 8, 278(32), 29487 - 95 Epub 2003 May 14.
Deficient regulation of DNA double-strand break repair in Fanconi anemia fibroblasts; Donahue SL et al.; Fibroblasts from patients with Fanconi anemia (FA) display genomic instability, hypersensitivity to DNA cross-linking agents, and deficient DNA end joining . Fibroblasts from two FA patients of unidentified complementation group also had significantly increased cellular homologous recombination (HR) activity . Results described herein show that HR activity levels in patient-derived FA fibroblasts of groups A, C, and G were 10-fold greater than those seen in normal fibroblasts . In contrast, HR activity in group D2 fibroblasts was identical to that in normal cells . Western blot analysis revealed that the RAD51 protein was elevated 10-fold above normal levels in group A, C, and G fibroblasts, but was not altered in group D2 fibroblasts . HR activity levels in these former cells could be restored to near-normal levels by electroporation with anti-RAD51 antibody, whereas similar treatment of normal and complementation group D2 fibroblasts had no effect . These findings are consistent with a model in which FA proteins function to coordinate DNA double-strand break repair activity by regulating both recombinational and non-recombinational DNA repair . Interestingly, whereas positive regulation of DNA end joining requires the combined presence of all FA proteins thus far tested, suppression of HR, which is minimally dependent on the FANCA, FANCC, and FANCG proteins, does not require FANCD2.

J Biol Chem, 2003 Aug 8, 278(32), 29420 - 34 Epub 2003 May 14.
Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression; Bourguignon LY et al.; In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line) . Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo . The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1) . Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3 . Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g . tumor cell growth, survival and invasion) are up-regulated . Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g . AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002) . Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g . Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g . cell growth, survival, and invasion) . Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.

J Biol Chem, 2003 Aug 1, 278(31), 28508 - 15 Epub 2003 May 14.
The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme; Crevillen P et al.; ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants . ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large) . In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity . The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties . Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates . No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome . This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate . Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation . This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species . This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.

J Biol Chem, 2003 Aug 1, 278(31), 28501 - 7 Epub 2003 May 14.
HU protein of Escherichia coli has a role in the repair of closely opposed lesions in DNA; Hashimoto M et al.; Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation . Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis . In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil . In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions . The repair of dihydrouracil is initiated by E . coli endonuclease III and processed via the base excision repair pathway . HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil . In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E . coli DNA polymerase and E . coli DNA ligase, respectively . The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E . coli.

Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 131 - 6
Enhanced expression of the gene for beta-glycosidase of Thermus caldophilus GK24 and synthesis of galacto-oligosaccharides by the enzyme; Choi JJ et al.; The gene (bglT) encoding Tca beta-glycosidase (Thermus caldophilus GK24 beta-glycosidase) was overexpressed under the control of the trp promoter on a high-copy-number plasmid, pTRPES, in Escherichia coli W3110 . The purified Tca beta-glycosidase enzyme was used in a galactosyl-transfer reaction to synthesize galacto-oligosaccharides from lactose . The optimum temperature and pH for the enzyme to synthesize galacto-oligosaccharides from 30% (w/v) lactose were 80 degrees C and 6.0, respectively . The major product of the reaction was a trisaccharide . The thermostable Tca beta-glycosidase produced galacto-oligosaccharides efficiently during the hydrolysis of lactose.

Mol Biotechnol, 2003 Jun, 24(2), 105 - 10
Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms; Zhang J et al.; DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays . Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3' ends . A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy . Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension . By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis . The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.

Plant Physiol, 2003 May, 132(1), 390 - 9
Expression of the plastid-located glutamine synthetase of Medicago truncatula . Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids; Melo PM et al.; In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2) . A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids . Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs . Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ . Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2) . This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules . Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive . Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope . Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules . These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.

Plant Physiol, 2003 May, 132(1), 372 - 80
Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants; Uefuji H et al.; Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps . In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively . The bacterially expressed encoded proteins were characterized for their catalytic properties . CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a K(m) value of 78 microM, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K(m) of 251 microM, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a K(m) of 1,222 microM . The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine . As a consequence, when all three recombinant proteins and E . coli extract were combined, xanthosine was successfully converted into caffeine in vitro . Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits . These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine --> 7-methylxanthosine --> 7-methylxanthine --> theobromine --> caffeine.

