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| J Immunol, 2004 Feb 15, 172(4), 2001 - 5 Cutting edge: Toll-like receptor signaling in macrophages induces ligands for the NKG2D receptor; Hamerman JA et al.; Macrophages recognize the presence of infection by using the Toll-like receptor (TLR) family of proteins that detect ligands on bacterial, viral, and fungal pathogens . We show that murine macrophages stimulated with pathogen products known to signal through TLRs express ligands for the NKG2D receptor, found on NK cells, activated CD8(+) T cells and activated macrophages . TLR signaling, through the MyD88 adaptor, up-regulates transcription of the retinoic acid early inducible-1 (RAE-1) family of NKG2D ligands, but not H-60 or murine UL16-binding protein-like transcript-1 . RAE-1 proteins are found on the surface of activated, but not resting, macrophages and can be detected by NKG2D on NK cells resulting in down-regulation of this receptor both in vitro and in vivo . RAE-1-NKG2D interactions provide a mechanism by which NK cells and infected macrophages communicate directly during an innate immune response to infection. J Biol Chem, 2004 Apr 16, 279(16), 16638 - 45 Epub 2004 Feb 05. Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft; Ng KK et al.; Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking . To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations . Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms . The location of this carboxyl-terminal segment appears to interfere with the binding of double-stranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins. Bioinformatics, 2004 May 1, 20(7), 1053 - 9 Epub 2004 Feb 05. Good spaced seeds for homology search; Choi KP et al.; MOTIVATION: Filtration is an important technique used to speed up local alignment as exemplified in the BLAST programs . Recently, Ma et al . discovered that better filtering can be achieved by spacing out the matching positions according to a certain pattern, instead of contiguous positions to trigger a local alignment in their PatternHunter program . Such a match pattern is called a spaced seed . RESULTS: Our numerical computation shows that the ranks of spaced seeds (based on sensitivity) change with the sequences similarity . Since homologous sequences may have diverse similarity, we assess the sensitivity of spaced seeds over a range of similarity levels and present a list of good spaced seeds for facilitating homology search in DNA genomic sequences . We validate that the listed spaced seeds are indeed more sensitive using three arbitrarily chosen pairs of DNA genomic sequences. Bioinformatics, 2004 May 1, 20(7), 1110 - 8 Epub 2004 Feb 05. Confirmation of data mining based predictions of protein function; King RD et al.; MOTIVATION: A central problem in bioinformatics is the assignment of function to sequenced open reading frames (ORFs) . The most common approach is based on inferred homology using a statistically based sequence similarity (SIM) method, e.g . PSI-BLAST . Alternative non-SIM based bioinformatic methods are becoming popular . One such method is Data Mining Prediction (DMP) . This is based on combining evidence from amino-acid attributes, predicted structure and phylogenic patterns; and uses a combination of Inductive Logic Programming data mining, and decision trees to produce prediction rules for functional class . DMP predictions are more general than is possible using homology . In 2000/1, DMP was used to make public predictions of the function of 1309 Escherichia coli ORFs . Since then biological knowledge has advanced allowing us to test our predictions . RESULTS: We examined the updated (20.02.02) Riley group genome annotation, and examined the scientific literature for direct experimental derivations of ORF function . Both tests confirmed the DMP predictions . Accuracy varied between rules, and with the detail of prediction, but they were generally significantly better than random . For voting rules, accuracies of 75-100% were obtained . Twenty-one of these DMP predictions have been confirmed by direct experimentation . The DMP rules also have interesting biological explanations . DMP is, to the best of our knowledge, the first non-SIM based prediction method to have been tested directly on new data . AVAILABILITY: We have designed the "Genepredictions" database for protein functional predictions . This is intended to act as an open repository for predictions for any organism and can be accessed at http://www.genepredictions.org Bioinformatics, 2004 May 1, 20(7), 1087 - 96 Epub 2004 Feb 05. Large-scale assessment of the utility of low-resolution protein structures for biochemical function assignment; Arakaki AK et al.; MOTIVATION: Several protein function prediction methods employ structural features captured in three-dimensional (3D) descriptors of biologically relevant sites . These methods are successful when applied to high-resolution structures, but their detection ability in lower resolution predicted structures has only been tested for a few cases . RESULTS: A method that automatically generates a library of 3D functional descriptors for the structure-based prediction of enzyme active sites (automated functional templates, 593 in total for 162 different enzymes), based on functional and structural information automatically extracted from public databases, has been developed and evaluated using decoy structures . The applicability to predicted structures was investigated by analyzing decoys of varying quality, derived from enzyme native structures . For 35% of decoy structures, our method identifies the active site in models having 3-4 A coordinate root mean square deviation from the native structure, a quality that is reachable using state of the art protein structure prediction algorithms . AVAILABILITY: See http://www.bioinformatics.buffalo.edu/resources/aft/ Neurogastroenterol Motil, 2004 Feb, 16(1), 125 - 33 Endogenous endothelin increases gallbladder tone and leads to acute cholecystitis in the Australian possum; Al-Jiffry BO et al.; Endothelins are bioactive peptides produced by gallbladder epithelial cells . We aimed to determine the role of endothelins in acute cholecystitis . Escherichia coli lipopolysaccharide vs saline (sham) was instilled into the gallbladder lumen of Australian possums . Some animals received the non-selective endothelin antagonist, tezosentan . At 4 or 24 h, plasma and gallbladder endothelins and white blood cell count (WBCC) were determined . Acute cholecystitis was assessed using a histopathology score . In other animals gallbladder tone was determined . At 4h, a dose-dependent 60-fold increase in gallbladder endothelin level occurred (P = 0.001) but other parameters remained comparable with sham animals . Epithelial cells were endothelin-immunoreactive . At 24 h, the WBCC rose (P < 0.007), and severe cholecystitis developed . Gallbladder but not plasma endothelin levels remained elevated . Tezosentan pre-treatment resulted in a histologically normal gallbladder, but the WBCC and gallbladder endothelin levels were elevated . Lipopolysaccharide or saline instillation also caused a time-dependent increase in gallbladder tone over 4 h (P < 0.001), but not in control animals . This increase was reduced by tezosentan treatment . Gallbladder endothelin production is an early event in acute cholecystitis, increases gallbladder tone and plays a crucial role in the inflammatory process. Cell Microbiol, 2004 Mar, 6(3), 289 - 301 Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture; Schuller S et al.; Shiga toxins (Stx) produced by Escherichia coli are associated with systemic complications such as haemolytic-uraemic syndrome . The mechanism of Stx translocation across the epithelial barrier is unknown as human intestinal epithelium lacks receptor Gb3 . In this study, we have examined the interaction of purified Stx1 and 2 with Caco-2 (Gb3+) and T84 (Gb3-) cell lines, and determined the effects of Stx on human intestine using in vitro organ culture (IVOC) . Stx exposure caused inhibition of protein synthesis and apoptosis in Caco-2 but not in T84 cells . However, both Stx1 and 2 were transported to the endoplasmic reticulum, and the Stx1 A-subunit was cleaved in a furin-dependent manner in both cell lines . Thus, a Gb3-independent retrograde transport route exists in T84 cells for Stx that does not induce cell damage . IVOC demonstrated increased epithelial cell extrusion in response to exposure to Stx2, but not Stx1, in both small intestine and colon . Pretreatment of Stx2 with Stx2-specific antibody abrogated this effect . Overlaying frozen sections with Stx showed lamina propria, but not epithelial, cell binding that paralleled Gb3 localization, and included endothelium and pericryptal myofibroblasts . This indicates that human intestinal epithelium may evince Stx2-induced damage in the absence of Gb3 receptors, by an as yet unrecognized mechanism. Cell Microbiol, 2004 Mar, 6(3), 243 - 54 Enterohaemorrhagic and enteropathogenic Escherichia coli use different mechanisms for actin pedestal formation that converge on N-WASP; Lommel S et al.; Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E . coli (EHEC), two closely related diarrhoeagenic pathogens, induce actin rearrangements at the surface of infected host cells resulting in the formation of pseudopod-like structures termed pedestals beneath intimately attached bacteria . We have shown previously that N-WASP, a key integrator of signalling pathways that regulate actin polymerization via the Arp2/3 complex, is essential for pedestal formation induced by EPEC using N-WASP-defective cell lines . Here we show that actin pedestal formation initiated by EHEC also depends on N-WASP . Amino acid residues 226-274 of N-WASP are both necessary and sufficient to target N-WASP to sites of EHEC attachment . The recruitment mechanism thus differs from that used by EPEC, in which amino-terminal sequences of N-WASP mediate recruitment . For EPEC, recruitment of N-WASP downstream of Nck has been postulated to be mediated by WIP . However, we find a direct interaction of N-WASP with WIP to be dispensable for EPEC-induced pedestal formation and present data supporting an F-actin-dependent localization of WIP to actin pedestals induced by both EPEC and EHEC . In summary, our data show that EPEC and EHEC use different mechanisms to recruit N-WASP, which is essential for actin pedestal formation induced by both pathogens. Eur J Biochem, 2004 Feb, 271(4), 724 - 33 The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm; Banerjee R et al.; In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end . We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins . In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively . On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS . In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm . Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain . Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site. Eur J Biochem, 2004 Feb, 271(4), 694 - 702 The unusual methanogenic seryl-tRNA synthetase recognizes tRNASer species from all three kingdoms of life; Bilokapic S et al.; The methanogenic archaea Methanococcus jannaschii and M . maripaludis contain an atypical seryl-tRNA synthetase (SerRS), which recognizes eukaryotic and bacterial tRNAsSer, in addition to the homologous tRNASer and tRNASec species . The relative flexibility in tRNA recognition displayed by methanogenic SerRSs, shown by aminoacylation and gel mobility shift assays, indicates the conservation of some serine determinants in all three domains . The complex of M . maripaludis SerRS with the homologues tRNASer was isolated by gel filtration chromatography . Complex formation strongly depends on the conformation of tRNA . Therefore, the renaturation conditions for in vitro transcribed tRNASer(GCU) isoacceptor were studied carefully . This tRNA, unlike many other tRNAs, is prone to dimerization, possibly due to several stretches of complementary oligonucleotides within its sequence . Dimerization is facilitated by increased tRNA concentration and can be diminished by fast renaturation in the presence of 5 mm magnesium chloride. Mol Microbiol, 2004 Feb, 51(4), 1205 - 17 The function of RNase G in Escherichia coli is constrained by its amino and carboxyl termini; Deana A et al.; RNase G is a homologue of the essential Escherichia coli ribonuclease RNase E . Whereas RNase E plays a key role in the degradation of mRNA and the processing of tRNA and rRNA in E . coli, the biological functions of RNase G appear more limited . We report here that this difference in function is not merely a consequence of the significantly lower cellular concentration of RNase G, but also reflects differences in the intrinsic properties of these ribonucleases, as overproducing wild-type RNase G at a level up to 20 times the usual cellular concentration of RNase E cannot normally compensate for the absence of RNase E in E . coli . Instead, RNase G can sustain significant growth of RNase E-deficient E . coli cells only when it bears an unnatural extension at its amino terminus (e.g . MRKGINM) or carboxyl terminus (e.g . GHHHHHH) . These extensions presumably enable RNase G to cleave critically important cellular RNAs whose efficient processing or degradation ordinarily requires RNase E . That extending the amino terminus of RNase G restores growth to E . coli cells lacking RNase E without detectably improving tRNA processing suggests that RNase E is not essential for tRNA production and is required for cell growth because it plays an indispensable role in the maturation or decay of essential E . coli RNAs other than tRNA. Mol Microbiol, 2004 Feb, 51(4), 1185 - 92 Intein harbouring large tandem repeats in replicative DNA helicase of Trichodesmium erythraeum; Yang J et al.; Protein splicing inteins can be small as approximately 130 aa or up to approximately 600 aa when harbouring an endonuclease domain . Here we report the identification and characterization of an unusually large intein, 1650 aa long and the largest of known inteins, encoded by the replicative DNA helicase gene dnaB of the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum . This Ter DnaB-1 intein co-exists with a 177-aa mini-intein in the same host protein and harbours large tandem repeats in which an 84-aa sequence is repeated 16 times . Comparison between this tandem repeats and the recently reported tandem repeats of Ter DnaE-1 intein revealed differences and similarities . The two tandem repeats, residing in different inteins of different host proteins, differ by 50% in size and have little sequence similarity . Tandem repeats in the Ter DnaB-1 intein were required for the protein splicing activity when tested in Escherichia coli, in contrast to tandem repeats of the Ter DnaE-1 intein that inhibited protein splicing . On the other hand, tandem repeats of both inteins are located in the same corresponding region of the intein sequence and have the same number of repeating units . These suggest that the two tandem repeats could be related but have diverged greatly in size, sequence and effect on protein splicing . Alternatively, they could have independent origins but evolved certain similarities because of common constraints in structure and maintenance. Mol Microbiol, 2004 Feb, 51(4), 937 - 48 Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation; Burke ME et al.; Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res) . Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III . 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E . coli . Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid . Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase . Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse . The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured. Biochem J, 2004 Jun 1, 380(Pt 2), 409 - 17 Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein); Bajaj K et al.; The protein CcdB (controller of cell division or death B) is an F-plasmid-encoded toxin that acts as an inhibitor of Escherichia coli DNA gyrase . The stability and aggregation state of CcdB have been characterized as a function of pH and temperature . Size-exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0 . CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer . Hence intersubunit interactions are not required for folding of individual subunits . The stability of both forms was characterized by isothermal denaturant unfolding and calorimetry . The free energies of unfolding were found to be 9.2 kcal x mol(-1) (1 cal approximately 4.184 J) and 21 kcal x mol(-1) at 298 K for the monomer and dimer respectively . The denaturant concentration at which one-half of the protein molecules are unfolded (C(m)) of the dimer is dependent on protein concentration, whereas the C(m) of the monomer is independent of protein concentration, as expected . Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdmCl (guanidinium chloride) . Differential scanning calorimetry in the presence of low concentrations of GdmCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein . The value of Delta C (p) (representing the change in excess heat capacity upon protein denaturation) is 2.8+/-0.2 kcal x mol(-1) x K(-1) for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration. Biochem J, 2004 May 1, 379(Pt 3), 849 - 55 Putrescine biosynthesis in mammalian tissues; Coleman CS et al.; L-ornithine decarboxylase provides de novo putrescine biosynthesis in mammals . Alternative pathways to generate putrescine that involve ADC (L-arginine decarboxylase) occur in non-mammalian organisms . It has been suggested that an ADC-mediated pathway may generate putrescine via agmatine in mammalian tissues . Published evidence for a mammalian ADC is based on (i) assays using mitochondrial extracts showing production of 14CO2 from {1-14C}arginine and (ii) cloned cDNA sequences that have been claimed to represent ADC . We have reinvestigated this evidence and were unable to find any evidence supporting a mammalian ADC . Mitochondrial extracts prepared from freshly isolated rodent liver and kidney using a metrizamide/Percoll density gradient were assayed for ADC activity using L-{U-14C}-arginine in the presence or absence of arginine metabolic pathway inhibitors . Although 14CO2 was produced in substantial amounts, no labelled agmatine or putrescine was detected . {14C}Agmatine added to liver extracts was not degraded significantly indicating that any agmatine derived from a putative ADC activity was not lost due to further metabolism . Extensive searches of current genome databases using non-mammalian ADC sequences did not identify a viable candidate ADC gene . One of the putative mammalian ADC sequences appears to be derived from bacteria and the other lacks several residues that are essential for decarboxylase activity . These results indicate that 14CO2 release from {1-14C}arginine is not adequate evidence for a mammalian ADC . Although agmatine is a known constituent of mammalian cells, it can be transported from the diet . Therefore L-ornithine decarboxylase remains the only established route for de novo putrescine biosynthesis in mammals. Biotechnol Prog, 2004 Jan-Feb, 20(1), 330 - 7 Structural and thermal stability characterization of Escherichia coli D-galactose/D-glucose-binding protein; D'Auria S et al.; The effect of temperature and glucose binding on the structure of the galactose/glucose-binding protein from Escherichia coli was investigated by circular dichroism, Fourier transform infrared spectroscopy, and steady-state and time-resolved fluorescence . The data showed that the glucose binding induces a moderate change of the secondary structure content of the protein and increases the protein thermal stability . The infrared spectroscopy data showed that some protein stretches, involved in alpha-helices and beta strand conformations, are particularly sensitive to temperature . The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein is well represented by a three-exponential model and that in the presence of glucose the protein adopts a structure less accessible to the solvent . The new insights on the structural properties of the galactose/glucose-binding protein can contribute to a better understanding of the protein functions and represent fundamental information for the development of biotechnological applications of the protein. Biotechnol Prog, 2004 Jan-Feb, 20(1), 284 - 8 New cationic exchanger support for reversible immobilization of proteins; Fuentes M et al.; New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g . MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads) . More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5 . This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 degrees C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions) . Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature) . Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp . beta-galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates . After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles. Biotechnol Prog, 2004 Jan-Feb, 20(1), 277 - 83 Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin; Kweon DH et al.; Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification . In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme . Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid-phase refolding method that utilizes a charged adsorbent as a supporting material . Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C-terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea-denatured state . When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range . The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl . The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase . Several factors that are important in improving the refolding efficiency are explored . Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield . The solid-phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance . Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme. Biotechnol Prog, 2004 Jan-Feb, 20(1), 44 - 50 Keratinocyte growth factor-2 production in an ompT-deficient Escherichia coli K-12 mutant; Laird MW et al.; A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110 . When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein . This cleavage resulted in a truncated KGF-2 species (S69-S208) . To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity . This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl . To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation . Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein . Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser) . From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E . coli host nearly abolished the N-terminal cleavage issue. J Antibiot (Tokyo), 2003 Nov, 56(11), 950 - 6 pTOYAMAcos, pTYM18, and pTYM19, actinomycete-Escherichia coli integrating vectors for heterologous gene expression; Onaka H et al.; A novel shuttle integration cosmid vector (pTOYAMAcos), based on pKU402, and shuttle integration vectors (pTYM18 and pTYM19) were constructed for the cloning of actinomycete DNA and its heterologous expression . These vectors contain oriT of an IncP transmissible plasmid in order to transfer genes by conjugation from Escherichia coli to actinomycetes, and they also contain int derived from actinophage phiC31 in order to integrate site-specifically into the chromosomal DNA . pTOYAMAcos contains the lambdacos site to promote packaging of vectors containing 35 to approximately 45-kb DNA fragments into lambda particles . pTYM18 and pTYM19 contain kanamaycin and thiostrepton resistance genes, respectively, and have multiple cloning sites including EcoRI and HindIII sites, which are available for blue/white screening in E . coli . To demonstrate the utility of these vectors, we expressed the entire gene cluster for rebeccamycin biosynthesis from Lechevalieria aerocolonigenes using pTOYAMAcos and detected rebeccamycin production in transformed S . lividans . In addition, we demonstrated the utility of pTYM 19 in a gene-disruption complementation test . L . aerocolonigenes deltarebC strain, which is defective in rebeccamycin production because of a rebC deletion, was restored to rebeccamycin production by complemention by rebC cloned in pTYM 19. Gastroenterology, 2004 Feb, 126(2), 511 - 9 Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor; Thiagarajah JR et al.; BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) provides an important apical route for Cl(-) secretion across intestinal epithelia . A thiazolidinone-type CFTR blocker (CFTR(inh)-172) reduced cholera toxin-induced fluid accumulation in mouse intestinal loops . Here, we characterize the efficacy and pharmacodynamics of CFTR(inh)-172 in blocking cAMP and cGMP induced Cl(-)/fluid secretion in rodent and human intestine . METHODS & RESULTS: CFTR(inh)-172 inhibited cAMP and cGMP agonist induced short-circuit current by >95% in T84 colonic epithelial cells (K(I) approximately 3 micromol/L) and in mouse and human intestinal sheets (K(I) approximately 9 micromol/L) . A single intraperitoneal injection of CFTR(inh)-172 (200 microg) blocked intestinal fluid secretion in a rat closed-loop model by >90% for cholera toxin and >70% for STa Escherichia coli toxin . In mice, CFTR(inh)-172 (20 microg) inhibited cholera toxin-induced intestinal fluid secretion by 90% (persistence t(1/2) approximately 10 hours, K(I) approximately 5 microg) and STa toxin by 75% (K(I) approximately 10 microg) . Tissue distribution and pharmacokinetic studies indicated intestinal CFTR(inh)-172 accumulation facilitated by enterohepatic circulation . An oral CFTR(inh)-172 preparation reduced fluid secretion by >90% in a mouse open-loop cholera model . CONCLUSIONS: A small molecule CFTR blocker markedly reduced intestinal ion and fluid secretion caused by cAMP/cGMP-dependent bacterial enterotoxins . CFTR inhibition may thus reduce fluid secretion in infectious secretory diarrheas. J Dairy Sci, 2004 Feb, 87(2), 316 - 20 Growth responses of Escherichia coli to immunoglobulin G from cows immunized with ferric citrate receptor, FecA; Takemura K et al.; Effects of purified immunoglobulin (Ig) G from cows immunized with ferric citrate receptor, FecA, on the in vitro growth of Escherichia coli were investigated . Twenty-one cows were assigned to one of 3 treatments: 1) FecA immunization, 2) E . coli J5 bacterin immunization, and 3) unimmunized control . FecA was derived from E . coli UT5600/pSV66 . Immunoglobulin G was purified from pooled colostral whey for each treatment group . The IgG from FecA immunized cows had higher titers against FecA compared with other treatment groups . Bacterial isolates tested were 14 E . coli from intramammary infections and E . coli UT5600/pSV66 . Iron depletion decreased the growth of E . coli compared with growth in Fe-replete medium . The presence of IgG further decreased the growth compared with the growth under iron restriction alone . Bacterial growth did not differ among IgG sources nor between IgG concentrations . Replenishing media with exogenous iron overrode the inhibitory effects of the Fe-depletion and IgG . Vaccinating cows with FecA had little effect on the growth inhibitory properties of IgG toward E . coli mastitis isolates cultured in Fe-deplete media. J Bacteriol, 2004 Feb, 186(4), 1205 - 12 A temperature-sensitive mutation in the dnaE gene of Caulobacter crescentus that prevents initiation of DNA replication but not ongoing elongation of DNA; Lo T et al.; A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant . Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the alpha-catalytic subunit of DNA polymerase III holoenzyme . Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein . This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins . Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation. J Bacteriol, 2004 Feb, 186(4), 1197 - 9 PriA is essential for viability of the Escherichia coli topoisomerase IV parE10(Ts) mutant; Grompone G et al.; The parE10(Ts) mutation, which renders Escherichia coli thermosensitive for growth by inactivation of the essential E . coli topoisomerase topo IV, is lethal at all temperatures when PriA, the main replication restart protein, is absent . This lethality is suppressed by the activation of a PriA-independent replication restart pathway (dnaC809 mutation) . This result suggests that topo IV acts prior to full-chromosome replication completion. J Bacteriol, 2004 Feb, 186(4), 1029 - 37 Molecular characterization of a high-affinity xylobiose transporter of Streptomyces thermoviolaceus OPC-520 and its transcriptional regulation; Tsujibo H et al.; Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an alpha-L-arabinofuranosidase (StxIV) in the presence of xylan . Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular beta-xylosidase (BxlA) . A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA) . The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA . Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA . The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon . The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides . The protein showed high-level affinity for xylobiose (K(d) = 8.75 x 10(-9) M) and for xylotriose (K(d) = 8.42 x 10(-8) M) . Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S . thermoviolaceus membranes . The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins . These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters . In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli . The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators . The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the -10 region of the bxl operon . The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose . This site was also present in the region located between stxI and stxIV and in the upstream region of stxII . BxlR specifically bound to the regions containing the inverted sequence . These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S . thermoviolaceus OPC-520. J Biol Chem, 2004 Apr 16, 279(16), 15994 - 9 Epub 2004 Feb 03. The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor; Santamaria-Araujo JA et al.; The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase . Moco is synthesized by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation . The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enzymes . Here we report the spectroscopic characterization and structure elucidation of precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial precursor Z synthesis . We have shown that purified precursor Z is as active as precursor Z present in E . coli cell extracts, demonstrating that no modifications during the purification procedure have occurred . High resolution electrospray ionization mass spectrometry afforded a {M + H}+ ion compatible with a molecular formula of C10H15N5O8P . Consequently 1H NMR spectroscopy not allowed structural characterization of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characterized non-productive follow-up product compound Z . The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1' position. J Biol Chem, 2004 Apr 23, 279(17), 17723 - 30 Epub 2004 Feb 02. Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct; Gros L et al.; Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts . E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders . E-adducts are repaired by the base excision repair pathway . Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA . Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA . We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity . Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells . Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I . These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo . The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer. J Biol Chem, 2004 Apr 30, 279(18), 18776 - 82 Epub 2004 Feb 02. Viral evolution as a tool to improve the tetracycline-regulated gene expression system; Das AT et al.; We present viral evolution as a novel and powerful method to optimize non-viral proteins . We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells . The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system . Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system . We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA . We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased . This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system . Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus. J Biol Chem, 2004 Apr 16, 279(16), 16301 - 10 Epub 2004 Feb 02. The binding of C10 oligomers to Escherichia coli transcription termination factor Rho; Chen X et al.; The binding of C10 RNA oligomers to wild type and mutant Escherichia coli transcription termination factor Rho provides a model for the enzyme-RNA interactions that lead to transcription termination . One surprising finding is that wild type Rho binds between five and six C10 oligomers per hexamer with KD = 0.3 microm, and five to six additional C10 molecules with KD = 7 microm . Previously, approximately half this number of oligomer-binding sites was reported (Wang, Y., and von Hippel, P . H . (1993) J . Biol . Chem . 268, 13947-13955); however, the E155K mutant form of Rho, thought at the time to be wild type, was used in that work . The present results with E155K Rho agree with the earlier work . C10 binding with mutant forms of Rho that are altered in RNA interactions, bearing amino acid changes F62S, G99V, F232C, T286A, or K352E, indicate that the higher affinity binding sites constitute what has been termed the primary RNA site, and the lower affinity sites constitute the secondary sites . The binding data together with the crystal structures for wild type Rho (Skordalakes, E., and Berger, J . M . (2003) Cell 114, 135-146) support structurally distinct locations on Rho for the two classes of C10-binding sites . The results are consistent with participation of residues 33 A apart in secondary site RNA interactions . The data further indicate that not all RNA sites on Rho must be filled for full ATPase and transcription termination activity, and suggest a model in which RNA binding to the higher affinity sites leads to a protein conformation change that exposes the previously hidden lower affinity sites. Int J Radiat Biol, 2004 Jan, 80(1), 21 - 7 Excision by the human methylpurine DNA N-glycosylase of cyanuric acid, a stable and mutagenic oxidation product of 8-oxo-7,8-dihydroguanine; Dherin C et al.; PURPOSE: 1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-cyanuric acid (cyanuric acid nucleoside or dCa) has been shown to be formed upon exposure of 8-oxo-7,8-dihydroguanine- (8-oxoG) containing oligodeoxyribonucleotides (ODN) to oxidizing agents . When present in DNA, cyanuric acid (Ca) is readily bypassed by Escherichia coli DNA polymerases, which preferentially incorporate 2'-deoxyadenosine-5'-monophosphate (dAMP) opposite to the lesion . Therefore, Ca could be a mutagenic DNA lesion yielding G.C to T.A transversions like 8-oxoG . These results call attention to the potential importance of secondary oxidation products of 8-oxoG . The present study investigates the capability of several DNA N-glycosylases to remove the Ca lesion in DNA . MATERIALS AND METHODS: A site-specifically modified 22-mer ODN containing a single Ca residue was hybridized with complementary sequences yielding four DNA duplexes harbouring Ca opposite each of the regular DNA bases . The four Ca.N duplexes were used as substrates for nine DNA N-glycosylases from bacterial, yeast or human origin . RESULTS: The results show that the human methylpurine DNA N-glycosylase (Mpg) can remove Ca from DNA duplexes . Interestingly, oxidized base-specific DNA N-glycosylases, Fpg, Nth, Ntg1, Ntg2, Ogg1, hNth1 and hOgg1, cannot repair Ca in DNA . Furthermore, the removal of Ca by Mpg varied markedly depending on the opposite DNA base, the rank being Ca.C=Ca.T>Ca.G=Ca.A . CONCLUSIONS: 8-OxoG-derived lesions in DNA such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), oxaluric acid (Oa), oxazolone (Oz) and Ca are substrates of base excision repair DNA N-glycosylases . Most of them, Sp, Gh, Oa and Oz, are substrates of the oxidized bases-specific enzymes such as Nth or Fpg . In contrast, Ca is substrate of the human methylpurine DNA N-glycosylase (Mpg). Zhonghua Xue Ye Xue Za Zhi, 2003 Dec, 24(12), 644 - 7 {Activating effects of protein transduction domain mediated BCR/ABL protein on CML T cells}; Liu Q et al.; OBJECTIVE: To study the activating effect of protein transduction domain (PTD) mediated BCR/ABL protein on T cells from CML patients . METHODS: The plasmid containing PTD and b3a2 bcr/abl of CML was constructed by genetic engineering and expressed in E . coli . The peripheral blood mononuclear cells from CML patients were stimulated in vitro with purified PTD-BCR/ABL protein and the expression of the early activation antigen CD(69) on CD(8)(+) and CD(4)(+) T cells was detected by flow cytometry (FCM) . RESULTS: The optimal concentration of PTD-BCR/ABL protein for activating CD(8)(+) T cells in vitro was 100 micro g/ml, CD(69) expression peaked in three days stimulation . CD(8)(+) T cells were activated in 10 of 15 CML patients, the expression rate of CD(69) was (15.01 +/- 3.75)% . CD(4)(+) T cells were activated in 4 of 15 patients, the expression rate of CD(69) was (10.32 +/- 3.08)% . Both CD(8)(+) and CD(4)(+) T cells were activated simultaneously in 3 of them . However, neither CD(4)(+) nor CD(8)(+) T cells was activated by stimulation with BCR/ABL protein in all 15 specimens, the expression rate of CD(69) on CD(8)(+) and CD(4)(+) T cells was (1.36 +/- 0.31)% and (1.41 +/- 0.43)%, respectively . There was no difference compared with that of PBS control group (P > 0.05) . CONCLUSION: By using a PTD-mediated antigen delivering system, exogenous BCR/ABL protein can be delivered into APC, processed and presented onto surface of APC to activate Ag-specific CD(8)(+) and CD(4)(+) T cells in vitro. Mol Genet Genomics, 2004 Mar, 271(2), 171 - 9 Epub 2004 Jan 31. The roles of different regions of the CycH protein in c-type cytochrome biogenesis in Sinorhizobium meliloti; Cinege G et al.; Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c . The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery . We propose that these two modules of CycH play roles in different functions of the protein . The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis . Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein . TPR domains are known to mediate protein-protein interactions . Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr-) and nitrogen-fixing (Fix-) phenotypes . We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr+ phenotype . In contrast, only the full-length protein confers the ability to fix nitrogen. Pediatr Emerg Care, 2004 Feb, 20(2), 108 - 11 Acute presentation of infected urachal cysts: case report and review of diagnosis and therapeutic interventions; Allen JW et al.; Urachal remnants, although relatively rare, masquerade as a large number of diverse disorders leading to a high rate of misdiagnosis . A typical case is reported in which a 10-year-old boy presented to the Emergency Department twice before being incorrectly diagnosed with a pelvic or lower abdominal periappendiceal abscess . Definitive diagnosis and treatment of an infected urachal cyst were made intraoperatively . A review and discussion of urachal remnants is presented, and a diagnostic algorithm and treatment plan is offered for this entity. Biol Pharm Bull, 2004 Feb, 27(2), 219 - 21 Expression of the soluble extracellular domain of human thrombopoietin receptor using a maltose-binding protein-affinity fusion system; Zhang Q et al.; The thrombopoietin (TPO) receptor (Mpl) belongs to the family of ligand-dependent cytokine receptors and plays a functional role in regulating platelet production . The signaling capacity largely depends on the binding of TPO to the extracellular domains of the TPO receptor (Mpl-EC) . Because the expression level of Mpl in human tissue is very low, studies on the functional and spatial characteristics of its ligand-binding sites have been limited . In the present study, we report the expression and purification of Mpl-EC as a fusion with the maltose-binding protein (MBP), designated MBP-Mpl-EC . MBP-Mpl-EC was expressed in the cytoplasm of Escherichia coli as a soluble fusion protein . Specific binding of TPO to purified MBP-Mpl-EC was demonstrated by a dot-blot assay and surface plasmon resonance . We conclude that bacterial expression of MBP-Mpl-EC yields large amounts of protein with correct folding and that it can be used for further structure and function analyses. Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1543 - 7 Epub 2004 Feb 02. The metabolic world of Escherichia coli is not small; Arita M; To elucidate the organizational and evolutionary principles of the metabolism of living organisms, recent studies have addressed the graph-theoretic analysis of large biochemical networks responsible for the synthesis and degradation of cellular building blocks {Jeong, H., Tombor, B., Albert, R., Oltvai, Z . N . & Barabasi, A . L . (2000) Nature 407, 651-654; Wagner, A . & Fell, D . A . (2001) Proc . R . Soc . London Ser . B 268, 1803-1810; and Ma, H.-W . & Zeng, A.-P . (2003) Bioinformatics 19, 270-277} . In such studies, the global properties of the network are computed by considering enzymatic reactions as links between metabolites . However, the pathways computed in this manner do not conserve their structural moieties and therefore do not correspond to biochemical pathways on the traditional metabolic map . In this work, we reassessed earlier results by digitizing carbon atomic traces in metabolic reactions annotated for Escherichia coli . Our analysis revealed that the average path length of its metabolism is much longer than previously thought and that the metabolic world of this organism is not small in terms of biosynthesis and degradation. J Biol Chem, 2004 May 7, 279(19), 19486 - 93 Epub 2004 Feb 02. Potential role of methionine sulfoxide in the inactivation of the chaperone GroEL by hypochlorous acid (HOCl) and peroxynitrite (ONOO-); Khor HK et al.; GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress . This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function . Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (.NO) or hydrogen peroxide (H(2)O(2)), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl) . For the exposure of 17.5 microm GroEL to 100-250 microm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A) . In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A . In contrast, HOCl produced only negligible yields of 3-chlorotyrosine . A remarkable finding was the conversion of Met(111) and Met(114) to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL . The high sensitivity of GroEL toward HOCl and ONOO(-) suggests that this protein may be a target for bacterial killing by phagocytes. J Biol Chem, 2004 Apr 16, 279(16), 15897 - 907 Epub 2004 Feb 02. C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized through C-1alpha or C-24 hydroxylation; Kamao M et al.; Recently, it was revealed that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring . We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites . When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively . 25(OH)D3 was at least as sensitive to C-3 epimerization as 1alpha, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction . Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3 . We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3 . The cultured cells metabolized 3-epi-25OHD3 and 3-epi-1alpha,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1alpha,24,25(OH)3D3, respectively . In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1alpha,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems . 3-Epi-25(OH)D3, 3-epi-1alpha,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1alpha,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1alpha,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60) . These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3. J Biol Chem, 2004 Apr 16, 279(16), 16863 - 74 Epub 2004 Feb 02. An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions; Ali V et al.; We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer . A genomewide search showed that E . histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU . Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E . histolytica . The EhNifS protein expressed in E . coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms . Eh-NifU protein existed as a tetramer and contained one stable {2Fe-2S}2+ cluster per monomer, revealed by spectroscopic and iron analyses . Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase . Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions . This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes. J Cell Biol, 2004 Feb 2, 164(3), 407 - 16 Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly; Campellone KG et al.; Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals . One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly . The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation . To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components . Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment . Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation . Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts . Copyright The Rockefeller University Press Blood Cells Mol Dis, 2004 Jan-Feb, 32(1), 176 - 81 A single amino acid change in the binding pocket alters specificity of an anti-integrin antibody AP7.4 as revealed by its crystal structure; Vasudevan S et al.; Monoclonal antibody (mAb) AP7.4 is an anti-integrin antibody recombinantly expressed in Escherichia coli specific to alphavbeta3 . It is known that in a variety of RGD-containing molecules, ligand specificity is regulated by structural determinants within the immediate vicinity of the RGD sequence . To better understand the role of the RGD sequence in integrin specificity, we report here the three-dimensional structure of Fab of mAb AP7.4 to a resolution of 2.25 A . The crystals belong to a triclinic space group P1 and the volume of the unit cell is consistent with the presence of two Fab molecules in it . The RGD sequence is located at the tip of a flexible loop in the complementary determining region (CDR-3) of the heavy chain . It has been shown that specific recognition of RGD ligands by their receptors is influenced mainly by the conformation of the tripeptide RGD and the amino acid residues flanking it on either side . Hence, the flexibility of the RGD-carrying loop observed in the crystal structure may stem from the fact that the antibody molecule mimics the function of these cell adhesion molecules. FEMS Microbiol Lett, 2004 Jan 30, 230(2), 215 - 25 In silico analysis of the sigma54-dependent enhancer-binding proteins in Pirellula species strain 1; Studholme DJ et al.; The planctomycetes are a phylogenetically distinct group of bacteria, widespread in aquatic and terrestrial environments . Their cell walls lack peptidoglycan and their compartmentalised cells undergo a yeast-like budding cell division process . Many bacteria regulate a subset of their genes by an enhancer-dependent mechanism involving the alternative sigma factor sigma54 (RpoN, sigmaN) in association with sigma54-dependent transcriptional activators known as enhancer-binding proteins (EBPs) . The sigma54-dependent regulon has previously been studied in several groups of bacteria, but not in the planctomycetes . We wished to exploit the recently published complete genome sequence of Pirellula species strain 1 to predict and analyse the sigma54-dependent regulon in this interesting group of bacteria . The genome of Pirellula species strain 1 encodes one homologue of sigma54, and 16 sigma54-dependent EBPs, including 10 two-component response regulators and a homologue of Escherichia coli RtcR . Two EBPs contain forkhead-associated domains, representing a novel protein domain combination not previously observed in bacterial EBPs and suggesting a novel link between the enhancer-dependent regulon and 'eukaryotic-like' protein phosphorylation in bacterial signal transduction . We identified several potential sigma54-dependent promoters upstream of genes and operons including two homologues of csrA, which encodes the global regulator CsrA, and rtcBA, encoding a RNA 3'-terminal phosphate cyclase . Phylogenetic analysis of EBP sequences from a wide range of bacterial taxa suggested that planctomycete EBPs fall into several distinct clades . Also the phylogeny of the sigma54 factors is broadly consistent with that of the host organisms . These results are consistent with a very ancient origin of sigma54 within the bacterial lineage . The repertoire of functions predicted to be under the control of the sigma54-dependent regulon in Pirellula shares some similarities (e.g . rtcBA) as well as exhibiting differences with that in other taxonomic groups of bacteria, reinforcing the evolutionarily dynamic nature of this regulon. FEMS Microbiol Lett, 2004 Jan 30, 230(2), 203 - 8 Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization; Sorsa LJ et al.; To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis . The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E . coli K-12 strain MG1655 . We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases . We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E . coli isolates, and 40 non-virulent E . coli strains from stool samples . From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits . Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC. Biochim Biophys Acta, 2004 Jan 28, 1660(1-2), 171 - 99 Metabolism and function of coenzyme Q; Turunen M et al.; Coenzyme Q (CoQ) is present in all cells and membranes and in addition to be a member of the mitochondrial respiratory chain it has also several other functions of great importance for the cellular metabolism . This review summarizes the findings available to day concerning CoQ distribution, biosynthesis, regulatory modifications and its participation in cellular metabolism . There are a number of indications that this lipid is not always functioning by its direct presence at the site of action but also using e.g . receptor expression modifications, signal transduction mechanisms and action through its metabolites . The biosynthesis of CoQ is studied in great detail in bacteria and yeast but only to a limited extent in animal tissues and therefore the informations available is restricted . However, it is known that the CoQ is compartmentalized in the cell with multiple sites of biosynthesis, breakdown and regulation which is the basis of functional specialization . Some regulatory mechanisms concerning amount and biosynthesis are established and nuclear transcription factors are partly identified in this process . Using appropriate ligands of nuclear receptors the biosynthetic rate can be increased in experimental system which raises the possibility of drug-induced upregulation of the lipid in deficiency . During aging and pathophysiological conditions the tissue concentration of CoQ is modified which influences cellular functions . In this case the extent of disturbances is dependent on the localization and the modified distribution of the lipid at cellular and membrane levels. Biochim Biophys Acta, 2004 Jan 28, 1660(1-2), 106 - 17 Loop X/XI, the largest cytoplasmic loop in the membrane-bound melibiose carrier of Escherichia coli, is a functional re-entrant loop; Ding PZ; The melibiose carrier of Escherichia coli is a membrane-bound sugar-cation cotransporter consisting of 12 transmembrane helices connected by cytoplasmic and periplasmic loops, with both N- and C-terminus on the cytoplasmic side . Using a functional cysteine-less carrier, cysteine was substituted individually for residues 347-378 that comprise the largest cytoplasmic loop X/XI . The majority of the cysteine mutants have good protein expression levels . The cysteine mutants were studied for their transport activities, and the inhibitory effects of two sulfhydryl reagents, PCMBS (7-A long) and BM (29-A long) . Cysteine substitution resulted in substantial loss of transport in 12 mutants . While PCMBS caused significant inhibition in only two mutants, T373C and V376C, from the periplasmic side (in a substrate-protective manner), more extensive inhibition pattern was observed from the cytoplasmic side, in seven mutants: V353C, Y358C, V371C, Q372C, T373C, V376C and G378C, suggesting that these residues are along the sugar pathway in the aqueous channel, close to the cytoplasmic side . Furthermore, the inhibitory effect of BM on the inside-out vesicles of the above mutants was clearly less than that of PCMBS, suggesting channel space limitation to large molecules, consistent with those residues being inside the channel . Three second-site revertants (A350C/F268L, A350C/I22S, and A350C/I22N) were selected . They may suggest proximities between loop X/XI and helices I and VIII, in agreement with a re-entrant loop structure . Self thiol cross-linkings of the cysteine mutants on loop X/XI failed to form dimers, suggesting that most of the loop is not surface-exposed from cytoplasmic side . Together, these results strongly indicated a functional re-entrant loop mechanistically important in Na+-coupled transporters. Biochim Biophys Acta, 2004 Jan 28, 1660(1-2), 66 - 74 Glutamate 87 is important for menaquinol binding in DmsC of the DMSO reductase (DmsABC) from Escherichia coli; Geijer P et al.; Escherichia coli dimethylsulfoxide (DMSO) reductase is a trimeric enzyme with a catalytic dimer (DmsAB) and an integral membrane anchor (DmsC) . Using site-directed mutagenesis, we examined six residues in the periplasmic loop between helices two and three, potentially involved in menaquinol binding in DmsC . Mutants were characterised for growth, enzyme expression and activity, and 2-n-heptyl-4-hydroxoquinoline N-oxide (HOQNO) inhibitor binding . Mutations of leucine 66, glycine 67, arginine 71, phenylalanine 73 and serine 75 had no effect on menaquinol binding . Only a glutamate residue (E87) located in helix three was important for menaquinol binding . E87 was replaced with lysine, glutamine and aspartate . All three mutants were assembled into the membrane . Neither the lysine nor the glutamine mutant enzymes were able to support anaerobic growth on glycerol/DMSO minimal media or oxidise lapachol . The glutamine mutant bound the inhibitor with lower affinity compared to wild-type, whereas in the lysine mutant, binding was almost abolished . The aspartate mutant behaved as a wild-type enzyme . The data shows that E87 is important for menaquinol binding and oxidation and is likely to act as a proton acceptor in the menaquinol binding site. Toxicon, 2003 Dec, 42(7), 753 - 61 Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin; Peng LS et al.; Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin . Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids . Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively . Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33 . The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E . coli . After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained . These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences . LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively . Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually . The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed. Domest Anim Endocrinol, 2004 Mar, 26(2), 111 - 26 Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis; Schmitz S et al.; During mammary gland infection, non-specific responses are the predominant ones . The goal of this study was to investigate the mRNA expression of various soluble immune components and of the major milk proteins during the acute phase of mammary inflammation . Five healthy lactating cows were intramammary infused in one quarter with 100 microg Escherichia coli-endotoxin (lipopolysaccharide, LPS) and the contralateral quarter with saline (9 g/l) serving as control . Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of various factors was quantified via real-time RT-PCR . Blood samples for determination of leukocyte number were taken simultaneously with the biopsy samples and rectal temperature was measured at 1-h intervals . Rectal temperature increased until 5h (P < 0.05) after LPS administration and remained elevated until 9 h after LPS inoculation . Blood leukocyte number decreased (P < 0.05) from 0 to 3 h from 7.7 +/- 1.1 x 10(9)l(-1) to 5.7 +/- 1.0 x 10(9)l(-1) and thereafter recovered to pre-treatment levels until 12 h after LPS challenge . In LPS-treated quarters, tumor necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P < 0.05) to highest values at 3h after LPS challenge . Lactoferrin, lysozyme, inducible nitric oxide synthase increased (P < 0.05) and peaked at 6 h after challenge, and platelet-activating factor acetylhydrolase-mRNA expression tended to increase (P = 0.07) . mRNA expression of insulin-like growth factor-I and of alphaS1-casein (CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change significantly, whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin decreased (P < 0.05) in both quarters and that of kappa-CN only in the LPS quarter . mRNA expression of some investigated factors (tumor necrosis factor-alpha, lysozyme, 5-lipoxygenase, alpha-lactalbumin) changed in control quarters, however in all respective factors less than in the LPS quarters (P < 0.05) . In conclusion, mRNA expression of most inflammatory factors increased within hours, whereas that of most milk proteins remained unchanged. J Mol Biol, 2004 Feb 13, 336(2), 313 - 8 Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations; Godoy-Ruiz R et al.; Native proteins are marginally stable . Low thermodynamic stability may actually be advantageous, although the accumulation of neutral, destabilizing mutations may have also contributed to it . In any case, once marginal stability has been reached, it appears plausible that mutations at non-constrained positions become fixed in the course of evolution (due to random drift) with frequencies that roughly reflect the mutation effects on stability ("pseudo-equilibrium hypothesis") . We have found that all glutamate-->aspartate mutations in wild-type Escherichia coli thioredoxin are destabilizing, as well as most of the aspartate-->glutamate mutations . Furthermore, the effect of these mutations on thioredoxin thermodynamic stability shows a robust correlation with the frequencies of occurrence of the involved residues in several-hundred sequence alignments derived from a BLAST search . These results provide direct and quantitative experimental evidence for the pseudo-equilibrium hypothesis and should have general consequences for the interpretation of mutation effects on protein stability, as they suggest that residue environments in proteins may be optimized for stabilizing interactions to a remarkable degree of specificity . We also provide evidence that such stabilizing interactions may be detected in sequence alignments, and briefly discuss the implications of this possibility for the derivation of structural information (on native and denatured states) from comparative sequence analyses. Mol Microbiol, 2004 Jan, 51(2), 539 - 49 Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413; Zahalak M et al.; Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo-nitrogenase . The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth . Two of the three nitrogenases in this strain are Mo-dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain . High-affinity transport of molybdate in A . variabilis was mediated by an ABC-type transport system encoded by the products of modA and modBC . The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli . The deduced ModC part of the fused gene lacked a recognizable molybdate-binding domain . Expression of modA and modBC was induced by starvation for molybdate . Mutants in modA or modBC were unable to grow using nitrate or Mo-nitrogenase . Growth using the alternative V-nitrogenase was not impaired in the mutants . A high concentration of molybdate (10 microM) supported normal growth of the modBC mutant using the Nif1 Mo-nitrogenase, indicating that there was a low-affinity molybdate transport system in this strain . The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations . However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low-affinity molybdate transport system in this strain. Mol Microbiol, 2004 Jan, 51(2), 461 - 9 Kinetics of plasmid segregation in Escherichia coli; Gordon S et al.; Low copy-number bacterial replicons occupy specific locations in their host cells . Production of a GFP-Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid-cell, whereas larger cells tend to have two foci, one at each quarter-cell position . Duplication of the central focus is presumed to represent active partition of plasmid copies . We report here our investigation by time-lapse microscopy of the subsequent movement of these copies to the quarter positions . Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter-cell destinations, where they remained until the next cell cycle . The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation . Aberrant positioning of mini-F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process . From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules . The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed. Mol Microbiol, 2004 Jan, 51(2), 385 - 93 Host processing of branched DNA intermediates is involved in targeted transposition of IS911; Loot C et al.; A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends . We show here that these IR-targeted events occur by an unusual mechanism . In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction . IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure . The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction . SET intermediates must be processed by the bacterial host to obtain the final insertion products . Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another . These sequence changes cannot be explained by the classic transposition pathway . A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication . This pathway resembles those described for processing other branched DNA structures such as stalled replication forks. Mol Microbiol, 2004 Jan, 51(2), 349 - 58 Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability; Simmons LA et al.; Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained . In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal . By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC . Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause . When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability . The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA . We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell. Plant J, 2004 Feb, 37(4), 617 - 25 Glycerol-insensitive Arabidopsis mutants: gli1 seedlings lack glycerol kinase, accumulate glycerol and are more resistant to abiotic stress; Eastmond PJ; The aim of this study was to investigate the process of glycerol catabolism in germinating Arabidopsis seed . A genetic screen was performed to isolate glycerol-insensitive (gli) mutant seedlings . Three separate mutant loci were identified (gli1, gli2 and gli3) . Of these, only gli1 is unable to utilise glycerol . Following germination, gli1 seedlings transiently accumulate glycerol derived from the breakdown of storage oil and are more resistant to hyperosmotic stress, salt stress, oxidative stress, freezing and desiccation . Enzyme assays revealed that gli1 lacks glycerol kinase activity . GLI1 mapped to chromosome 1 near the putative glycerol kinase gene NHO1 . Mutations in this gene were identified in three independent gli1 alleles . A cDNA encoding GLI1 was cloned and its function was proven by complementation of an Escherichia coli glycerol kinase (glpK) deletion strain . Quantitative RT-PCR analysis showed that GLI1 is expressed in all tissues, but is transiently upregulated during early post-germinative growth and leaf senescence . These data show that glycerol kinase is required for glycerol catabolism in Arabidopsis and that the accumulation of glycerol can enhance resistance to a variety of abiotic stresses associated with dehydration. Biochemistry, 2004 Feb 10, 43(5), 1386 - 92 FhuF, part of a siderophore-reductase system; Matzanke BF et al.; FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane . E . coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments . Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores . A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome . Mossbauer spectra revealed that FhuF in its {Fe(2+)-Fe(3+)} state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function . This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates. Biochemistry, 2004 Feb 10, 43(5), 1213 - 22 Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis; Sandmark J et al.; The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA . The Y17F, Y144F, and D147N mutations in the active site were constructed independently . The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme . Crystallographic analyses of these mutants do not show significant changes in the structure of the active site . The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis . The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction . Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site . The D147N mutant is catalytically inactive in both half-reactions . The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role . Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases . This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose . Decarboxylated SAM {S-adenosyl-(5')-3-methylthiopropylamine} reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor. Plant Mol Biol, 2003 Sep, 53(1-2), 189 - 99 Epoxide hydrolase: a mRNA induced by the fungal pathogen Alternaria alternata on rough lemon (Citrus jambhiri Lush); Gomi K et al.; An expression profile of genes induced by non-pathogenic Alternaria alternata on rough lemon leaves was obtained by sequencing 500 subtractive PCR clones generated from mRNA of leaves inoculated with the fungus after subtraction with that of non-inoculated leaves . About 6% of the cDNA sequences had homology to known putative defense-related genes including epoxide hydrolase . A full-length cDNA (951 bp) from rough lemon that encoded epoxy hydrolase was isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR, and designated as RlemEH . The product of this gene expressed with an in vitro translation system with Escherichia coli also had activity of a soluble type of epoxide hydrolase . The transcript of rough lemon RlemEH was not detected in flowers, fruits, stems or leaves, but was induced after inoculation of leaves with conidia of Alternaria alternata, wounding, or treatment with C6 volatiles, including trans-2-hexenol and cis-3-hexenol, and methyl jasmonate . The response of the epoxide hydrolase gene correlated well with the activation of defense mechanisms induced in plant-fungus interactions. Undersea Hyperb Med, 2003 Winter, 30(4), 305 - 11 Lack of toxic side effects in neutrophils following hyperbaric oxygen; Juttner B et al.; Conflicting data have been reported about the impact of repeated HBO2 exposure on the production of superoxide radicals during the neutrophil respiratory burst (RB) and on phagocytosis . In this study we wanted to see if exposure to hyperoxia would affect human neutrophil RB and phagocytosis . Short- and long-term effects after single or repetitive HBO2 exposure of 2.5 atmospheres absolute over a period of 90 min were studied in 40 healthy volunteers . The RB was measured by the intracellular oxidation of dihydrorhodamine after induction by Escherichia coli (E . coli), or priming with recombinant tumour necrosis factor alpha (TNF-alpha), followed by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation . The phagocytic activity was determined by the intake of FITC-labelled opsonized E . coli . No differences could be found between RB and phagocytic activity before and after HBO2 therapy, regardless of short- or long-term exposure . These findings indicate that exposure to hyperoxia does not impair these two important functions of the human innate host defense. Arch Pharm (Weinheim), 2004 Jan, 337(1), 15 - 9 Synthesis of new pyrrolo{2, 3-b}pyridines as a potent inhibitor of tumour necrosis factor alpha; Hilmy KM; The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and 1L-1beta . Accordingly, new pyrrolo{2, 3-}pyridine derivatives 5a-d were prepared from 2-amino-3-cyanopyrroles 3a-d via the intermediate propenylaminopyrroles 4a-d . Then the compounds 5a-d were tested for their ability to inhibit the production of TNF-alpha in vivo in rats . The most potent compounds 5a and 5b possess enhanced ability to inhibit the production of TNF-alpha stimulated with bacterial lipopolysaccharide. Bioorg Med Chem, 2004 Feb 15, 12(4), 675 - 82 A new linker for glucuronylated anticancer prodrugs; Rivault F et al.; The synthesis, enzymatic hydrolysis and self decomposition of model glucuronylated prodrugs, incorporating a new linker with different aryl substituents, have been studied . Determination of kinetic parameters (V(max), K(m) and t(1/2)) showed the important role of aromatic substitution in enzymatic recognition and linker decomposition. Arch Biochem Biophys, 2004 Feb 15, 422(2), 221 - 9 Single chain antibodies that recognize the N-glycosylation site; Kikuchi M et al.; We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity . We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library . Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val . However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides . The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state . These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition . These features are likely to correlate with the known binding specificity of OT. FEBS Lett, 2004 Jan 30, 558(1-3), 39 - 44 Functional properties of soybean nodulin 26 from a comparative three-dimensional model; Biswas S; A model of the nodulin 26 channel protein has been constructed based on comparative modeling and molecular dynamics simulations . Structural features of the protein indicate a selectivity filter that differs from those of the known structures of Escherichia coli glycerol facilitator and mammalian aquaporin 1 . The model structure also reveals important roles of Ser207 and Phe96 in ligand binding and transport. FEBS Lett, 2004 Jan 30, 558(1-3), 13 - 8 Location of the Escherichia coli RNA polymerase alpha subunit C-terminal domain at an FNR-dependent promoter: analysis using an artificial nuclease; Barnard AM et al.; The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain . Using preparations of E . coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter . We conclude that one alpha subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E . coli transcription activator that is related to FNR . In complementary experiments, we show that the second alpha subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR. Mol Cell, 2004 Jan 30, 13(2), 191 - 200 Kinetic determinants of high-fidelity tRNA discrimination on the ribosome; Gromadski KB et al.; The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading . Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally . Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex . tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate. Mol Cell, 2004 Jan 30, 13(2), 157 - 68 Reprogrammed genetic decoding in cellular gene expression; Namy O et al.; Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation . These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine . This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes. Zhonghua Er Ke Za Zhi, 2003 Feb, 41(2), 128 - 30 {Genetic diversity of human Parvovirus B19 VP1 unique region}; Qian XH et al.; OBJECTIVE: Human Parvovirus B19 (HPV B19) is a small (23 nm), non-enveloped DNA virus found in 1974 . It has been proved that HPV B19 is associated with a variety of childhood diseases, such as erythema infectious, transient aplastic crisis, aplastic anemia, idiopathic thrombocytopenic purpura and arthropathy, etc . There have been no any effective vaccines to prevent HPV B19 infection so far . The HPV B19 genome is composed of 5.6 kb single strand DNA . This genome encodes a nonstructural protein NS1, two structural proteins VP1 and VP2 . Most neutralizing linear epitopes of HPV B19 cluster in the VP1 unique and VP1-VP2 junction regions . Only proteins encoded by genes of the VP1 unique and VP1-VP2 junction regions can stimulate bodies to produce protective antibodies . Aim of the present study was to get the VP1 unique region gene of HPV B19 and to analyze the genetic diversity so as to further study its function and application . METHODS: The VP1 unique region gene of HPV B19 was amplified from the serum of a child with idiopathic thrombocytopenic purpura by PCR . The purified PCR product was cloned into pGEM-T easy vector and transfected into the host strain E . coli (DH5 alpha) . Positive clones were chosen and then the target gene was sequenced . RESULTS: The target gene sequence of HPV B19 VP1 unique region was amplified and cloned successfully . It had 705 nucleotides . Compared with the relevant sequences published in Genbank, the sequencing results were revealed with two nucleotides changes in the HPV B19 VP1 unique region, but their coding amino acid were not changed . CONCLUSION: It is suggested that genetic diversity exists in the VP1 unique region of HPV B19 . Construction of the recombinant plasmid of HPV B19 VP1 unique region gene might benefit to further study. J Agric Food Chem, 2004 Feb 11, 52(3), 577 - 80 Enzymatic synthesis of gamma-glutamylvaline to improve the bitter taste of valine; Suzuki H et al.; The taste of several bitter amino acids is reduced, sourness produced, and preference increased by gamma-glutamylization . An enzymatic method for synthesizing gamma-Glu-Val involving bacterial gamma-glutamyltranspeptidase (GGT) was developed . The optimum reaction conditions for the synthesis of gamma-Glu-Val were 20 mM Gln, 300 mM Val, and 0.04 U/ml GGT, pH 10 . After 3-hr incubation at 37 degrees C, 17.6 mM gamma-Glu-Val was obtained, with the yield being 88% . gamma-Glu-Val was purified on a Dowex 1 x 8 column and then identified by NMR. In Vivo, 2003 Nov-Dec, 17(6), 577 - 81 Comparison of cytotoxicity and radical scavenging activity between tea extracts and Chinese medicines; Okayasu H et al.; Three hot water extracts of black tea, green tea and powdered green tea and five Chinese medicines (Shosaiko-tou, Orengedoku-tou, Goshuyu-tou, Choto-san, Keishininjinn-tou) were investigated for their ability to modify nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells, and for their cytotoxicity, radical intensity and scavenging activity . All eight materials significantly reduced the extracellular concentration of NO in the LPS-stimulated Raw 264.7 cells . ESR spectroscopy shows that tea extracts, which had higher cytotoxicity, generated higher amounts of radicals, and more efficiently scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by Fenton reaction) and NO (generated by 1-hydroxyl-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene) than Chinese medicines . Close association between the radical intensity and radical scavenging activity suggests their bimodal (anti-oxidant and pro-oxidant) action . Pretreatment of mice with tea extracts significantly reduced the lethality of Escherichia coli-infection . All tea extracts showed no apparent anti-HIV activity . The present study demonstrates, for the first time, several attractive features of tea extracts in comparison with Chinese medicines, suggesting the possible application of the tea extracts for radical-mediated diseases. Genesis, 2004 Jan, 38(1), 39 - 50 General method for the modification of different BAC types and the rapid generation of BAC transgenic mice; Sparwasser T et al.; Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA . As a consequence, several thousand BAC clones are now mapped to the human and mouse genome . It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially . Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice . We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice . Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available . Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice . genesis 38:39-50, 2004 . Rapid Commun Mass Spectrom, 2004, 18(3), 319 - 24 Rapid screening for S-adenosylmethionine-dependent methylation products by enzyme-transferred isotope patterns analysis; Wan W et al.; We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products . In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution . Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components . To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay . This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis . Biopolymers, 2004 Feb 15, 73(3), 340 - 7 Structural motifs in ribosomal RNAs: implications for RNA design and genomics; Zorn J et al.; The various motifs of RNA molecules are closely related to their structural and functional properties . To better understand the nature and distributions of such structural motifs (i.e., paired and unpaired bases in stems, junctions, hairpin loops, bulges, and internal loops) and uncover characteristic features, we analyze the large 16S and 23S ribosomal RNAs of Escherichia coli . We find that the paired and unpaired bases in structural motifs have characteristic distribution shapes and ranges; for example, the frequency distribution of paired bases in stems declines linearly with the number of bases, whereas that for unpaired bases in junctions has a pronounced peak . Significantly, our survey reveals that the ratio of total (over the entire molecule) unpaired to paired bases (0.75) and the fraction of bases in stems (0.6), junctions (0.16), hairpin loops (0.12), and bulges/internal loops (0.12) are shared by 16S and 23S ribosomal RNAs, suggesting that natural RNAs may maintain certain proportions of bases in various motifs to ensure structural integrity . These findings may help in the design of novel RNAs and in the search (via constraints) for RNA-coding motifs in genomes, problems of intense current focus . Biotechnol Bioeng, 2004 Feb 20, 85(4), 422 - 33 Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media; Whiffin VS et al.; A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli . The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption . In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation . In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction . Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process . The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome . Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1519 - 24 Epub 2004 Jan 30. In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state; Elbaz Y et al.; EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins . Here, we show its synthesis in a cell-free system in a fully functional form . The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamicro(H)(+)-dependent fashion . Here, we used the cell-free system to study the oligomeric properties of EmrE . EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally . Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer . An additional interaction between dimers has been detected by using crosslinking between unique Cys residues . This finding implies the existence of a dimer of dimers. J Biol Chem, 2004 Apr 9, 279(15), 15376 - 84 Epub 2004 Jan 30. The N-terminal metal-binding site 2 of the Wilson's Disease Protein plays a key role in the transfer of copper from Atox1; Walker JM et al.; The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver . Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease . The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP . We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process . The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe . Such selectivity is not observed when free copper is added to N-WNDP . Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper . The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites . Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process . X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1 . Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1 . The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed. Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Dec;68(6 Pt 1):061911 . Epub 2003 Dec 24. Reparametrizing the loop entropy weights: effect on DNA melting curves; Blossey R et al.; Recent advances in the understanding of the melting behavior of double-stranded DNA with statistical mechanics methods lead to improved estimates of the weight factors for the dissociation events of the chains, in particular for interior loop melting . So far, in the modeling of DNA melting, the entropy of denaturated loops has been estimated from the number of configurations of a closed self-avoiding walk . It is well understood now that a loop embedded in a chain is characterized by a loop closure exponent c which is higher than that of an isolated loop . Here we report an analysis of DNA melting curves for sequences of a broad range of lengths (from 10 to 10(6) base pairs) calculated with a program based on the algorithms underlying MELTSIM . Using the embedded loop exponent we find that the cooperativity parameter is one order of magnitude bigger than current estimates . We argue that in the melting region the double helix persistence length is greatly reduced compared to its room temperature value, so that the use of the embedded loop closure exponent for real DNA sequences is justified. Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Dec;68(6 Pt 1):061910 . Epub 2003 Dec 24. Universality and Shannon entropy of codon usage; Frappat L et al.; The distribution functions of codon usage probabilities, computed over all the available GenBank data for 40 eukaryotic biological species and five chloroplasts, are best fitted by the sum of a constant, an exponential, and a linear function in the rank of usage . For mitochondria the analysis is not conclusive . These functions are characterized by parameters that strongly depend on the total guanine and cytosine (GC) content of the coding regions of biological species . It is predicted that the codon usage is the same in all exonic genes with the same GC content . The Shannon entropy for codons, also strongly dependent on the exonic GC content, is computed. J Bone Miner Res, 2004 Jan, 19(1), 89 - 99 Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts; Hu YS et al.; Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation . We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption . OCIL shows promise as a new antiresorptive . INTRODUCTION: Murine and rat osteoclast inhibitory lectins (mOCIL and rOCIL, respectively) are type II membrane C-type lectins expressed by osteoblasts and other extraskeletal tissues, with the extracellular domain of each, expressed as a recombinant protein, able to inhibit in vitro osteoclast formation . MATERIALS AND METHODS: We isolated the human homolog of OCIL (hOCIL) from a human fetal cDNA library that predicts a 191 amino acid type II membrane protein, with the 112 amino acid C-type lectin region in the extracellular domain having 53% identity with the C-type lectin sequences of rOCIL and mOCIL . The extracellular domain of hOCIL was expressed as a soluble recombinant protein in E . coli, and its biological effects were determined . RESULTS AND CONCLUSIONS: The hOCIL gene is 25 kb in length, comprised of five exons, and is a member of a superfamily of natural killer (NK) cell receptors encoded by the NK gene complex located on chromosome 12 . Human OCIL mRNA expression is upregulated by interleukin (IL)-1alpha and prostaglandin E2 (PGE2) in a time-dependent manner in human osteogenic sarcoma MG63 cells, but not by dexamethasone or 1,25 dihydroxyvitamin D3 . Soluble recombinant hOCIL had biological effects comparable with recombinant mOCIL on human and murine osteoclastogenesis . In addition to its capacity to limit osteoclast formation, OCIL was also able to inhibit bone resorption by mature, giant-cell tumor-derived osteoclasts . Thus, a human homolog of OCIL exists that is highly conserved with mOCIL in its primary amino acid sequence (C-lectin domain), genomic structure, and activity to inhibit osteoclastogenesis. J Chromatogr A, 2004 Jan 23, 1024(1-2), 95 - 104 Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system; Kepka C et al.; The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system {(50% ethylene oxide-50% propylene oxide)-Dextran T 500} . The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis . The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase . Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved . By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer . The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA. Abdom Imaging, 2003 Nov-Dec, 28(6), 887 - 8 Residual contrast medium in the cortex of the kidney around an infected renal cyst on CT: case report; Tsuruta T et al.; We present a case of a patient with an infected renal cyst . Delayed computed tomography showed residual contrast medium in the cortex of the kidney around it . Delayed computed tomography might be useful to identify an infected renal cyst. J Biol Chem, 2004 Apr 9, 279(15), 14945 - 53 Epub 2004 Jan 28. End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon; Bibillo A et al.; The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5'-end of one RNA template (the donor) to the 3'-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates . These jumps between RNA templates have similarity to the end-to-end template jumps described for the RNA-directed RNA polymerases encoded by certain RNA viruses . Here we describe for the first time the mechanism by which such end-to-end template jumps can occur . Most template jumps by the R2 reverse transcriptase are brought about by the enzyme's ability to add nontemplated (overhanging) nucleotides to the cDNA when it reaches the end of the donor RNA . The enzyme then anneals these overhanging nucleotides to sequences at the 3'-end of the acceptor RNA . The annealing is most efficient if it involves the terminal nucleotide(s) of the acceptor RNA but can occur to sites at least 5 nucleotides from the 3'-end . These end-to-end jumps are similar to steps proposed to be part of the integration reaction of non-long terminal repeat retrotransposons and can explain chimeric integration products derived from multiple RNA templates. J Biol Chem, 2004 Apr 9, 279(15), 14496 - 501 Epub 2004 Jan 28. Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition; Birgander PL et al.; Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme . Activity is increased by ATP binding and decreased by dATP binding . To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein . All mutant proteins retained about 70% of wild type enzymatic activity . We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response . We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine . Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins . Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins . We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction. Nucleic Acids Res, 2004 Jan 29, 32(2), 570 - 8 Print 2004. Catalytic and DNA-binding properties of the human Ogg1 DNA N-glycosylase/AP lyase: biochemical exploration of H270, Q315 and F319, three amino acids of the 8-oxoguanine-binding pocket; van der Kemp PA et al.; The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA . In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1 . Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity . The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type . The results show that hOgg1 mutated at H270 (H270A and H270L) or F319 (F319A) exhibits greatly reduced (50- to 1000-fold) DNA glycosylase activity, whereas the AP lyase activity is only moderately affected (<4-fold) . The affinity of the hOgg1 mutants (H270A, H270L and F319A) for 8-oxoG.C-containing DNA is also greatly reduced (>30-fold), whereas their affinity for THF.C-containing DNA is only moderately reduced (<7-fold) . The results also show that hOgg1 mutated at Q315 (Q315A) exhibits catalytic and DNA-binding properties similar to those of the wild-type . Therefore, H270 and F319 are essential to form the functional 8-oxoG-binding pocket, whereas Q315 is less crucial . In contrast, H270, Q315 and F319 are not required for efficient binding of THF.C and cleavage of AP sites . Finally, hOgg1 mutant proteins with a substitution of H270A or F319A are members of a new type of hOgg1 that is deficient in DNA glycosylase but proficient in AP lyase. Nucleic Acids Res . 2004 Jan 29;32(2):e22. Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates; Hale MB et al.; We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector . The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites . As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity. J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Mar 5, 801(2), 331 - 7 Application of short monolithic columns for fast purification of plasmid DNA; Branovic K et al.; Anion-exchange chromatography is one of the most important methods in downstream processing of plasmid DNA, both as a process and as an analytical technique . Separation of plasmid DNA on traditional particle-based anion-exchange supports is usually slow . Moreover, such supports have a low capacity for plasmid DNA due to the steric exclusion effects . In this work, the separation of plasmid DNA using short monolithic columns, Convective Interaction Media, will be presented . It will be demonstrated that plasmid DNA can be purified from bacterial cells using alkaline lysis followed by chromatography on a very short weak anion-exchange chromatographic columns-disks-with good purity and quality within a short time . Furthermore, the separation of plasmid DNA from cell RNA can be carried out without the need of adding RNAse . Fast and efficient method for in-process control of the purified plasmid will be described as well. Phytochemistry, 2004 Feb, 65(3), 307 - 12 Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves; Shah K et al.; A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel . Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing . AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases . An expression study showed that it is expressed in root, stem, flower and leaf . It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate . The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed. Anal Biochem, 2004 Feb 15, 325(2), 308 - 16 High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints; Fischer E et al.; 13C-constrained flux balancing analysis based on gas chromatography-mass spectrometry data is presented here as a simple and robust method for the estimation of intracellular carbon fluxes . In this approach, the underdetermined system of metabolite balances deduced from stoichiometric relations and measured extracellular rates is complemented with 13C constraints from metabolic flux ratio analysis . Fluxes in central carbon metabolism of exponentially growing Escherichia coli were estimated by 13C-constrained flux balancing from three different 13C-labeled glucose experiments . The best resolution of the network was achieved using 13C constraints derived from {U-13C}glucose and {1-13C}glucose experiments . The corresponding flux estimate was in excellent agreement with a solution that was independently obtained with a comprehensive isotopomer model . This new methodology was also demonstrated to faithfully capture the intracellular flux distribution in E . coli shake flasks and 1-ml deep-well microtiter plates . Due to its simplicity, speed, and robustness, 13C-constrained metabolic flux balancing is promising for routine and high-throughput analysis on a miniaturized scale. Biochem Biophys Res Commun, 2004 Feb 20, 314(4), 984 - 7 A novel inhibitor protein of N-myristoyltransferase from Escherichia coli; Gowda S et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions . Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer . Attenuation of NMT activity may prove a novel therapeutic protocol for cancer . We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h . The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography . The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration . It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM . The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity . Therefore, we conclude that the inhibitor protein acts directly on NMT. Genes Cells, 2003 Dec, 8(12), 941 - 50 Suppression of spontaneous and hydrogen peroxide-induced mutations by a MutT-type nucleotide pool sanitization enzyme, the Escherichia coli Orf135 protein; Kamiya H et al.; BACKGROUND: We recently found that the Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolysed 2-hydroxy-dATP (2-OH-dATP), and less efficiently, 8-hydroxy-dGTP . RESULTS: In this study, we examined the effects of the absence of the orf135 gene . Frequencies of spontaneous and H2O2-induced mutations were two- to three-fold higher in the orf135- strain than in the wild-type strain . These mutations include various mutations involving a G:C-->T:A transversion, the same type of mutation elicited by 2-OH-dATP . Over-expression of the Orf135 protein suppressed mutations even in the wild-type strain, as well as in the orf135- strain . CONCLUSIONS: The mutator phenotype of bacteria lacking the Orf135 protein suggests that this protein is involved in the suppression of mutations induced by oxidized deoxynucleotides in vivo and that various MutT-type enzymes contribute to nucleotide pool sanitization. J Org Chem, 2004 Feb 6, 69(3), 593 - 600 Active site mapping of 2-deoxy-scyllo-inosose synthase, the key starter enzyme for the biosynthesis of 2-deoxystreptamine . Mechanism-based inhibition and identification of lysine-141 as the entrapped nucleophile; Nango E et al.; A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics including neomycin, kanamycin, gentamicin, etc . is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes the carbocycle formation from d-glucose-6-phosphate to 2-deoxy-scyllo-inosose (DOI) . To clarify its precise reaction mechanism and crucial amino acid residues in the active site, we took advantage of a mechanism-based inhibitor carbaglucose-6-phosphate (pseudo-dl-glucose, C-6-P) with anticipation of its conversion to a reactive alpha,beta-unsaturated carbonyl intermediate . It turned out that C-6-P clearly showed time- and concentration-dependent inhibition against DOIS, and the molecular mass of the resulting modified-DOIS with C-6-P was 160 mass units larger than that of native DOIS . Thus, the expected alpha,beta-unsaturated intermediate appeared to trap a specific nucleophilic group in the active site through the Michael-type 1,4-addition . The covalently modified amino acid residue was determined to be Lys-141 by means of enzymatic digestion and subsequent LC/MS and LC/MS/MS of the digest . Also discussed are the role of Lys-141 in the substrate recognition and the reaction pathway and comparison with evolutionary related dehydroquinate synthase. Scand J Gastroenterol, 2003 Dec, 38(12), 1256 - 61 Phagocytosis and LPS-stimulated production of cytokines and prostaglandin E2 is different in Kupffer cells isolated from the periportal or perivenous liver region; Bykov I et al.; BACKGROUND: Kupffer cells can release pro-inflammatory mediators and contribute to damage, which often appears in a zonated fashion . METHODS: To assess position-associated functional differences, functions of intact Kupffer cells isolated from either the periportal or perivenous acinar region of rat liver were compared . RESULTS: Kupffer cells from the periportal region phagocytosed 2-3 times more FITC-labelled zymosan particles than corresponding perivenous cells, as determined by confocal microscopy and fluorescence assay . Periportal cells also produced more TNF-alpha and IL-1beta, but less NO and PGE2, compared to perivenous cells and the stimulation by addition of lipopolysaccharides (LPS) was moderate . In contrast, after overnight culture LPS dramatically increased TNF-alpha release and significantly more so in perivenous Kupffer cells (26-fold) than in periportal cells (11-fold) . CONCLUSION: Our study suggests that periportal Kupffer cells are responsible for a major part of phagocytosis by the liver . The stronger LPS response of recovered perivenous Kupffer cells suggests a dominant role of these cells in pro-inflammatory events that ultimately may contribute to development of damage in this region. Plant Mol Biol, 2003 Oct, 53(3), 411 - 22 Tomato EF-Ts(mt), a functional mitochondrial translation elongation factor from higher plants; Benichou M et al.; Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Ts(mt) that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts) . Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Ts(mt)-GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Ts(mt) to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein . Escherichia coli recombinant LeEF-Ts(mt) co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E . coli, indicating an interaction with bacterial EF-Tu . Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Ts(mt) fraction . The purified LeEF-Ts(mt) stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu . Furthermore, LeEF-Ts(mt) was capable of catalysing the nucleotide exchange reaction with E . coli EF-Tu . Altogether, these data demonstrate that LeEF-Ts(mt) encodes a functional mitochondrial EF-Ts . LeEF-Ts(mt) represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants. Nature, 2004 Jan 29, 427(6973), 415 - 8 Summing up the noise in gene networks; Paulsson J; Random fluctuations in genetic networks are inevitable as chemical reactions are probabilistic and many genes, RNAs and proteins are present in low numbers per cell . Such 'noise' affects all life processes and has recently been measured using green fluorescent protein (GFP) . Two studies show that negative feedback suppresses noise, and three others identify the sources of noise in gene expression . Here I critically analyse these studies and present a simple equation that unifies and extends both the mathematical and biological perspectives. Exp Mol Med, 2003 Dec 31, 35(6), 578 - 85 Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain; Kim KS et al.; The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis . The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor . Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo . The recombinant u-ATF from E . coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E . coli-derived UK1 showed no binding by Biacore analysis . In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM) . Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane . These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain. Plant Cell Physiol, 2004 Jan, 45(1), 114 - 7 Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs; Hirose T et al.; Many of the chloroplast mRNAs possess Shine-Dalgarno (SD)-like sequences (typically GGAGG) in the 5'-untranslated regions, but the position is highly variable . Using a homologous in vitro translation system, we assessed the role for translation of SD-like sequences in four tobacco chloroplast mRNAs . The rbcL mRNA has a typical SD-like sequence at a position similar to the conserved position (-12 to -4 with respect to the start codon) observed in E . coli, and this sequence was found to be essential for translation . This was also the case for the atpE mRNA . However, SD-like sequences in the rps12 mRNA and in the petB mRNA is located far from (-44 to -42) and too close to (-5 to -2) the initiation codon, respectively, and these sequences were not essential for translation . These results indicate that functional SD-like sequences are located around 10 nucleotides upstream from the translational start codon . Competition assays confirmed that a functional SD-like sequence interacts with the 3' terminus of chloroplast 16S rRNA. Nucleic Acids Res . 