Plant Physiol, 2003 May, 132(1), 243 - 55
Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato; Tu CJ et al.; Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins . The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains . Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides . Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program . LapN was a single-copy gene encoding a rare-class transcript . A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A . Comparison of LAP-N with other plant LAPs identified 28 signature residues that classified LAP proteins as LAP-N or LAP-A like . Overexpression of a His(6)-LAP-N fusion protein in Escherichia coli demonstrated distinct differences in His(6)-LAP-N and His(6)-LAP-A activities . Similar to LapA, the LapN RNA encoded a precursor protein with a molecular mass of 60 kD . The 5-kD presequence had features similar to plastid transit peptides, and processing of the LAP-N presequence could generate the mature 55-kD LAP-N . Unlike LapA, the LapN transcript contained a second in-frame ATG, and utilization of this potential initiation codon would yield a 55-kD LAP-N protein . The localization of LAP-N could be controlled by the balance of translational initiation site utilization and LAP-N preprotein processing.

Plant Physiol, 2003 May, 132(1), 137 - 45
STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis, encodes disproportionating enzyme . Further evidence for a function of alpha-1,4 glucanotransferases during starch granule biosynthesis in green algae; Wattebled F et al.; In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure . This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS) . The mutants of STA11 were showed to lack D-enzyme, a plant alpha-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase . We have cloned and characterized both the cDNA and gDNA corresponding to the C . reinhardtii D-enzyme . We now report allele-specific modifications of the D-enzyme gene in the mutants of STA11 . These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodes D-enzyme . MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutant C . reinhardtii cells . We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.

J Biol Chem, 2003 Jul 18, 278(29), 26862 - 9 Epub 2003 May 13.
The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p; Janas E et al.; The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae . To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity . The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a Km of 0.6 mm and did not show cooperativity in ATPase activity . However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1.7), indicating that the functional state is a dimer . Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD . The nucleotide composition of trapped intermediate states was determined using {alpha-32P}ATP and {gamma-32P}ATP . Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs . Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode . Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides.

J Biol Chem, 2003 Aug 1, 278(31), 29016 - 23 Epub 2003 May 13.
Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase; Casals N et al.; This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina . Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type . We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure . The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity . The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site . We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

J Biol Chem, 2003 Aug 8, 278(32), 29478 - 86 Epub 2003 May 13.
Global iron-dependent gene regulation in Escherichia coli . A new mechanism for iron homeostasis; McHugh JP et al.; Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores . Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes . This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance . Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E . coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways . Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced . Many of these genes encode iron-rich respiratory complexes . This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions . This mechanism thus accounts for the low iron contents of fur mutants and explains how E . coli can modulate its iron requirements . Analysis of 55Fe-labeled E . coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data . In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

J Biol Chem, 2003 Aug 1, 278(31), 29000 - 8 Epub 2003 May 13.
Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a); Kim JS et al.; Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain . Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo . However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated . To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli . The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo . The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis . Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice . Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth . Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

J Biol Chem, 2003 Aug 1, 278(31), 28455 - 61 Epub 2003 May 13.
The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate; Hou Z et al.; ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo . We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II) . In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA . The site of phosphorylation was Asp-436, as expected from sequence alignments . We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I) . Thus, ZntA is specific for a broad range of divalent soft metal ions . The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II) . Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain . By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA . The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions . Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.

Br J Pharmacol, 2003 May, 139(1), 35 - 48
Downregulation of mdr1a expression in the brain and liver during CNS inflammation alters the in vivo disposition of digoxin; Goralski KB et al.; 1 . Inflammation is a pathophysiological event that has relevance for altered drug disposition in humans . Two functions of P-glycoprotein (P-gp) are hepatic drug elimination and prevention of drug entry into the central nervous system (CNS) . Our objective was to investigate if localized CNS inflammation induced by Escherichia coli lipopolysaccharide (LPS) would modify mdr1a/P-gp expression and function in the brain and liver . 2 . Our major finding was that the CNS inflammation in male rats produced a loss in the expression of mdr1a mRNA in the brain and liver that was maximal 6 h after intracranial ventricle (i.c.v.) administration of LPS . When (3)H-digoxin was used at discrete time points, as a probe for P-gp function in vivo, an increase in brain and liver (3)H-radioactivity and plasma level of parent digoxin was produced 6 and 24 h following LPS treatment compared to the saline controls . Digoxin disposition was similarly altered in mdr1a(+/+) mice but not in mdr1a(-/-) mice 24 h after administering LPS i.c.v . 3 . In male rats, the biliary elimination of parent digoxin was reduced at 24 h (60%) and 48 h (40%) after LPS treatment and was blocked by the P-gp substrate cyclosporin A . An observed loss in CYP3A1/2 protein and organic anion transporting polypeptide 2 mRNA in the liver may make a minor contribution to digoxin elimination in male rats after LPS treatment . 4 . Conditions which impose inflammation in the CNS produce dynamic changes in mdr1a/P-gp expression/function that may alter hepatic drug elimination and the movement of drugs between the brain and the periphery . The use of experimental models of brain inflammation may provide novel insight into the regulation of P-gp function in that organ.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 121 - 7
Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions; Milardi D et al.; The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another . Specifically, whether copper and disulfide contributions to protein stability are additive has been examined . To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry . A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 degrees C and T=67 degrees C . Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability . These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 67 - 73
cDNA cloning, expression, and functional characterization of a zebrafish SULT1 cytosolic sulfotransferase; Sugahara T et al.; Using the reverse transcriptase-polymerase chain reaction technique, a full-length cDNA encoding a novel zebrafish sulfotransferase was cloned and sequenced . Sequence analysis indicated that this zebrafish sulfotransferase belongs to the SULT1 cytosolic sulfotransferase gene family . The recombinant form of the zebrafish sulfotransferase, purified from Escherichia coli cells, displayed sulfating activities toward a number of endogenous compounds, in particular dopamine and thyroid hormones, in addition to xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds . The zebrafish sulfotransferase exhibited substrate dependence in pH optimum . In comparison with those determined with dopamine as substrate, the zebrafish sulfotransferase displayed much lower K(m) and higher V(max) with n-propyl gallate as substrate . A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 43 degrees C . Among 10 divalent metal cations tested, Hg(2+), Co(2+), Zn(2+), Cd(2+), Cu(2+), and Pb(2+) exhibited dramatic inhibitory effects on the activity of the zebrafish sulfotransferase.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 40 - 50
Purification and characterization of T cell protein tyrosine phosphatase reveals significant functional homology to protein tyrosine phosphatase-1B; Romsicki Y et al.; We have developed a protocol for rapid purification of T cell protein tyrosine phosphatase (TCPTP) and the structurally related protein tyrosine phosphatase-1B (PTP-1B) from bacterial cells . The pH profile for TCPTP was bell-shaped with an optimum of 5.5 . The catalytic domain and full-length versions of TCPTP bound a potent inhibitor with affinities similar to those of PTP-1B . The K(m) values for the catalytic domains of TCPTP and PTP-1B increased with increasing ionic strength, whereas the k(cat) values remained unchanged . Arrhenius plots revealed that TCPTP and PTP-1B possess similar activation energies of 25.3+/-1.2 and 18.4+/-3.0 kJ/mol, respectively . Increasing solvent microviscosity (up to 40% (w/v) sucrose) did not affect k(cat)/K(m) of either enzyme . However, high sucrose concentrations protected both enzymes from thermal inactivation . These studies show that, although they share a 72% amino acid sequence identity within their catalytic domains, TCPTP and PTP-1B are functionally very similar in vitro.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 34 - 9
Adenosyl coenzyme and pH dependence of the {4Fe-4S}2+/1+ transition in lysine 2,3-aminomutase; Hinckley GT et al.; 5'-{N-{(3S)-3-Amino-carboxypropyl}-N-methylamino}-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the {4Fe-4S}(2+) center in lysine 2,3-aminomutase to the +1 oxidation state . azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM . The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5 . Reduction of the {4Fe-4S}(2+) center in the presence of this analog gives a 10K {4Fe-4S}(1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH) . The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the {4Fe-4S}(2+) center . The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the {4Fe-4S}(2+) center . The signal shape of the low-temperature EPR signal for the {4Fe-4S}(1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra . Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised . These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.

Vaccine, 2003 Jun 2, 21(19-20), 2548 - 55
Murine antibody response to intranasally administered enterotoxigenic Escherichia coli colonization factor CS6; de Lorimier AJ et al.; Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea worldwide and is an important cause of infant morbidity and mortality in developing nations . ETEC colonization factors (CF) are virulence determinants that appear to be protective antigens in humans and are the major target of vaccine efforts . One of the most prevalent CF, CS6, is expressed by about 30% of ETEC worldwide . This study was designed to compare the immunogenicity between encapsulated CS6 (CS6-PLG) and unencapsulated CS6 . Recombinant CS6 was purified and encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLG) microspheres using current Good Manufacturing Practices (cGMP) . CS6-PLG and CS6 were administered intranasally (IN) to BALB/c mice in three vaccinations 4 weeks apart . Enzyme linked immunosorbent assay (ELISA) was used to measure the anti-CS6 response in serum and mucosal secretions following each of the three inoculations . Mice vaccinated with two or three doses of CS6-PLG demonstrated a significantly greater rise in serum anti-CS6 IgG and mucosal IgA titer values than those immunized with two or three doses of CS6 alone . Three doses of CS6-PLG led to anti-CS6 serum IgG and mucosal IgA titer values 14-fold and 4.4-fold greater, respectively, than three doses of CS6 (P<0.02) . IN administered CS6 to mice is safe and highly immunogenic either alone or when encapsulated in microspheres . PLG microsphere encapsulation of CS6 significantly augments the antibody response to that antigen when administered to a mucosal surface.

Vaccine, 2003 Jun 2, 21(19-20), 2516 - 22
The domain III fragment of Japanese encephalitis virus envelope protein: mouse immunogenicity and liposome adjuvanticity; Wu SC et al.; The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts . In this study, the domain III protein of the attenuated strain CH2195LA was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli . The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E . coli . The purified domain III fusion protein (TrxD3) was emulsified in Freund's adjuvant (FA) as well as in different charged liposomes for immunization in mice . Immunization of TrxD3 fusion protein emulsified in Freund's adjuvant and only the cationic liposome resulted in eliciting neutralizing antibodies and protective immunity in ICR mice . The cationic liposome can serve not only as a safer but also an effective adjuvant for the TrxD3 protein immunization . These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.

Vaccine, 2003 Jun 2, 21(19-20), 2500 - 5
A single amino acid substitution in a recombinant G protein vaccine drastically curtails protective immunity against respiratory syncytial virus (RSV); Huang Y et al.; Recent studies have indicated a dominant T cell epitope located approximately between amino acids 184 and 203 on the respiratory syncytial virus (RSV) G protein . Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128-229) of the RSV G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids on vaccine efficacy . By changing two amino acids Arg 188 and Lys 192 to alanine, the ability of the Trx-G 128-229 fusion protein to protect mice against RSV challenge was virtually abolished . Mice immunized with the double mutant protein showed low levels of neutralizing antibodies and no pulmonary eosinophilic infiltrate, in contrast to that observed in mice immunized with wild type protein prior to RSV challenge . While less effective than the double mutant, mutation of either Arg 188 or Lys 192 to Ala drastically impaired the ability of immunized Trx-G 128-229 to induce neutralizing antibodies and to elicit pulmonary eosinophilia associated with RSV challenge . Despite low levels of virus-neutralizing antibodies, G protein-specific antibodies were detected by Western blotting in the sera from mice immunized with either of the single mutants (Arg 188 or Lys 192) but not the double mutant . Finally, immunization of mice with truncated forms of the Trx-G protein, showed partial protection against RSV challenge with Trx-G 128-188 but not with Trx-G 189-229 . Taken together, the results indicate an important role for Arg 188 and Lys 192 in the induction of protective immunity and priming for eosinophilia against RSV . Furthermore, while the dominant protective linear epitope on the RSV G protein requires an intact sequence around Arg 188, there are additional, but less potent, protective epitopes upstream of Arg 188.

Vaccine, 2003 Jun 2, 21(19-20), 2485 - 91
OM-174, a new adjuvant with a potential for human use, induces a protective response when administered with the synthetic C-terminal fragment 242-310 from the circumsporozoite protein of Plasmodium berghei; Meraldi V et al.; The goal of this project was the evaluation of a novel immunomodulatory adjuvant for human use, OM-174, which is a soluble adjuvant derived from Escherichia coli lipid A . For this study, we used a synthetic peptide, known for its safety and reproducibility and the murine model of BALB/c mice . The long peptide (PbCS 242-310) used corresponds to the C-terminal region of the circumsporozoite protein (CSP) that is the major protein on the surface of Plasmodium sporozoites . Subcutaneous injections of PbCS 242-310 in combination with soluble adjuvant OM-174 induced long lasting peptide-specific antibody titres comparable to those obtained by immunization with incomplete Freund's adjuvant (IFA) . The ex vivo evaluation of the CD8(+) T cell response by IFN-gamma ELISPOT assay revealed that the injection of polypeptide with OM-174 adjuvant induced, compared to IFA, a similar and an eight-fold increased frequency of peptide-specific lymphocytes in the draining lymph-nodes and in the spleen, respectively . The CD8(+) T-cells are specific for the sequence PbCS 245-253, a well-known H-2K(d)-restricted CTL epitope, and are cytotoxic as shown in a chromium release assay . Immunization of BALB/c mice with this polypeptide in combination with adjuvant OM-174 conferred a protection after challenge with live Plasmodium berghei sporozoites.The strong antibody and CTL responses observed to a synthetic peptide in mice, the safety profile of the adjuvant and its extensive physico-chemical characterization suggest that OM-174 has a potential use in vaccine formulations for humans.

Vaccine, 2003 Jun 2, 21(19-20), 2383 - 93
Antigen entrapped in the escheriosomes leads to the generation of CD4(+) helper and CD8(+) cytotoxic T cell response; Syed FM et al.; In previous study, we demonstrated the potential of Escherichia coli (E . coli) lipid liposomes (escheriosomes) to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells including professional antigen presenting cells . Our present study demonstrates that antigen encapsulated in escheriosomes could be successfully delivered simultaneously to the cytosolic as well as endosomal processing pathways of antigen presenting cells, leading to the generation of both CD4(+) T-helper and CD8(+) cytotoxic T cell response . In contrast, encapsulation of same antigen in egg phosphatidyl-choline (egg PC) liposomes, just like antigen-incomplete Freund's adjuvant (IFA) complex, has inefficient access to the cytosolic pathway of MHC I-dependent antigen presentation and failed to generate antigen-specific CD8(+) cytotoxic T cell response . However, both egg PC liposomes as well as escheriosomes-encapsulated antigen elicited strong humoral immune response in immunized animals but antibody titre was significantly higher in the group of animals immunized with escheriosomes-encapsulated antigen . These results imply usage of liposome-based adjuvant as potential candidate vaccine capable of eliciting both cell-mediated as well as humoral immune responses . Furthermore, antigen entrapped in escheriosomes stimulates antigen-specific CD4(+) T cell proliferation and also enhances the level of IL-2, IFN-gamma and IL-4 in the immunized animals.

Shock, 2003 May, 19(5), 433 - 9
Endotoxin-induced endothelial cell proinflammatory phenotypic differentiation requires stress fiber polymerization; Cuschleri J et al.; Endotoxin-induced intercellular adhesion molecule-1 (ICAM-1) and interleukin 8 (IL-8) production in endothelial cells, which is mediated by Toll-receptor signaling, is essential for optimal neutrophil recruitment and migration during sepsis . Endotoxin also causes stress fiber polymerization that has recently been shown to affect intracellular signaling . However, the role of this polymerization process on endothelial-induced neutrophil adhesion and migration is unknown . Human umbilical vein endothelial cells (HUVEC) were stimulated with lipopolysaccharide (LPS) . Selected cells were pretreated with cytochalasin D (CD) or lactrunculin A (LA), agents that disrupt actin polymerization . Cellular protein was extracted and analyzed by Westem blot for the phosphorylated form of IL-1-associated kinase (IRAK) and production of ICAM-1 . Extracted nuclear protein was analyzed by Western blot and electrophoretic mobility shift assay (EMSA) for nuclear translocation and activity of NF-kappaB . IL-8 production was determined by enzyme-linked immunoabsorbant assay (ELISA) . Neutrophil adhesion was assayed fluorometrically using calcein-AM-labeled neutrophils on treated endothelial cells . LPS treatment led to phosphorylation of IRAK, and subsequent NF-kappaB translocation and activation . This cellular signaling was followed by ICAM-1 expression and IL-8 production . Pretreatment of cells with CD or LA led to a significant inhibition of IRAK phosphorylation, and NF-kappaB nuclear translocation and activation . Actin depolymerization also significantly inhibited LPS-induced ICAM-1 and IL-8 production . HUVEC pretreated with CD or LA demonstrated significant inhibition of LPS-induced neutrophil adhesion . Endotoxin-induced actin polymerization is essential for optimal intracellular signaling through IRAK and NF-kappaB . Failure of these signaling events is associated with a marked reduction in adhesion molecule production, IL-8 production, and neutrophil adhesion . These findings support the necessity of stress fiber polymerization for optimal recruitment of neutrophils during sepsis.

Shock, 2003 May, 19(5), 427 - 32
Neuroprotective effects of coenzyme Q10 at rostral ventrolateral medulla against fatality during experimental endotoxemia in the rat; Chuang YC et al.; Coenzyme Q10 (CoQ10, ubiquinone) is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant . We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome using a novel water-soluble formulation of this quinone derivative . Experiments were conducted in adult male Sprague-Dawley rats that were maintained under propofol anesthesia . Intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensuing within 4 h . The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; "pro-life" phase) and a secondary decrease (Phase III; "pro-death" phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure signals . Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 microg) into the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension . CoQ10 pretreatment also significantly prolonged the duration of and augmented the total power density of the vasomotor components of systemic arterial pressure signals in Phase II endotoxemia . The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted . We conclude that CoQ10 provides neuroprotection against fatality during experimental endotoxemia by reducing superoxide anion production at the RVLM, whose neuronal activity is intimately related to the "life-and-death" process.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 281 - 8
Characterization and expression analysis of genes encoding phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase of Lotus japonicus, a model legume; Nakagawa T et al.; Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK) . We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules . One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules . The other gene is expressed in shoots and roots at a low level . Both forms have the putative phosphorylation site, Ser11 . We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L . japonicus . The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro . The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant . In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules . When L . japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2003 Mar, 20(1), 64 - 7
{Recombinant human brain myelin basic protein and its antibody}; Liu J et al.; We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector . The recombinant vector p5TMP was transformed into E . coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside) . A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE . Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning . Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP . The titer was obtained at 1:16 after 5 injections . The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.

Diabetologia, 2003 May, 46(5), 689 - 98 Epub 2003 May 13.
Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage; Gao G et al.; AIMS/HYPOTHESIS: Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin) . It specifically binds to tissue kallikrein and inhibits kallikrein activity . Our study was designed to test its effects on retinal neovascularization and vascular permeability . METHODS: Endothelial cell proliferation was determined by {(3)H} thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry . Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model . Vascular permeability was assayed by the Evans blue method . Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA . RESULTS: Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells . Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model . Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR . Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production . KBP competed with (125)I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model . CONCLUSION/INTERPRETATION: Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system . Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP.

J Allergy Clin Immunol, 2003 May, 111(5), 1106 - 10
Immediate-type hypersensitivity reaction to ingestion of mycoprotein (Quorn) in a patient allergic to molds caused by acidic ribosomal protein P2; Hoff M et al.; BACKGROUND: Quorn is the brand name for a line of foods made with so-called "mycoprotein," which springs from the mold Fusarium venenatum . Since the introduction on the food market, there have been complaints from consumers reporting adverse gastrointestinal reactions after ingestion of mycoprotein . To date, it is not clear whether the reported symptoms are IgE-mediated . OBJECTIVE: The aim of the study was to describe for the first time a case history of an asthmatic patient with severe hypersensitivity reactions to ingested mycoprotein and to identify and characterize the potential allergen that might be responsible for this . METHODS: The sensitization pattern of the asthmatic subject was characterized, and food allergy to mycoprotein was assessed by double-blinded placebo-controlled food challenge . Afterward, specific IgE antibodies of the serum of this patient were used to screen a Fusarium culmorum cDNA expression library . The coding sequence of one enriched cDNA-clone was expressed in Escherichia coli to produce a recombinant protein that was further purified and immunologically characterized . RESULTS: The patient showed high sensitization to many known aeroallergens but apart from Quorn not to any other tested food samples . The deduced amino acid sequence of the enriched cDNA-clone (Fus c 1) showed large identity to the 60S acidic ribosomal protein P2 which is highly conserved among several species and also described as minor allergen in other mold species . The frequency of IgE reactivity of sera from F culmorum -sensitized subjects to rFus c 1 was approximately 35% . By enzyme allergosorbent test inhibition, we found 65% inhibition of mycoprotein IgE reactivity by rFus c 1 . On the opposite we found reduced IgE reactivity of rFus c 1 of 68% by using mycoprotein as inhibitor . CONCLUSIONS: Sensitization to mold allergens by the respiratory tract and subsequent oral ingestion of cross-reactive proteins may lead to severe food-allergic reactions . Thus, the 60S acidic ribosomal protein P2 of F venenatum probably is the reason for the described severe hypersensitivity reactions of the patient to Quorn-mycoprotein because of its potential cross-reactivity to the F culmorum allergen Fus c 1.

J Virol, 2003 Jun, 77(11), 6450 - 65
Development and application of a reverse genetics system for Japanese encephalitis virus; Yun SI et al.; Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children . Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone . We describe here the development of such a clone . A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome . This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5' and 3' ends . The synthetic RNAs thus produced were highly infectious in susceptible cells (>10(6) PFU/ micro g), and these cells rapidly generated a high titer of synthetic viruses (>5 x 10(6) PFU/ml) . The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity . Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli . A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated . Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types . This novel reverse genetics system for JEV will greatly facilitate research into JEV biology . It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

J Biol Chem, 2003 Jul 25, 278(30), 28252 - 7 Epub 2003 May 12.
Crystal structure of the intrinsically flexible addiction antidote MazE; Loris R et al.; A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE . Only 45% of the polypeptide chain is found ordered in the crystal . The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain . A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets . This hydrophobic core consists exclusively of short aliphatic residues . The folded part of MazE contains a novel DNA binding motif . A model for DNA binding that is consistent with the available biochemical data is presented.

J Cell Biol, 2003 May 12, 161(3), 471 - 6
Modeling network dynamics: the lac operon, a case study; Vilar JM et al.; We use the lac operon in Escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular networks . We integrate three different levels of description (molecular, cellular, and that of cell population) into a single model, which seems to capture many experimental aspects of the system.

J Neuroimmunol, 2003 May, 138(1-2), 49 - 55
Maternal infection regulates BDNF and NGF expression in fetal and neonatal brain and maternal-fetal unit of the rat; Gilmore JH et al.; Maternal infection during pregnancy is associated with increased risk for neurodevelopmental disorders . Lipopolysaccharide (LPS) or saline was administered to rats to model maternal infection, and levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in maternal plasma, placenta, amniotic fluid, fetal liver/spleen, fetal brain, and cerebral cortex after birth were determined by ELISA or semiquantitative Western blot analysis . BDNF expression was significantly increased in the fetal brain (p=0.039); NGF expression was significantly increased in neonatal cortex (p=0.0009) . Neurotrophic factor expression was also altered in other tissues of the maternal-fetal unit . Abnormal expression of neurotrophic factors represents a potential mechanism through which maternal infection increases risk for neurodevelopmental disorders.

Mol Biochem Parasitol, 2003 May, 128(2), 195 - 204
Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine; Angov E et al.; Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites . We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli . The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins . The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards . It was structurally correct based on binding conformation-dependent mAbs, and was stable . Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42) . FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.

Domest Anim Endocrinol, 2003 May, 24(4), 341 - 51
Effect of menhaden fish oil supplementation and lipopolysaccharide exposure on nursery pigs . I . Effects on the immune axis when fed diets containing spray-dried plasma; Carroll JA et al.; The objective of the present study was to evaluate the potential immunological benefit of adding menhaden fish oil to the diet of weaned pigs . Twenty-four crossbred male pigs were weaned at approximately 18 days of age and placed on a complex nursery diet containing 30% lactose and 7% plasma protein with 6% corn oil as the fat source (Cont, n=12) or with 5% menhaden fish oil and 1% corn oil as the fat source (MFO, n=12) for a period of 15 days . Body weights did not differ (P>0.78) between dietary groups either at the beginning or end of the 15 days feeding period . On day 15, all pigs were non-surgically fitted with an indwelling jugular catheter . On d 16, pigs received an i.v . injection of either saline (n=6/dietary group) or lipopolysaccharide (LPS; 150 microg/kg body weight; n=6/dietary group) and blood samples were collected at 30 min intervals for a period of 5h . Serum was harvested and stored at -80 degrees C for analysis of cortisol (CS), corticosteroid-binding globulin (CBG), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) . There was no significant effect of diet on basal concentrations (Time 0) of any of the blood parameters analyzed . A Time x Treatment x Diet interaction (P<0.03) was observed for serum CS such that those pigs which consumed the MFO diet followed by LPS treatment had a reduced CS response as compared to the LPS-treated pigs on the Cont diet . A Time x Treatment interaction (P<0.01) was observed for serum CBG such that LPS treatment reduced circulating CBG as compared to the saline-treated pigs . Time x Treatment x Diet interactions were also observed for serum concentrations of TNF-alpha (P=0.084) and IFN-gamma (P=0.022) such that both the TNF-alpha and IFN-gamma response to the LPS challenge was lower in those pigs receiving the MFO diet as compared to the LPS-treated pigs on the Cont diet . Overall, serum CS was negatively correlated with the CBG response (r=-0.40, P<0.001), however, the strongest negative correlation was observed in the LPS-treated pigs which consumed the MFO diet (r=-0.63, P<0.001) . While further studies are needed to evaluate the immunological response of including MFO in the nursery pig diet, the present study demonstrates that supplementation with MFO does indeed alter the immunological response to an LPS challenge.

J Mol Biol, 2003 May 23, 329(1), 121 - 34
The allosteric transition of GroEL induced by metal fluoride-ADP complexes; Inobe T et al.; To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL . The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition . The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence . The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible . The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL . Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model . From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol . However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is structurally equivalent to the allosteric state induced by ATP . These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL . It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand.

J Mol Biol, 2003 May 23, 329(1), 15 - 33
An atomic model for actin binding by the CH domains and spectrin-repeat modules of utrophin and dystrophin; Sutherland-Smith AJ et al.; Utrophin and dystrophin link cytoskeletal F-actin filaments to the plasmalemma . Genetic strategies to replace defective dystrophin with utrophin in individuals with muscular dystrophy requires full characterization of these proteins . Both contain homologous N-terminal actin-binding motifs composed of a pair of calponin-homology (CH) domains (CH1 and CH2) that are connected by spectrin-repeat modules to C-terminal membrane-binding sequences . Here, electron microscopy and 3D reconstruction of F-actin decorated with utrophin and dystrophin actin-binding constructs were performed using Utr261 (utrophin's CH domain pair), Utr416 (utrophin's CH domains and first spectrin-repeat) and Dys246 (dystrophin's CH domain pair) . The lozenge-like utrophin CH domain densities localized to the upper surface of actin subdomain 1 and extended azimuthally over subdomain 2 toward subdomains 3 and 4 . The cylinder-shaped spectrin-repeat was located at the end of the CH domain pair and was aligned longitudinally along the cleft between inner and outer actin domains, where tropomyosin is present when on thin filaments . The connection between the spectrin-repeat module and the CH domains defined the orientation of CH1 and CH2 on actin . Resolution of utrophin's CH domains and spectrin-repeats permitted docking of crystal structures into respective EM densities, leading to an atomic model where both CH and spectrin-domains bind actin . The CH domain-actin interaction for dystrophin was found to be more complex than for utrophin . Binding assays showed that Utr261 and Utr416 interacted with F-actin as monomers, whereas Dys246 appeared to associate as a dimer, consistent with a bilobed Dys246 structure observed on F-actin in electron microscope reconstructions . One of the lobes was similar in shape, position and orientation to the monomeric CH domains of Utr261, while the other lobe apparently represented a second set of CH domains in the dimeric Dys246 . The extensive contact made by dystrophin on actin may be used in vivo to help muscles dissipate mechanical stress from the contractile apparatus to the extracellular matrix.

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