2004 Jan 28;32(2):e21. Selective DNA amplification from complex genomes using universal double-sided adapters; Callow MJ et al.; There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species . Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers . This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules . The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event . As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites . Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest. Biotechnol Bioeng, 2004 Feb 5, 85(3), 351 - 8 Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents; Seng Wong T et al.; Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles . For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range . Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency . Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents . By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO . The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction . Biotechnol Bioeng, 2004 Feb 5, 85(3), 293 - 7 Affinity purification of plasmid DNA by temperature-triggered precipitation; Kostal J et al.; This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs) . Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger . In one purification step, plasmid DNA was purified from E . coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins . This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines . Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 379 - 81 Epub 2004 Jan 23. Crystallization and preliminary X-ray crystallographic analysis of the RecR protein from Deinococcus radiodurans, a member of the RecFOR DNA-repair pathway; Lee BI et al.; The RecR protein plays a key role in the RecFOR pathway of recombination, which is necessary for the repair of ssDNA gaps . RecR from Deinococcus radiodurans has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 1000 as a precipitant . X-ray diffraction data to 2.90 A resolution have been collected at 100 K using Cu Kalpha X-rays from a mercury-soaked crystal . The crystal belongs to space group C222(1), with unit-cell parameters a = 106.96, b = 122.25, c = 156.01 A . The asymmetric unit contains four monomers of RecR, with a crystal volume per protein weight (V(M)) of 2.57 A(3) Da(-1) and a solvent content of 51.0%. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 374 - 5 Epub 2004 Jan 23. Crystallization and preliminary X-ray crystallographic studies on recombinant rat choline acetyltransferase; Lian W et al.; Choline acetyltransferase (ChAT) catalyzes the biosynthesis of the neurotransmitter acetylcholine from acetyl-CoA and choline in cholinergic neurons . Rat ChAT (rChAT) was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized . Diffraction data were collected from a single crystal under cryoconditions at the F1 beamline at the Cornell High Energy Synchrotron Source, with a maximal useful diffraction pattern to 1.55 A resolution . The crystals were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 138.97, b = 77.67, c = 59.67 A and a scaling R(sym) of 0.054 for 72 446 unique reflections . Packing considerations indicate there to be one molecule per asymmetric unit . It is expected that in the near future the structure of rChAT will be obtained using molecular-replacement methods . Elucidation of the structure of rChAT will aid in the development of therapeutic agents for Alzheimer's disease. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 371 - 3 Epub 2004 Jan 23. Purification, crystallization and preliminary X-ray diffraction analysis of the catalytic domain of adenylyl cyclase Rv1625c from Mycobacterium tuberculosis; Ketkar AD et al.; The Rv1625c gene product is an adenylyl cyclase identified in the genome of Mycobacterium tuberculosis strain H37Rv . It shows sequence similarity to the mammalian nucleotide cyclases and functions as a homodimer, with two substrate-binding sites at the dimer interface . A mutant form of the catalytic domain of this enzyme, K296E/F363R/D365C (KFD-->ERC), was overexpressed in Escherichia coli cells in a soluble form . Crystals were obtained using the hanging-drop vapour-diffusion method with PEG 8000 as a precipitant . The protein crystallized in space group P4(1), with unit-cell parameters a = b = 71.25, c = 44.51 A . X-ray diffraction data were collected to a resolution of 3.4 A and the structure has been solved by the molecular-replacement method using a previously built theoretical model of the protein as the search molecule. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 365 - 7 Epub 2004 Jan 23. Crystallization and preliminary X-ray crystallographic analysis of a ytfG gene product from Escherichia coli; Kim IK et al.; The Escherichia coli ytfG gene product, with NAD(P)H:quinone oxidoreductase activity, was crystallized by the hanging-drop vapour-diffusion method at 296 K . A 1.78 A data set has been collected using synchrotron radiation at Pohang Light Source, South Korea . The crystal belongs to the primitive trigonal system, with unit-cell parameters a = b = 81.7, c = 76.8 A . Analysis of the packing density shows that the asymmetric unit probably contains one monomer, with a solvent content of 48.8%. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 353 - 6 Epub 2004 Jan 23. Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen; Zhao Y et al.; Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method . Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant . The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A . Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively . Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69% . An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 350 - 2 Epub 2004 Jan 23. Expression, purification and crystallization of human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1; Harjes S et al.; 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is used to incorporate sulfate into biomolecules . The human PAPS synthetase 1 catalyzes two steps leading from adenosine triphosphate (ATP) and sulfate to PAPS . The ATP sulfurylase domain catalyzes the formation of the intermediate adenosine-5'-phosphosulfate (APS) . The APS kinase domain then adds a phosphate group to the 3'-ribose and releases PAPS . In this article, the recombinant expression, purification and crystallization of the full-length protein is described . In Escherichia coli the protein is only partly soluble and copurifies with GroEL . The pure protein migrates as a dimer in gel-filtration chromatography . It is moderately active, forming 25 nmol PAPS per minute per milligram . Crystals grow to 100 x 100 x 300 micro m and diffract to 1.75 A. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 340 - 1 Epub 2004 Jan 23. Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp; Ip DT et al.; A novel orange fluorescent protein, with excitation and emission maxima at 548 and 565 nm, respectively, from the Cnidaria tube anemone Cerianthus sp . has been cloned and overexpressed in Escherichia coli . The orange fluorescent protein has been crystallized by the sitting-drop vapour-diffusion method at 290 K using polyethylene glycol 3350 as a precipitant . A complete set of diffraction data was collected to 2.0 A resolution at 100 K . The crystals belong to the space group R3, with hexagonal unit-cell parameters a = b = 216.947, c = 51.839 A . There are four protein molecules in the asymmetric unit, giving a Matthews coefficient of 2.3 A(3) Da(-1) and a solvent content of 47%. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 334 - 6 Epub 2004 Jan 23. Expression, crystallization and preliminary X-ray studies of the recombinant PTB domain of mouse dok1 protein; Shi N et al.; The PTB domain of mouse dok1 fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study . Crystals have been obtained using the vapour-diffusion method and belong to space group P2(1)2(1)2(1) . X-ray diffraction data were collected in-house to 2.5 A resolution . A selenomethionine (SeMet) dok1 PTB fusion-protein derivative was expressed using the same expression system, purified in a reductive environment and crystals were obtained under similar conditions . Subsequently, three different wavelength data sets from the derivative crystal were collected to 2.5 A resolution at SPring-8. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 304 - 9 Epub 2004 Jan 23. Structure of circularly permuted DsbA(Q100T99): preserved global fold and local structural adjustments; Manjasetty BA et al.; The thiol-disulfide oxidoreductase DsbA is required for efficient formation of disulfide bonds in the Escherichia coli periplasm . The enzyme is the strongest oxidant of the family of thioredoxin-like proteins and three-dimensional structures of both oxidized and reduced forms are known . DsbA consists of a catalytic thioredoxin-like domain and a helical domain that is inserted into the thioredoxin motif . Here, the X-ray structure of a circularly permuted variant, cpDsbA(Q100T99), is reported in which the natural termini are joined by the pentapeptide linker GGGTG, leading to a continuous thioredoxin domain, and new termini that have been introduced in the helical domain by breaking the peptide bond Thr99-Gln100 . cpDsbA(Q100T99) is catalytically active in vivo and in vitro . The crystal structure of oxidized cpDsbA(Q100T99), determined by molecular replacement at 2.4 A resolution, was found to be very similar to that of wild-type DsbA . The lower thermodynamic stability of cpDsbA(Q100T99) relative to DsbA is associated with small structural changes within the molecule, especially near the new termini and the circularizing linker . The active-site helices and adjacent loops display increased flexibility compared with oxidized DsbA. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 275 - 80 Epub 2004 Jan 23. The impact of Lys-->Arg surface mutations on the crystallization of the globular domain of RhoGDI; Czepas J et al.; The potential of rational surface mutagenesis for enhanced protein crystallization is being probed in an ongoing effort . In previous work, it was hypothesized that residues with high conformational entropy such as Glu and Lys are suitable targets for surface mutagenesis, as they are rarely incorporated in crystal contacts or protein-protein interfaces . Previous experiments using Lys-->Ala, Glu-->Ala and Glu-->Asp mutants confirmed that mutated proteins were more likely to crystallize . In the present paper, the usefulness of Lys-->Arg mutations is studied . Several mutations of the globular domain of human RhoGDI were generated, including the single mutants K105R, K113R, K127R, K138R and K141R, the double mutants K(98,99)R and K(199,200)R and the triple mutants K(98,99,105)R and K(135,138,141)R . It is shown that Lys-->Arg mutants are more likely to crystallize than the wild-type protein, although not as likely as Lys-->Ala mutants . Out of the nine mutants tested, five produced diffracting crystals, including the K(199,200)R double mutant, which crystallized in a new space group and exceeded by approximately 1.0 A the resolution of the diffraction of the wild-type crystal . Major crystal contacts in the new lattice were created by the mutated epitope. J Virol, 2004 Feb, 78(4), 2057 - 61 Protection of Penaeus monodon against white spot syndrome virus by oral vaccination; Witteveldt J et al.; White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry . No adequate treatments against this virus are available . It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates . As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins . Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28 . Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection . To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination . A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%) . This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P . monodon . These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry. J Virol, 2004 Feb, 78(4), 1817 - 30 Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression; White EA et al.; The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections . We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions . Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359 . The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles . The IE2 86 Delta 356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC . This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant . Both exhibit detectable upregulation of major immediate-early transcripts at early times . The IE2 86 Delta 427-435 and IE2 86 Delta 505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter . These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection . We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression. J Biol Chem, 2004 Apr 23, 279(17), 17707 - 14 Epub 2004 Jan 27. Divergence in noncognate amino acid recognition between class I and class II lysyl-tRNA synthetases; Levengood J et al.; Lysine insertion during coded protein synthesis requires lysyl-tRNA(Lys), which is synthesized by lysyl-tRNA synthetase (LysRS) . Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria . To compare amino acid recognition between the two forms of LysRS, the effects of l-lysine analogues on aminoacylation were investigated . Both enzymes showed stereospecificity toward the l-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine) . Lysine analogues containing substitutions at other positions were generally most effective as inhibitors of LysRS2 . For example, the K(i) values for aminoacylation of S-(2-aminoethyl)-l-cysteine and l-lysinamide were over 180-fold lower with LysRS2 than with LysRS1 . Of the other analogues tested, only gamma-aminobutyric acid showed a significantly higher K(i) for LysRS2 than LysRS1 . These data indicate that the lysine-binding site is more open in LysRS2 than in LysRS1, in agreement with previous structural studies . The physiological significance of divergent amino acid recognition was reflected by the in vivo resistance to growth inhibition imparted by LysRS1 against S-(2-aminoethyl)-l-cysteine and LysRS2 against gamma-aminobutyric acid . These differences in resistance to naturally occurring noncognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis . In addition, the specific inhibition of LysRS1 indicates it is a potential drug target. Biophys J, 2004 Feb, 86(2), 690 - 704 Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues; Varma S et al.; To understand ion permeation, one must assign correct ionization states to titratable amino acid residues in protein channels . We report on the effects of physical and methodological assumptions in calculating the protonation states at neutral bulk pH of titratable residues lining the lumen of the native Escherichia coli OmpF channel, and five mutants . We systematically considered a wide range of assumed protein dielectric constants and all plausible combinations of protonation states for electrostatically interacting side chains, and three different levels of accounting for solute shielding: 1), full nonlinear Poisson-Boltzmann; 2), linearized Poisson-Boltzmann; and 3), neglect of solute shielding . For this system we found it acceptable to neglect solute shielding, a result we postulate to be generalizable to narrow lumens of other protein channels . For the large majority of residues, the protonation state at neutral bulk pH was found to be independent of the assumed dielectric constant of the protein, and unambiguously determined by the calculation; for native OmpF only Asp-127 has a protonation state that is sensitive to the assumed protein dielectric constant . Our results are significant for understanding two published experimental observations: the structure of the narrow part of the channel, and the ionic selectivity of OmpF mutants. Mol Biochem Parasitol, 2004 Mar, 134(1), 65 - 73 The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo; Morales FC et al.; The complete sequence of SmCys, a cystatin expressed by Schistosoma mansoni, was obtained . Constructs of SmCys consisting of deletions of 10 and 20 amino acid residues from the N-terminal of the full length recombinant protein, were cloned in the pQE-30 vector, expressed in Escherichia coli and assayed for inhibitory activity against papain . Kinetic analysis showed that SmCys -10 and SmCys -20 had K(i) values of 0.7391 and 4.9154, respectively, as compared to 0.0647, displayed by the full length recombinant . Protease inhibition by SmCys was also observed in vivo . When the recombinant products were incubated during 7 days with live schistosomula in the presence of red blood cells, only the full length product could completely inhibit the formation of haemozoin, a dark pigment formed as a by-product of haemoglobin digestion . The sequence data of the recombinant SmCys proteins were used for the construction of molecular models, which were then subjected to molecular dynamics for 2ns . In comparison to the full length, the models corresponding to the truncated constructs, showed a distinctive change on the surface charge distribution . This parameter was more pronounced in SmCys -20, which also displayed a significant displacement of the inhibitory domain, a result which could explain the kinetic data in terms of the loss of attachment sites . These changes correlated well with the progressive lack of inhibition observed for the recombinant deletion constructs, in vitro and in vivo. Vet Parasitol, 2004 Jan 30, 119(2-3), 237 - 45 Effect of acaricides on the activity of a Boophilus microplus glutathione S-transferase; da Silva Vaz I Jr et al.; In the present study, we report the effect of several acaricides on the enzyme activity of a Boophilus microplus recombinant glutathione S-transferase (rGST) . GST was expressed in Escherichia coli and was purified with glutathione (GSH) affinity column chromatography . The kinetic constants were determined by reacting GST with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione . We report the effect of several acaricides on the enzyme activity of rGST . Some acaricides (ethion, amitraz, chlorpyrifos, DDT, cypermethrin, diazinon, ivermectin, deltamethrin and flumethrin) inhibited rGST . Contrarily, coumaphos had an activating effect . Although the accurate mechanisms of the B . microplus resistance to acaricides remain elusive, this work helps in understanding how acaricides can interact with GST. Biochim Biophys Acta, 2004 Jan 20, 1676(2), 182 - 92 Molecular characterization of a light-responsive gene, breast basic conserved protein 1 (OsiBBC1), encoding nuclear-localized protein homologous to ribosomal protein L13 from Oryza sativa indica; Jain M et al.; Rice (Oryza sativa L . subsp . indica) cDNA for the gene OsiBBC1, encoding homologue of the breast basic conserved protein 1 (BBC1), similar to ribosomal protein L13, has been identified and characterized . OsiBBC1 codes for a 24 kDa highly basic protein with two potential bipartite nuclear localization signals (NLS) and a transcriptional activation domain (TAD) . The structural part of the gene is interrupted by four introns . The OsiBBC1 gene is represented by two copies in the rice genome and both of them are expressed . Northern analysis showed that OsiBBC1 is expressed more in the young root, post-fertilized influorescence, leaf base and callus tissue, which are comprised of actively dividing cells, indicating its role in cell division . The OsiBBC1 transcript accumulated more in the root of light-grown seedlings as compared to the shoot while its levels were higher in the shoot as compared to root of the etiolated seedlings, indicating its down-regulation by light . The western analysis, carried out using antibodies raised against a recombinant fusion protein, 6xHis-OsiBBC1, corroborated its tissue-specific expression profile observed by northern analysis . In addition, OsiBBC1/RPL13 protein could be targetted to the nucleus by particle bombardment of OsiBBC1::GUS fusion construct in the onion epidermal cells. Vet Res, 2003 Nov-Dec, 34(6), 721 - 36 The effect of milk production level on host resistance of dairy cows, as assessed by the severity of experimental Escherichia coli mastitis; Kornalijnslijper E et al.; This study investigated the possible effects of milk production level on the host resistance of dairy cows . High (n = 18) and low (n = 18) producing cows on a research farm, which respectively produced 11 443 and 7 727 kg milk in their previous lactation, were compared . To enhance the possible differences in host resistance between high and low producing cows, the animals in both groups were metabolically stressed by overfeeding during the dry period or were fed according to requirements, resulting in four groups of nine cows . The metabolic status was monitored from two weeks pre-partum until 2.5-4.5 weeks post-partum . Host resistance was assessed by measuring the severity of experimentally induced Escherichia coli mastitis . Pre-partum blood glucose levels tended to be higher in overfed cows than in cows fed according to requirements . The post-partum energy balance was significantly more negative in high producing cows than in low producers, and tended to be more negative in overfed cows compared to cows fed according to the requirements . Post-partum plasma glucose, NEFA, beta-OH-butyrate and urea concentrations were similar in the four groups . Plasma glucose concentrations were significantly lower and liver triacylglycerol concentrations were significantly higher in third than in second parity cows . Host resistance was not affected by the production level or feeding regimen . There were no significant correlations between the metabolic status and the severity of experimental E . coli mastitis, except for the relatively more severe mastitis in the cows with beta-OH-butyrate concentrations above 1.4 mmol/L . In conclusion, milk production level did not affect host resistance in dairy cows, as measured by the severity of experimental E . coli mastitis . Even in a situation where cows were metabolically stressed by overfeeding, high producers were as able as low producers to cope with the demands of milk production, without consequences for host resistance. Lett Appl Microbiol, 2004, 38(2), 135 - 9 Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli; Vessoni Penna TC et al.; AIMS: The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity . METHODS AND RESULTS: GFPuv was expressed by Escherichia coli, extracted by the three-phase partitioning method and purified by elution through an hydrophobic interaction column . The collected fractions were further diluted in Tris-HCl-EDTA (pH 8.0) and subjected to continuous heating at set temperatures (45-95 degrees C) . From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (microg ml-1) . To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (Ea) were calculated . CONCLUSIONS: For temperatures between 45 and 70 degrees C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 degrees C, the average decrease in GFPuv fluorescence was between 72 to 83% . SIGNIFICANCE AND IMPACT OF THE STUDY: The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam. J Am Chem Soc, 2004 Feb 4, 126(4), 1055 - 62 Versatile protein biotinylation strategies for potential high-throughput proteomics; Lue RY et al.; We present intein-mediated approaches for efficient biotinylation of proteins site-specifically . The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin . Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion . Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization . In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways . For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system . Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications . They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo. C R Biol, 2003 Dec, 326(12), 1175 - 84 Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo; Pochet S et al.; No backbone motif other than phospho-ribose and phospho-deoxyribose has been found in natural nucleic acids, currently restricting the molecular types of replicable biopolymers to DNA and RNA . With the aim of propagating and expressing a third type of nucleic acid in vivo, we assessed the replicability of polynucleotides with a phospho-hexitol backbone (HNA) in vivo and in vitro . Faithful polymerisation of up to four deoxynucleotides templated by hexitol oligonucleotides was established in vitro using DNA polymerase from Escherichia coli (PolA Klenow exo-fragment) and Thermus aquaticus (Taq polymerase) . Condensation of up to three successive hTTPs (hexitol thymidine triphosphate) in responses to a pentameric hexitol template (hA)5 could also be demonstrated in vitro . Such a marginal HNA-dependent HNA polymerase activity of natural polymerases may be evolved in the future to catalyse in vitro amplification of HNA . The transmission of a two-codon-long genetic message carried on a hexameric hexitol template was also established using a selection screen for restoring thymidylate synthase activity in E . coli . These results exemplify the potential that can be explored by converting artificial substrates with natural enzymes in the field of informational polymer synthesis. Bioessays, 2004 Feb, 26(2), 111 - 5 Expansion of the genetic code in yeast: making life more complex; Davis BK; Proteins account for the catalytic and structural versatility displayed by all cells, yet they are assembled from a set of only 20 common amino acids . With few exceptions, only 61 nucleotide triplets also direct incorporation of these amino acids . Endeavors to expand the genetic code recently progressed to nucleus-containing cells, after Chin et al.1 transferred Escherichia coli genes for a mutant tyrosine-adaptor molecule and its synthetase into Saccharomyces cerevisiae . Transformed yeast cells were produced that exhibit efficient site-specific incorporation of non-biotic amino acids into proteins . This makes it likely that code complexity can be elevated experimentally in mammals . Arch Virol, 2004 Feb, 149(2), 215 - 23 Epub 2003 Oct 30. Identification of a collagen-like protein gene from white spot syndrome virus; Li Q et al.; The most unique feature of white spot syndrome virus (WSSV) is the presence of a collagen-like protein (termed as WSSV-CLP) . In this report, the N-terminal fragment of WSSV-CLP (CLPn) was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified . Specific antibody was then raised against the purified fusion protein (GST-CLPn) . Temporal analysis showed that the WSSV collagen gene was an early viral gene . Immunogold localization using specific antibody revealed that the gold particles, under a transmission electron microscope, were presented along the outer envelope of WSSV virions . This experiment suggested that the collagen gene encoded an envelope protein of WSSV . Using immuno-affinity chromatography, the WSSV-CLP was purified from crudely purified WSSV virions . The WSSV-CLP was N-glycosylated, as indicated by the increased migration in SDS-PAGE after treatment with N-linked glycosidase F. Appl Microbiol Biotechnol, 2004 Aug, 65(3), 295 - 300 Epub 2004 Jan 24. Orally administrable enterotoxigenic Escherichia coli vaccine encapsulated by ethylcellulose powder dispersion; Liao CW et al.; To overcome the limitations of injection administration to vaccinate neonatal piglets against diarrheal disease, an oral vaccine needs to be developed . Enteric microspheres of oral vaccines were developed by a co-spray drying process based on formalin-inactivated enterotoxigenic Escherichia coli antigens with various encapsulating materials . The encapsulating efficiencies of ECN7m, ECN14m and ECN22m (vaccine microsphere formulations) tested by extraction procedure are high, more than 85% . To assess enteric characteristics, an in vitro dissolution test was performed with microspheres . Formulations with ethylcellulose ECN14m and ECN22m allow controlled release in a neutral or basic environment and resisted acid damage . In all cases, 95% of the E . coli protein was released within 2 h at pH 6.8-7, but there was no release at pH 1.5-2 . However, ECN7m was less acid-resistant and had lower release at low pH . In animal immunization tests, oral immunization with microspheres of formulations ECN14 and ECN22m effectively evoked both systemic IgG and mucosal IgA responses against E . coli whole cell antigens in mice . In the mice challenge test, orally administrable ECNm14 (12 mg) or ECN22m (12.6 mg) vaccine (i.e., encapsulating 3.0x10(9) cfu inactive bacterial mass) provided good protection from infection in animals. Eur J Nucl Med Mol Imaging, 2004 Mar, 31(3), 433 - 8 Epub 2004 Jan 27. Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside; Lee KH et al.; There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest . The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene . We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase . We showed that {(125)I}iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein . We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity . COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in {(125)I}iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001) . Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001) . These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene . Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene . Compared with control tumours, lacZ-expressing tumours were slightly better visualised on {(123)I}iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05) . The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels . Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression. Biosci Biotechnol Biochem, 2004 Jan, 68(1), 253 - 6 Introduction of DPR, an enterostatin fragment peptide, into soybean beta-conglycinin alpha' subunit by site-directed mutagenesis; Takenaka Y et al.; DPR, a fragment peptide of enterostatin (VPDPR) having hypocholesterolemic activity, was introduced into the three homologous sites, EPR, DYR, and DPI, in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis . The modified beta-conglycinin was expressed in Escherichia coli and recovered in the soluble fraction . After purification on ion-exchange HPLC, the modified beta-conglycinin was digested by trypsin to release integrated DPR . The yield of DPR from 1 mole of the modified beta-conglycinin was 1.2 mole. Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1496 - 501 Epub 2004 Jan 26. Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids; Basset GJ et al.; It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates . In Escherichia coli, PABA is made from chorismate in two steps . First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC) . The PabC protein then mediates elimination of pyruvate and aromatization to give PABA . Fungi, actinomycetes, and Plasmodium spp . also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB . These bipartite proteins are commonly called "PABA synthases," although it is unclear whether they produce PABA or ADC . Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide . These cDNAs encode functional enzymes, as demonstrated by complementation of an E . coli pabA pabB double mutant and a yeast PABA-synthase deletant . The partially purified recombinant Arabidopsis protein did not produce PABA unless the E . coli PabC enzyme was added, indicating that it forms ADC, not PABA . The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates . When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial . In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol.g(-1) fresh weight. J Biol Chem, 2004 Apr 9, 279(15), 15368 - 75 Epub 2004 Jan 26. Ribosome stalling during translation elongation induces cleavage of mRNA being translated in Escherichia coli; Sunohara T et al.; Recently, it has been found that ribosome pausing at stop codons caused by certain nascent peptides induces cleavage of mRNA in Escherichia coli cells (1, 2) . The question we addressed in the present study is whether mRNA cleavage occurs when translation elongation is prevented . We focused on a specific peptide sequence (AS17), derived from SecM, that is known to cause elongation arrest . When the crp-crr fusion gene encoding CRP-AS17-IIA(Glc) was expressed, cAMP receptor protein (CRP) proteins truncated around the arrest sequence were efficiently produced, and they were tagged by the transfer-messenger RNA (tmRNA) system . Northern blot analysis revealed that both truncated upstream crp and downstream crr mRNAs were generated along with reduced amounts of the full-length crp-crr mRNA . The truncated crp mRNA dramatically decreased in the presence of tmRNA due to rapid degradation . The 3' ends of truncated crp mRNA correspond well to the C termini of the truncated CRP proteins . We conclude that ribosome stalling by the arrest sequence induces mRNA cleavage near the arrest point, resulting in nonstop mRNAs that are recognized by tmRNA . We propose that the mRNA cleavage induced by ribosome stalling acts in concert with the tmRNA system as a way to ensure quality control of protein synthesis and possibly to regulate the expression of certain genes. Biochemistry, 2004 Feb 3, 43(4), 1082 - 92 Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors; Ikushiro H et al.; Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine . We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli . The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate . On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex . Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT . Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion . The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme . Beta-chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner . The detailed mechanisms for the SPT inactivation were discussed . This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments. Biochemistry, 2004 Feb 3, 43(4), 1075 - 81 Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A; Branson JP et al.; The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation . Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes . Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase . Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction . The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form . Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA . C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects . The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond . Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity. Biochemistry, 2004 Feb 3, 43(4), 1054 - 64 Binding of the b-subunit in the ATP synthase from Escherichia coli; Diez M et al.; The rotary mechanism of ATP synthase requires a strong binding within stator subunits . In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli . The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant of 1.8 microM . The binding of b-subunit monomeric and dimeric forms to the isolated F(1) part was investigated by fluorescence correlation spectroscopy and steady-state fluorescence . The mutants b(34-156)T62C and EF(1)-gammaT106C were labeled with several fluorophores . Fluorescence correlation spectroscopy was used to measure translational diffusion times of the labeled b-subunit, labeled F(1), and a mixture of the labeled b-subunit with unlabeled F(1) . Data analysis revealed a dissociation constant of 0.2 nM of the F(1)b(2) complex, yielding a Gibbs free energy of binding of DeltaG(o)= -55 kJ mol(-1) . In steady-state fluorescence resonance energy transfer (FRET) measurements it was found that binding of the b-subunit to EF(1)-gammaT106C-Alexa488 resulted in a fluorescence decrease of one-third of the initial FRET donor fluorescence intensity . The decrease of fluorescence was measured as a function of b-concentration, and data were described by a model including equilibria for dimerization of the b-subunit and binding of b and b(2) to F(1) . For a quantitative description of fluorescence decrease we used two different models: the binding of the first and the second b-subunit causes the same fluorescence decrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2) . Data evaluation revealed a dissociation constant for the F(1)b(2) complex of 0.6 nM (model 1) or 14 nM (model 2), giving DeltaG(o)= -52 kJ mol(-1) and DeltaG(o)= -45 kJ mol(-1), respectively . The maximal DeltaG observed for ATP synthesis in cells is approximately DeltaG= 55 kJ mol(-1) . Therefore, the binding energy of the b-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated in the elastic strain between rotor and stator subunits and then transduced to the catalytic site in one single step . Models in which energy transduction takes place in at least two steps are favored. Biochemistry, 2004 Feb 3, 43(4), 981 - 8 Selection of human cytochrome P450 1A2 mutants with enhanced catalytic activity for heterocyclic amine N-hydroxylation; Kim D et al.; Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo{4,5-f}quinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens . Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products . Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation . Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with >10-fold increased MeIQ activation based on the E . coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k(cat)/K(m)) in steady-state N-hydroxylation assays done with isolated membrane fractions . SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines . The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H(2)O(2) formation . Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate . In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein. Biochemistry, 2004 Feb 3, 43(4), 962 - 9 G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase; Connolly SA et al.; Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase . In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate . This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements . A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts . Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants . Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group . Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1 . Our studies also support an interaction previously proposed between Gln118 and C73 . Defining the RNA-protein interactions critical for efficient aminoacylation by E . coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity. Biochemistry, 2004 Feb 3, 43(4), 953 - 61 Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR; Perez E et al.; Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains . This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation . On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors . Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation . One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer . The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation . The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor . It is likely that this complementarity is involved in discriminating different methylation states of the receptors. Biochemistry, 2004 Feb 3, 43(4), 945 - 52 A positive charge at position 33 of thioredoxin primarily affects its interaction with other proteins but not redox potential; Lin TY et al.; Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif . The redox potentials vary over a wide range for these proteins . A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide . Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential . The substitution does not affect the protein's redox potential . The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium . However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type . The mutation mainly affects K(m), with little effect on k(cat) . The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half . Whether the mutant protein supports the growth of phages T3/7 and f1 was tested . The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered . The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C . The global structure of the mutant protein does not change when studied by CD and fluorescence spectra . Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins . Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential. Biochemistry, 2004 Feb 3, 43(4), 860 - 9 Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX); Tafer H et al.; On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized . (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N{(1)H}-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145 . For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA . The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains . In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster . Both nonrandom structures are present with an abundance of about 25% of the protein molecules . The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein. Mol Cells, 2003 Dec 31, 16(3), 385 - 91 Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini; Ryu J et al.; Poor membrane permeability of proteins is a major limitation of protein therapy . In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD . Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP . The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry . The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus . However, the same protein with PTDs at its both termini was taken up even more efficiently . All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells . Uptake was lower at 4 degrees C than at 37 degrees C . The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs. Mol Cells, 2003 Dec 31, 16(3), 377 - 84 The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction; Lee HJ et al.; The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site . This is followed by strand exchange and finally ligation of the ends of the recombined strands . Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process . We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner . Levels of Mg++ ion below 5 mM also slow strand exchange substantially . We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange. Int J Vitam Nutr Res, 2003 Nov, 73(6), 468 - 77 The effect of graded levels of dietary casein, with or without methionine supplementation, on glutathione concentration in unstressed and endotoxin-treated rats; Alhamdan AA et al.; Glutathione (GSH) concentration was measured in rats fed either graded levels of dietary casein (experiment 1; 180 g, 120 g, 80 g, or 60 g protein/kg diet) or graded levels of dietary casein, supplemented with methionine to equalize dietary sulfur amino acid content to that seen in an 180 g/kg casein diet supplemented with 0.3 g L-methionine/kg diet (experiment 2; 180 g protein +0.3 g L-methionine, 80 g protein +6.70 g L-methionine, or 60 g protein +7.45 g L-methionine/kg diet) . Rats were given an inflammatory challenge by intraperitoneal injection of endotoxin (lipopolysaccharide from Escherichia coli), and were compared with ad libitum and pair-fed controls . Glutathione concentration in various organs (liver, lung, spleen, and thymus) decreased in animals fed the low-protein diets (80 g or 60 g/kg diet) . Addition of the sulfur amino acid, methionine, to the low-protein diets restored glutathione concentrations in animals fed ad libitum and prevented the fall in GSH concentration, which occurred in lung, spleen, and thymus in response to the endotoxin . Despite the similarity in the amount of sulfur amino acid consumed between the groups fed the 180 g protein +0.3 g L-methionine and the 60 g protein +7.45 g L-methionine/kg diet, in experiment 2, hepatic GSH concentration significantly increased in the latter group, in animals fed ad libitum and in the endotoxin-treated animals, but not in the pair-fed controls. J Cell Biochem, 2004 Feb 1, 91(2), 250 - 7 The biological effects of N3-methyladenine; Fronza G et al.; The targeting of damage to DNA remains an attractive strategy to kill tumor cells . One of the serious side effects of alkylating agents is that they create both toxic (desired) and mutagenic (undesired) lesions . The result is that patients successfully treated for a primary cancer are at significant risk to develop cancer related to their therapy . To address this issue we have prepared agents that selectively methylate DNA at the N3-position of adenine . The presence of this lesion in DNA is thought to halt DNA polymerase, and this then initiates a cascade of events including cell death . The toxicity and mutagenicity of the compound, Me-lex, used to generate N3-methyladenine is discussed in bacterial, yeast, and mammalian systems . Mechanisms are proposed to explain the biological activities of N3-methyladenine . Nucleic Acids Res, 2004 Jan 23, 32(2), 477 - 87 Print 2004. Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts; Ichinoe A et al.; There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined . The level of type c mRNA is relatively high in brain . Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHalpha), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHbeta) . An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHalpha and 47 kDa MUTYHbeta, respectively . MUTYHalpha and MUTYHbeta were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice . Both MUTYHalpha and MUTYHbeta were mainly localized in the nuclei and some in mitochondria in wild-type ES cells . Recombinant MUTYHalpha and beta were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHalpha was partly soluble and thus could be purified . Recombinant MUTYHalpha possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity. Infect Immun, 2004 Feb, 72(2), 1216 - 20 The siderophore receptor IroN, but not the high-pathogenicity island or the hemin receptor ChuA, contributes to the bacteremic step of Escherichia coli neonatal meningitis; Negre VL et al.; Using a neonatal rat meningitis model, we examined the involvement of three iron uptake systems, namely, the high-pathogenicity island, the hemin receptor ChuA, and the siderophore receptor IroN, in the pathogenesis of Escherichia coli neonatal meningitis . Only IroN appeared to play a major role during the bacteremic step of the disease. Infect Immun, 2004 Feb, 72(2), 684 - 90 Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin; Pickett CL et al.; A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes . All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin . These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before . Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E . coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not . PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type . Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes. J Biol Chem, 2004 Apr 9, 279(15), 15067 - 75 Epub 2004 Jan 24. Hydrogen bonding in a model bacteriochlorophyll-binding site drives assembly of light harvesting complex; Kwa LG et al.; In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins . In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches . Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties . In contrast, replacement of both helices results in the loss of antenna complexes from the membrane . The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position -4 relative to the liganding histidine of the alpha-subunit . In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix . A hydrogen bond between the hydroxy group of the serine and the 13(1) keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices . The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane . Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site. Carcinogenesis, 2004 Jun, 25(6), 1045 - 51 Epub 2004 Jan 23. Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo; Alekseyev YO et al.; Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts . The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1) . The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms . Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER) . The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER . Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains) . We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair . This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo . Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine. Antimicrob Agents Chemother, 2004 Feb, 48(2), 608 - 11 Replacement of ParC alpha4 helix with that of GyrA increases the stability and cytotoxicity of topoisomerase IV-quinolone-DNA ternary complexes; Pfeiffer ES et al.; Replacement of the alpha4 helix of ParC with that of GyrA increased the stability of topoisomerase IV-quinolone-DNA ternary complexes . This mutant topoisomerase IV-mediated cell killing was more efficient than topoisomerase IV-mediated cell killing in Escherichia coli . Thus, the alpha4 helix plays critical roles in determining the stability and the cytotoxicity of ternary complexes. J Biotechnol, 2004 Feb 19, 108(1), 17 - 30 An improved beta-galactosidase reporter gene; Anson DS et al.; The coding sequence for the E . coli beta-galactosidase gene was codon-optimised for expression in mammalian cells . When expressed in mammalian cells the codon-optimised gene results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E . coli gene sequence . RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency . When used in a lentiviral construct the codon-optimised gene results in an approximately five-fold increase in apparent titre, as determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, in comparison to an analogous construct containing the native E . coli gene . Southern blot analysis shows this is due to an increased efficiency of detection of transduced cells . In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E . coli gene sequence . In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E . coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA . Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E . coli gene sequence . A variant of the codon-optimised beta-galactosidase gene sequence that includes an effective nuclear localisation signal was also made. Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 854 - 60 Profiling the allosteric response of an engineered beta-galactosidase to its effector, anti-HIV antibody; Ferraz RM et al.; Escherichia coli beta-galactosidase responds enzymatically to antiviral antibodies when a viral antigenic peptide, acting as receptor, is conveniently displayed in the vicinity of the active site . The allosteric response of a beta-galactosidase molecular sensor containing a B-cell epitope from HIV has been finely dissected upon binding of an effector monoclonal antibody, within a wide range of standard concentrations of both enzyme and substrate . The topography of the enzymatic activation reveals a wide set of conditions in which the enzymatic response renders a signal over threefold the background, that is suitable for analytical biosensing . Moreover, at discrete enzyme-substrate coordinates, the effector antibody promotes an enhanced activation factor up to fivefold . The insertion of the 37-mer viral peptide between beta-galactosidase residues 795 and 796 is observed as inducer of the structural flexibility required for molecular sensing, whose dynamics and efficiency are intimately associated with the concentrations of enzyme and substrate, the two partners in the signal transduction event. Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 810 - 6 Spectroelectrochemistry of cytochrome P450cam; Bistolas N et al.; The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described . The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite . Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded . Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes . A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined . The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis. Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 737 - 44 Replacement of 198MQMDII203 of mouse IRF-1 by 197IPVEVV202 of human IRF-1 abrogates induction of IFN-beta, iNOS, and COX-2 gene expression by IRF-1; Upreti M et al.; Interferon regulatory factor-1 (IRF-1) is a transcription factor exhibiting functional diversity because of its ability to activate transcription from promoters of several IRF-1-dependent genes . It is a modular protein, where the overall structure is not essential for function of its individual domains . A comparison of the mouse and human IRF-1 amino acid sequences enabled us to identify a stretch of six amino acids (198-203) within the transactivation domain of mouse IRF-1, 198MQMDII(203) to be different from that of the human IRF-1, 197IPVEVV(202) . This indicated a possible functional significance of the six amino acid stretches in the two IRF-1 molecules . The murine IRF-1 sequence at 198-203 (MQMDII) was replaced by IPVEVV . Recombinant wild type mouse IRF-1 with 198MQMDII(203) and its mutant form with 198IPVEVV(203), expressed as GST-IRF-1-fusion proteins, showed similar DNA-binding activity . However, ectopic expression of the wild type and mutant IRF-1 in the human embryonic kidney (HEK-293) cells showed the effect of replacement of this region on expression of a few chromosomal genes that are transcriptionally activated by IRF-1 viz . IFN-beta, iNOS, and COX-2 genes . In our study, expression of wild type IRF-1 activated these genes as judged by RT-PCR but the mutant IRF-1 did not show this effect . Thus, the MQMDII (198-203 a.a.) region of mouse IRF-1 has a functional context in relation to expression of IRF-1-inducible genes. Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 730 - 6 Mammalian D-aspartyl endopeptidase: a scavenger for noxious racemized proteins in aging; Kinouchi T et al.; The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis . Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP) . DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans . A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP . On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome. Biochim Biophys Acta, 2004 Jan 30, 1608(1), 1 - 9 The gross structure of the respiratory complex I: a Lego System; Friedrich T et al.; The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the entry point for electrons into the respiratory chains of many bacteria and mitochondria of most eucaryotes . It couples electron transfer with the translocation of protons across the membrane, thus providing the proton motive force essential for energy-consuming processes . Electron microscopy revealed the 'L'-shaped structure of the bacterial and mitochondrial complex with two arms arranged perpendicular to each other . Recently, we showed that the Escherichia coli complex I takes on another stable conformation with the two arms arranged side by side resulting in a horseshoe-shaped structure . This model reflects the evolution of complex I from pre-existing modules for electron transfer and proton translocation. FEBS Lett, 2004 Jan 16, 557(1-3), 81 - 7 Structural studies on delta(3)-delta(2)-enoyl-CoA isomerase: the variable mode of assembly of the trimeric disks of the crotonase superfamily; Mursula AM et al.; Subunits of the enzymes in the crotonase superfamily form tight trimeric disks . In most members of this protein superfamily these disks assemble further into hexamers . Here we report on the 2.1 A structure of a tight hexameric crystal form of the yeast peroxisomal delta(3)-delta(2)-enoyl-CoA isomerase (Eci1p) . A comparison of this structure to a previously solved crystal form of Eci1p and other structures of this superfamily shows that there is much variability with respect to the relative distance between the disks and their relative orientations . In particular helices H2 and H9 are involved in the inter-trimer contacts and there are considerable structural differences in these helices in this superfamily . Helices H2 and H9 are near the catalytic cavity and it is postulated that the observed structural variability of these helices, stabilized by the different modes of assembly, has allowed the evolution of the wide range of substrate and catalytic specificity within this enzyme superfamily. FEBS Lett, 2004 Jan 16, 557(1-3), 49 - 56 Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins; Umetsu M et al.; Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies . The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure . Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra . The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms . This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies . Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g . 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis . This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation . We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins. J Mol Biol, 2004 Feb 6, 336(1), 287 - 301 Association and dissociation kinetics for CheY interacting with the P2 domain of CheA; Stewart RC et al.; The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR) . The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics . One such feature could be the P2 domain of CheA . P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs . In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100 . Here we examined the kinetics of the binding interactions between CheY and P2 . The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited . Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Huckel shielding . The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation . Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA . However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems. J Mol Biol, 2004 Feb 6, 336(1), 131 - 44 The structure of Escherichia coli ATP-phosphoribosyltransferase: identification of substrate binding sites and mode of AMP inhibition; Lohkamp B et al.; ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme of the histidine pathway, is a complex allosterically regulated enzyme, which controls the flow of intermediates through this biosynthetic pathway . The crystal structures of Escherichia coli ATP-PRT have been solved in complex with the inhibitor AMP at 2.7A and with product PR-ATP at 2.9A (the ribosyl-triphosphate could not be resolved) . On the basis of binding of AMP and PR-ATP and comparison with type I PRTs, the PRPP and parts of the ATP-binding site are identified . These structures clearly identify the AMP as binding in the 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP)-binding site, with the adenosine ring occupying the ATP-binding site . Comparison with the recently solved Mycobacterium tuberculosis ATP-PRT structures indicates that histidine is solely responsible for the large conformational changes observed between the hexameric forms of the enzyme . The role of oligomerisation in inhibition and the structural basis for the synergistic inhibition by histidine and AMP are discussed. Vaccine, 2004 Jan 26, 22(5-6), 793 - 800 Sub-fragments of the envelope gene are highly protective against the Japanese encephalitis virus lethal infection in DNA priming--protein boosting immunization strategies; Wu HH et al.; The envelope (E) gene of Japanese encephalitis virus (JEV) plays a major protective role against JEV infection . In order to locate the part of E gene that is responsible for this protection, an N-terminal fragment EA (nucleotide number 933-1877 bp of JEV genome) and a C-terminal fragment EB (nucleotide number 1851-2330 bp of JEV genome) from E gene were prepared . Both of these fragments were used in the form of recombinant proteins (rEA and rEB) and plasmid DNA (pEA, pM15EA and pEB) for immunizations . Recombinant EA protein (rEA) was previously found to be non-protective because it was expressed in an insoluble form . Plasmid EA (pEA) was also found to be non-protective unless it is preceded by a 15 mer signal peptide derived from the very C-terminal of the membrane gene (M) of JEV to form pM15EA plasmid indicating the importance of the signal peptide in the expression of EA immunogenicity . Although pM15EA and pEB are both immunogenic and protective against JEV lethal infection, the protection by both fragments however is not optimal . Even when pM15EA and pEB were used together for immunization, maximum protection as those induced by control vaccine was not achieved . However, if individual fragments (EA or EB) were used in a DNA priming-protein boosting or protein priming-DNA boosting strategy, high levels of protection were achieved by both fragments . This was especially true for EA fragment where the level of protection against JEV lethal infection was equal to that induced by commercially available vaccine alone . The protection correlated very well with the neutralizing antibody titers and the T helper cell involved in this process in mainly the Th1 type. Vaccine, 2004 Jan 26, 22(5-6), 773 - 80 Immunization of colorectal cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryl lipid A in liposomal emulsion, with and without granulocyte-macrophage colony-stimulating factor; Neidhart J et al.; KSA (Ep-CAM) is highly expressed by colorectal cancers . The safety and immunologic effects of a vaccine consisting of recombinant baculovirus-derived KSA formulated with monophosphoryl lipid A (MPL) in liposomes and emulsified in mineral oil were evaluated, with and without co-administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) . Eleven patients with metastatic colorectal cancer received three subcutaneous (s.c.) injections of the vaccine at 4-week intervals . Six patients were randomized to also receive human recombinant GM-CSF (rGM-CSF) by subcutaneous injection daily for 4 days with each vaccination . Immunizations with and without rGM-CSF were well tolerated . Seven of the 11 patients developed significant KSA-specific cellular immune responses as assessed by lymphoproliferation and interferon-gamma (IFN-gamma) ELISPOT assays . All nine tested patients developed positive delayed type hypersensitivity reactions . Eight of the 11 patients developed KSA-specific antibody responses . The highest levels of cellular immune responses were observed in patients who received GM-CSF . Immunization with baculovirus-derived KSA formulated with monophosphoryl lipid A in liposomal emulsion is safe and can elicit KSA-specific immune responses . Co-administration of GM-CSF with this formulation is an effective method of generating KSA-specific T-helper (Th) 1-associated cellular immune responses. Vaccine, 2004 Jan 26, 22(5-6), 735 - 9 Efficient delivery of DNA to dendritic cells mediated by influenza virosomes; Cusi MG et al.; In an attempt to enhance the immunological efficacy of DNA-based vaccines, we have investigated a new biological means for delivering target gene DNA directly to professional antigen presenting cells (APC), such as the dendritic cells (DC), which are ultimately responsible for the antigen presentation and the primary activation of the immune system . For this purpose we investigated influenza virosomes (IRIV) with assembled DNA as a possible biological carrier for targeting the APC in vivo and in vitro . By cytofluorimetric analysis of the draining lymph nodes of Balb/c mice which had received (by intranasal (in.) administration) FITC-labeled DNA assembled with IRIV, we detected a significant labeled DNA uptake in a subset of lymph node deriving cells expressing DC surface markers . Subsequent mRNA analysis of these lymph nodes showed that the trans-gene delivered by the virosomes was effectively expressed as mRNA . Finally, a further cytofluorimetric analysis performed on human DC-enriched-PMBC, infected in vitro with labeled DNA/IRIV lead to the conclusion that the majority of APC (DC, B lymphocytes and CD16+ cells) are able to incorporate the labeled DNA transported by the construct . These findings suggest that the virosome is an efficient delivery system for testing infectious, as well as anti-cancer, DNA-based vaccine research. Clin Sci (Lond), 2004 Jun, 106(6), 577 - 81 Acute Escherichia coli endotoxaemia decreases the plasma l-arginine/asymmetrical dimethylarginine ratio in humans; Mittermayer F et al.; Acute inflammation impairs vascular function . Based on the association between endothelial dysfunction and plasma concentrations of L-arginine and the endogenous nitric oxide synthase inhibitor ADMA (asymmetrical dimethylarginine), we hypothesized that the ratio between L-arginine and ADMA could be affected by experimental inflammation . Plasma concentrations of L-arginine, ADMA and SDMA (symmetrical dimethylarginine) were studied at baseline and 3.5 h after intravenous administration of Escherichia coli endotoxin {LPS (lipopolysaccharide), 20 units/kg of body mass; n =8} or placebo ( n =9) in healthy males . L-Arginine and dimethylarginines were quantified after solid-phase extraction by reversed-phase HPLC . Body temperature, heart rate and leucocyte count increased after LPS administration ( P <0.01 for all) . LPS administration decreased plasma concentrations of L-arginine from 66 micromol/l {95% CI (confidence interval): 56, 88} at baseline to 48 micromol/l (CI: 40, 60) after 3.5 h ( P <0.02), but did not affect ADMA and SDMA concentrations . Consequently, the L-arginine/ADMA ratio declined significantly from a median of 159 (CI: 137, 193) to 135 (CI: 103, 146); a decrease of 25 (CI: -68, -13; P <0.02) . L-Arginine, ADMA, SDMA and the L-arginine/ADMA ratio remained constant over time in controls . Acute inflammation reduces the L-arginine/ADMA ratio which could contribute to impaired vascular function. J Chem Inf Comput Sci, 2004 Jan-Feb, 44(1), 154 - 60 Development of neural network QSPR models for Hansch substituent constants . 2 . Applications in QSAR studies of HIV-1 reverse transcriptase and dihydrofolate reductase inhibitors; Chiu TL et al.; In this paper, the applications of a Hansch substituent constant predictor(1) to Quantitative Structure-Activity Relationships (QSAR) studies of E . coli dihydrofolate reductase (DHFR) inhibitors 2,4-diamino-5-(substituted-benzyl)pyrimidines as well as HIV-1 reverse transcriptase (RT) inhibitors 1-{(2-hydroxyethoxy)methyl}-6-(phenylthio)thymine (HEPT) derivatives are demonstrated . Both data sets contain functional groups for which the substituent constants (pi, MR, F and R) could not be found in standard substituent constant tables . The substituent constant predictor allowed us to derive predicted pi, MR, F and R values for all substituents in both data sets, thus enabling the generation of easily interpretable QSAR models of comparable or better predictivity than previous models. J Bioenerg Biomembr, 2003 Oct, 35(5), 399 - 407 Overexpression, purification, and characterization of human and bovine mitochondrial ATPase inhibitors: comparison of the properties of mammalian and yeast ATPase inhibitors; Ichikawa N et al.; Mitochondrial ATP synthase (F1F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein . In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor . The human and bovine inhibitors inhibited bovine ATPase in a similar way . The yeast inhibitor also inhibited bovine F1F0-ATPase, although the activity was about three times lower than the mammalian inhibitors . All three inhibitors inhibited yeast F1F0-ATPase in a similar way . The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase . The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity . Common inhibitory sites of mammalian and yeast inhibitors are suggested. J Bioenerg Biomembr, 2003 Oct, 35(5), 389 - 97 Mutagenesis studies of the F1F0 ATP synthase b subunit membrane domain; Hardy AW et al.; A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase . Each b subunit has a single-membrane domain . The constraints on the membrane domain have been studied by systematic mutagenesis . Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase . However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex . These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity . Expression of the b(N2A,T6A,Q10A) subunit was also oxidative phosphorylation deficient, but the b(N2A,T6A,Q10A) protein was incorporated into an F1F0 complex . Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function . The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer. Am J Trop Med Hyg, 2003 Dec, 69(6), 652 - 6 Modulation of cytokine expression in human keratinocytes and fibroblasts by extracts of scabies mites; Arlian LG et al.; Sarcoptes scabiei lives in the stratum corneum of its mammalian host . Keratinocytes and fibroblasts are among the first cells to encounter the burrowing mite and its products . The aim of this study was to determine if molecules in an extract of S . scabiei modulate the expression of cytokines by keratinocytes and fibroblasts . Human keratinocytes and fibroblasts were exposed to an extract of S . scabiei var . canis in the absence or presence of Escherichia coli lipopolysaccharide . Cytokine expression was measured by an enzyme-linked immunosorbent assay . Components in the S . scabiei extract induced marked increases in secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) and slight increases in production of granulocyte-colony-stimulating factor (G-CSF) by keratinocytes . The scabies extract down-regulated keratinocyte secretion of IL-1 receptor antagonist, but did not influence the production of IL-1alpha or IL-1beta . In comparison, components in the scabies extract induced marked increases in the elaboration of IL-6, IL-8, G-CSF, and VEGF by fibroblasts . Neither cell type produced eotaxin, stem cell factor, or tumor necrosis factor-alpha under any of the conditions tested . This study demonstrates that components in an extract of the mite S . scabiei are able to influence cytokine expression by human keratinocytes and fibroblasts. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2003, (121), 34 - 9 {Study on a method for delivering scFv recombinant antibody into cultured cells}; Nakajima O et al.; We try to develop a method for delivering antibody from blood circulation through blood brain barrier to brain . In order to achieve this goal, antibody has to cross cellular membrane of brain capillary endothelial cells twice . As a first step of our study, we examined the ability for scFv antibody to cross cellular membrane of RBL-2H3 cells once and be delivered into the inside of the cultured cells with the help of TAT peptide . TAT peptide was originally found in Tat protein from the HIV-1 virus and known as one of protein transduction domains . First, oligonucleotide encoding TAT peptide was linked to 5' terminal of gene fragment of scFv antibody by PCR technology . TAT-linked scFv gene fragment was subcloned into pET-23b vector and successfully expressed in E . coli as inclusion body . After solubilization and purification, TAT-linked scFv recombinant protein was added to the culture of RBL-2H3 cells . TAT-linked scFv delivered into RBL-2H3 cells was detected by means of immunocytochemistry using fluorescence microscopy . TAT-linked scFv crossed cellular membrane more efficiently than scFv without TAT peptide. Planta, 2004 May, 219(1), 95 - 102 Epub 2004 Jan 22. NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin; Igamberdiev AU et al.; Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO . Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions . Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions . The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate . Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000- g membrane fraction . The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both) . It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins . The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin . Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells . The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios . They were increased under hypoxia and antimycin A treatment . Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates . There was a correspondence between NO degradation and nitrate formation . The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to the size of the hemoglobin dimer . The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor. Planta, 2004 May, 219(1), 84 - 94 Epub 2004 Jan 22. Cloning and functional expression of an ( E, E)-alpha-farnesene synthase cDNA from peel tissue of apple fruit; Pechous SW et al.; Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple ( Malus domestica Borkh.) fruit . The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood . Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, reverse transcription-polymerase chain reaction with RNA isolated from peel tissue of 'Law Rome' apples yielded an approx . 800-bp gene fragment . This was used to screen a cDNA library generated from the peel tissue mRNA . A full-length terpene synthase (TS) cDNA 1,931 nucleotides long was isolated . The 1,728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa . Sequence analysis of the apple TS showed it to be most similar to several monoterpene synthases . Oddly, the TS includes an RR(X(8))W motif near the N-terminus that is common among monoterpene synthases but it lacks the plastid transit peptide sequence typically associated with genes of that group . Expression of the apple TS gene in Escherichia coli gave myc-epitope-tagged and untagged proteins estimated at approx . 68 and approx . 66 kDa, respectively . In assays of sesquiterpene synthase activity, with farnesyl diphosphate as substrate, the untagged bacterially expressed TS gene product synthesized ( E, E)-alpha-farnesene almost exclusively . In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only ( E)-beta-ocimene, albeit at much reduced levels . Addition of a C-terminal myc tag appeared to completely prevent production of soluble protein under all of the expression conditions tested . This is the first report of an ( E, E)-alpha-farnesene synthase gene ( AFS1; GenBank accession number AY182241) from a flowering plant . RNA gel blots showed that AFS1 transcript increased about 4-fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5 degrees C . In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and fell to nearly undetectable levels by 8 weeks. Planta, 2004 Apr, 218(6), 1062 - 70 Epub 2004 Jan 23. Molecular cloning and expression analysis of three genes encoding starch synthase II in rice; Jiang H et al.; Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively . The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52-73% similarity with other members of the plant SSII family . The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli . Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms . Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice . The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed. Acta Biochim Pol, 2003, 50(4), 941 - 5 Non-random distribution of GATC sequences in regions of promoters stimulated by the SeqA protein of Escherichia coli; Strzelczyk B et al.; The SeqA protein of Escherichia coli is not only the main negative regulator of DNA replication initiation but also a specific transcription factor . It binds to hemimethylated GATC sequences and, with somewhat different specificity, to fully methylated GATC regions . Recently, a microarray analysis was reported, in which transcriptomes of wild-type and DeltaseqA strains were compared . Although in the seqA mutant the levels of some transcripts were significantly decreased while certain transcripts were evidently more abundant relative to wild-type bacteria, no correlation between the presence of GATC motifs in promoter sequences and transcription activity was found . However, here we show that when larger DNA fragments, encompassing positions from -250 to +250 relative to the transcription start site, are analyzed, some common features of GATC distribution near the promoters activated by SeqA can be demonstrated . Nevertheless, it seems that the GATC pattern is not the only determinant of SeqA-dependence of promoter activity. Acta Biochim Pol, 2003, 50(4), 921 - 39 UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication; Krwawicz J et al.; Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication . DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork . Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity . This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III) . We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E . coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g . somatic cells) . We show that in E . coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur . This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient . We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication . It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB) . Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant. Acta Biochim Pol, 2003, 50(4), 909 - 20 Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase; Kolasa IK et al.; A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation . They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers . At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments . To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase . We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand . A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated . A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter . All these findings are discussed in relation to recent structural and kinetic models of RPo formation. EMBO J, 2004 Jan 28, 23(2), 294 - 301 Epub 2004 Jan 22. Escherichia coli YidC is a membrane insertase for Sec-independent proteins; Serek J et al.; YidC is a recently discovered bacterial membrane protein that is related to the mitochondrial Oxa1p and the Alb3 protein of chloroplasts . These proteins are required in the membrane integration process of newly synthesized proteins that do not require the classical Sec machinery . Here we demonstrate that YidC is sufficient for the membrane integration of a Sec-independent protein . Microgram amounts of the purified single-spanning Pf3 coat protein were efficiently inserted into proteoliposomes containing the purified YidC . A mutant Pf3 coat protein with an extended hydrophobic region was inserted independently of YidC into the membrane both in vivo and in vitro, but its insertion was accelerated by YidC . These results show that YidC can function separately from the Sec translocase to integrate membrane proteins into the lipid bilayer. EMBO J, 2004 Jan 28, 23(2), 396 - 405 Epub 2004 Jan 22. Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator; Geissmann TA et al.; The Sm-like protein Hfq is involved in post-transcriptional regulation by small, noncoding RNAs in Escherichia coli that act by base pairing . Hfq stabilises the small RNAs and mediates their interaction with the target mRNA by an as yet unknown mechanism . We show here a novel chaperoning use of Hfq in the regulation by small RNAs . We analysed in vitro and in vivo the role of Hfq in the interaction between the small RNA RyhB and its sodB (iron superoxide dismutase) mRNA target . Hfq bound strongly to sodB mRNA and altered the structure of the mRNA, partially opening a loop . This gives access to a sequence complementary to RyhB and encompassing the translation initiation codon . RyhB binding blocked the translation initiation codon of sodB and triggered the degradation of both RyhB and sodB mRNA . Thus, Hfq is a critical chaperone in vivo and in vitro, changing the folding of the target mRNA to make it subject to the small RNA regulator. Virus Genes, 2004 Jan, 28(1), 19 - 39 Investigations on the ORF 167L of lymphocystis disease virus (Iridoviridae); Essbauer S et al.; The predicted open reading frame 167L (ORF 167L) of lymphocystis disease virus (LCDV, Iridoviridae ) isolated from plaice, dab and flounder was investigated . The ORF 167L corresponding genes of the three LCDV isolates were amplified, cloned and sequenced . A comparison of the LCDV strains showed that the nucleotide sequence of ORF 167L and its deduced amino acid sequence were highly conserved in the genus lymphocystivirus (a homology of 80% in dab and flounder/plaice, 97% in plaice and flounder) . The N-terminus protein predicted from the ORF 167L suggests similarities to the tumor necrosis factor receptor (TNFR)-family, and to TNFR-like proteins, which play an important role in various poxvirus species . Further, homology to the CUB-domain was shown at the C-terminus of the LCDV protein . Phylogenetic analyses of partial LCDV protein sequences identified two clusters: one cluster containing the flounder and plaice LCDV isolate (LCDV-1), and another cluster, containing the dab LCDV isolate (LCDV-2) . The ORF 167L of plaice LCDV was expressed in Escherichia coli, and in fish cells . The expressed ORF resulted in a 30-kDa cytoplasmic protein lacking a signal peptide . An established monoclonal antibody (mAb 18) was used to detect LCDV proteins in skin explants of flounders and cryosections of dab skin . Specific fluorescence was found in the cytoplasm of intact epitheloid cells of the lymphocystis capsule and in the epidermis skin covering the lymphocystic nodules . LCDV-specific labelling of mAb 18 was also shown in spleen and liver tissue of LCDV-positive flounders . The ORF 167L protein seemed not to have the extracellular receptor function predicted from the usual cellular TNFR . The myxomavirus M-T2 protein, a poxviral TNFR homologue, was also shown not to have TNFR-like functions but to be involved in the apoptosis signal cascade. Science, 2004 Jan 23, 303(5657), 534 - 7 Snapshots of DsbA in action: detection of proteins in the process of oxidative folding; Kadokura H et al.; DsbA, a thioredoxin superfamily member, introduces disulfide bonds into newly translocated proteins . This process is thought to occur via formation of mixed disulfide complexes between DsbA and its substrates . However, these complexes are difficult to detect, probably because of their short-lived nature . Here we show that it is possible to detect such covalent intermediates in vivo by a mutation in DsbA that alters cis proline-151 . Further, this mutant allowed us to identify substrates of DsbA . Alteration of the cis proline, highly conserved among thioredoxin superfamily members, may be useful for the detection of substrates and intermediate complexes in other systems. Protein Sci, 2004 Feb, 13(2), 370 - 80 An automated in vitro protein folding screen applied to a human dynactin subunit; Scheich C et al.; The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies) . Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives . However, it is difficult to assess the success of such a screen if no biological assay is available . We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy . The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E . coli . The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses. J Biol Chem, 2004 Apr 9, 279(15), 15281 - 8 Epub 2004 Jan 22. Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum; Hinsch KD et al.; Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum . The functions of ODF have not yet been clearly elucidated . We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide . This protein was analyzed by sequencing peptides derived following limited proteolysis . Peptide sequences were found to match VDAC2 and VDAC3 . VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes . In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing . Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits . Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3 . In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs . Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF . Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF . Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria. J Biol Chem, 2004 Apr 23, 279(17), 17004 - 12 Epub 2004 Jan 22. Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7; Kremer W et al.; Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure . In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7 . The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far . Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP . However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran . Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism . In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP . Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution . The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release . In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP. J Exp Bot, 2004 Feb, 55(396), 387 - 95 Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells; Yonamine I et al.; The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control . In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants . Expressions of both AtHAL3 genes are induced by salt stress . AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance . In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity . This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3 . To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified . NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined . Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells . NtHAL3 genes could complement the temperature-sensitive mutation in the E . coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway . Cells overexpressing NtHAL3a had an increased intracellular ratio of proline . Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells. Biochimie, 2003 Dec, 85(12), 1265 - 8 1964: The first model for the shape of a transfer RNA molecule . An account of an unpublished small-angle X-ray scattering study; Witz J; The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolecules in Strasbourg . The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule . The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink . This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D . dissertation (J . Witz, Etude de la structure de quelques polynucleotides en solution par diffusion centrale des rayons X, Ph.D . dissertation, University of Strasbourg, France, 1964) but has never been published in detail . It is the purpose of this note to recall this story. Vet Microbiol, 2004 Jan 14, 98(1), 45 - 53 Influence of porcine intestinal pH and gastric digestion on antigenicity of F4 fimbriae for oral immunisation; Snoeck V et al.; Newly weaned piglets can be orally immunised against F4+ enterotoxigenic Escherichia coli (ETEC) infection with F4 fimbriae . However, to efficiently develop a vaccine against ETEC induced postweaning diarrhoea, knowledge of the stability of the F4 fimbriae to different pH and gastric digestion is needed . The gastrointestinal pH in suckling and recently weaned piglets was measured and the stability of F4 fimbriae to different pH and to pepsin was assessed in vitro . In the stomach the lowest pH was found in the fundus gland region . Gastric pH values below 2.5 were not found in suckling piglets or at the day of weaning, in contrast to piglets 1 and 2 weeks postweaning . Along the first half of the small intestine and in the caecum, a negative correlation was found between pH and age . The F4 fimbriae were stable to pH 1.5 and 2 for 2 h, whereas longer incubation periods resulted in conversion of the multimeric forms into monomers . The F4 fimbriae were partially degraded by incubation for 15-30 min in simulated gastric fluid at pH 1.5 and 2, and completely digested from 3 h onwards . At pH 3, the fimbriae maintained their antigenicity for at least 4h . The results demonstrate that gastric digestion will only have a limited impact on oral immunisation since liquid passes through the stomach relatively quickly (50% within 2 h) . However, we previously demonstrated that the transit times are prolonged shortly after weaning . Shortly after weaning it could be necessary to protect the F4 fimbriae against gastric digestion to obtain efficient oral immunisation of the piglets. Curr Biol, 2004 Jan 20, 14(2), R78 - 80 Molecular chaperones: structure of a protein disaggregase; Mogk A et al.; The ring-forming molecular chaperone Hsp104/ClpB is a member of the AAA+ protein family which rescues proteins from aggregated states . The newly determined crystal structure of ClpB provides new insights into the mechanism of protein disaggregation, suggesting a crowbar activity mediated by a unique coiled-coil domain. Clin Exp Immunol, 2004 Feb, 135(2), 318 - 21 Immune responses to glutamic acid decarboxylase and insulin in patients with gestational diabetes; Fuchtenbusch M et al.; Pregnancy is a natural state of immunoprotection and tolerance . We studied subjects with gestational diabetes (GDM) to evaluate the influence of pregnancy on the humoral immune response to the autoantigen GAD and to injected insulin . Antibodies against glutamic acid decarboxylase (GADA) subclasses and epitope reactivity were determined in 34 GADA-positive pregnant patients with GDM, in 20 GADA-positive relatives of people with TID and in 25 GADA-positive patients with newly diagnosed TID . Partum levels of insulin antibodies (IA), IgG1- and IgG4-IA were measured in 131 women with GDM treated with human insulin from the time of diabetes diagnosis (including 22 with GADA) and were compared to 19 patients with TID after 3 months of insulin treatment . GADA titre and subclasses were similar among all groups . GADA in GDM patients bound fewer epitopes than GADA in relatives of patients with TID (all epitopes being present in 23%versus 65%, P < 0.01) . In particular, antibodies to the minor GADA epitopes GAD6596-249, GAD651-100 and GAD67 were less frequent in patients with GDM compared to relatives (P < 0.01) . Antibodies to insulin (IA) were found in 17% of patients with GDM . They were more frequent in GDM patients with GADA compared to GADA-negative patients (41%versus 12%, P < 0.005) . IgG1 was the dominant insulin antibody subclass response in both patients with GDM and TID but levels of IgG1-IA and IgG4-IA were significantly lower in patients with GDM compared to patients with TID (P < 0.004) . Antibody responses in women with gestational diabetes appear to be dampened and restricted, but without change in subclass usage. Chem Commun (Camb), 2004 Jan 21, (2), 182 - 3 Epub 2003 Dec 09. A fluorescent analogue of UDP-N-acetylglucosamine: application for FRET assay of peptidoglycan translocase II (MurG); Li JJ et al.; A direct continuous fluorescence assay for translocase II MurG based on fluorescence resonance energy transfer (FRET) has been developed using a 6-substituted fluorescent analogue of UDP-N-acetylglucosamine. Chem Commun (Camb), 2004 Jan 7, (1), 118 - 9 Epub 2003 Nov 14. The first SERRS multiplexing from labelled oligonucleotides in a microfluidics lab-on-a-chip; Docherty FT et al.; The first simultaneous detection of three dye-labelled oligonucleotides in a microfluidics chip by SERRS is reported. Proc Natl Acad Sci U S A, 2004 Feb 3, 101(5), 1165 - 70 Epub 2004 Jan 21. Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis; Greif DM et al.; The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM) . In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation . Using {(32)P}orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC . Phosphorylation of CaM by purified CK2 in vitro reduces the V(max) of immunopurified eNOS by a factor of 2 but has no effect on the K(A) for CaM or calcium . Additionally, {(32)P}orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM ("phosphonull" S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the "phosphomimetic" Ser-81 to aspartate (S81D) mutant is unchanged . Further studies using Escherichia coli-expressed and phenyl-Sepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved . Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS . Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue . We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses. J Biol Chem, 2004 Apr 9, 279(15), 14746 - 51 Epub 2004 Jan 21. A novel NADH-linked l-xylulose reductase in the l-arabinose catabolic pathway of yeast; Verho R et al.; An NADH-dependent l-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora . The enzyme is part of the yeast pathway for l-arabinose catabolism . A fungal pathway for l-arabinose utilization has been described previously for molds . In this pathway l-arabinose is sequentially converted to l-arabinitol, l-xylulose, xylitol, and d-xylulose and enters the pentose phosphate pathway as d-xylulose 5-phosphate . In molds the reductions are NADPH-linked, and the oxidations are NAD(+)-linked . Here we show that in A . monospora the pathway is similar, i.e . it has the same two reduction and two oxidation reactions, but the reduction by l-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme . The ALX1 gene encoding the NADH-dependent l-xylulose reductase is strongly expressed during growth on l-arabinose as shown by Northern analysis . The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized . The reversible enzyme converts l-xylulose to xylitol . It also converts d-ribulose to d-arabinitol but has no activity with l-arabinitol or adonitol, i.e . it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the l-configuration and the hydroxyl group of C-3 is in the d-configuration . It also has no activity with C-6 sugars or sugar alcohols . The K(m) values for l-xylulose and d-ribulose are 9.6 and 4.7 mm, respectively . To our knowledge this is the first report of an NADH-linked l-xylulose reductase. J Biol Chem, 2004 Apr 2, 279(14), 13825 - 32 Epub 2004 Jan 20. An R111C polymorphism in wild turkey cardiac troponin I accompanying the dilated cardiomyopathy-related abnormal splicing variant of cardiac troponin T with potentially compensatory effects; Biesiadecki BJ et al.; Cardiac muscle contraction is regulated by Ca(2+) through the troponin complex consisting of three subunits: troponin C (TnC), troponin T (TnT), and troponin I (TnI) . We reported previously that the abnormal splicing of cardiac TnT in turkeys with dilated cardiomyopathy resulted in a greater binding affinity to TnI . In the present study, we characterized a polymorphism of cardiac TnI in the heart of wild turkeys . cDNA cloning and sequencing of the novel turkey cardiac TnI revealed a single amino acid substitution, R111C . Arg(111) in avian cardiac TnI corresponds to a Lys in mammals . This residue is conserved in cardiac and skeletal muscle TnIs across the vertebrate phylum, implying a functional importance . In the partial crystal structure of cardiac troponin, this amino acid resides in an alpha-helix that directly contacts with TnT . Structural modeling indicates that the substitution of Cys for Arg or Lys at this position would not disrupt the global structure of troponin . To evaluate the functional significance of the different size and charge between the Arg and Cys side chains, protein-binding assays using purified turkey cardiac TnI expressed in Escherichia coli were performed . The results show that the R111C substitution lowered binding affinity to TnT, which is potentially compensatory to the increased TnI-binding affinity of the cardiomyopathy-related cardiac TnT splicing variant . Therefore, the fixation of the cardiac TnI Cys(111) allele in the wild turkey population and the corresponding functional effect reflect an increased fitness value, suggesting a novel target for the treatment of TnT myopathies. Br J Ophthalmol, 2004 Feb, 88(2), 273 - 5 Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor; Smith JR et al.; AIM: To investigate the role of arachidonic acid derived chemotactic factor, LTB(4), in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB(4) receptor . METHODS: BLT1 gene deficient and wild type BALB/c mice were injected intravitreally with Escherichia coli 055:B5 lipopolysaccharide (250 ng/2 microl) . Number of leukocytes invading the anterior chamber 24 hours later were counted on tissue cross sections . RESULTS: In all mice, EIU was characterised by a polymorphonuclear and mononuclear cell infiltrate . Numbers of infiltrating cells did not differ significantly between control and BLT1 gene knockout mice . CONCLUSION: Chemotactic factors other than LTB(4) are primarily responsible for leukocyte migration into the eye during murine EIU. J Biotechnol, 2004 Feb 5, 107(3), 265 - 73 A new method for measuring scouring efficiency of natural fibers based on the cellulose-binding domain-beta-glucuronidase fused protein; Degani O et al.; Cellulose-binding domains (CBDs) are characterized by their ability to strongly bind to different forms of cellulose . This study examined the use of a recombinant CBD fused to the reporter enzyme beta-glucuronidase (CBD-GUS) to determine the extent of removal of the water-repellent waxy component of cotton fiber cuticles following hydrolytic treatment, i.e., scouring . The CBD-GUS test displayed higher sensitivity and repeatability than conventional water absorb techniques applied in the textile industry . Increases in the levels of CBD-GUS bound to the exposed cellulose correlated to increases in the fabric's hydrophilicity as a function of the severity of the scouring treatment applied, clearly indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites . The binding of CBD-GUS also gave measurable and repeatable results when used on untreated or raw fabrics in comparison with conventional water drop techniques . The quantitative response of the reaction as bound enzyme activity was optimized for fully wettable fabrics . A minimal free enzyme concentration-to-swatch weight ratio of 75:1 was found to be necessary to ensure enzyme saturation (i.e., a linear response), corresponding to a free enzyme-to-bound enzyme ratio of at least 3:5. J Biotechnol, 2004 Feb 5, 107(3), 233 - 43 Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system; Sasaki Y et al.; Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions . In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined . In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination . The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system . Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown. J Immunol Methods, 2004 Jan, 284(1-2), 165 - 75 Production of a biotinylated single-chain antibody fragment in the cytoplasm of Escherichia coli; Santala V et al.; Biotinylated antibodies are commonly used reagents in research and molecular diagnostics . The traditional approach to biotinylate antibodies is to conjugate a chemically active biotin derivative to certain chemical groups on protein surface . An alternative method, which can be used for site-specific biotinylation of recombinant antibodies, takes advantage of the capability of the enzyme biotin ligase to catalyze the attachment of a biotin to a unique lysine residue in specific protein/peptide substrates that can be genetically linked to the antibody to generate a fusion protein . We describe here expression of functional scFv and concomitant enzymatic biotinylation of it in bacterial cytoplasm . The anti-thyroid-stimulating hormone (TSH) scFv was produced as an N-terminal fusion with the biotinylated domain of the biotin carboxyl carrier protein of Escherichia coli in the redox modified E . coli strain Origami B which has an oxidizing cytoplasmic environment . After optimization of the biotin concentration and expression temperature, this approach allowed the production of biotinylated and immunoreactive fusion protein with the yield of 1.4 mg/l/OD(600) (13.6 mg/l) in a simple shake flask culture . The biotinylated fusion protein released from disrupted cells can be directly used, for example, in immunoassay applications . This was proved by setting up a TSH immunoassay using the bio-scFv as a solid-phase capture antibody . The sensitivity of the assay was comparable with the currently used commercial immunoassays. J Immunol Methods, 2004 Jan, 284(1-2), 99 - 106 Direct comparison of traditional ELISAs and membrane protein arrays for detection and quantification of human cytokines; Copeland S et al.; Many labs wish to measure cytokines in an accurate, reproducible, and rapid manner . An antibody-based membrane array for measuring cytokines has been developed based on the same technology as the traditional ELISA . The aim of this study was to compare results obtained with the traditional ELISA method with those from the membrane array technology, a form of low-cost proteomics . Diluted human whole blood was stimulated with live bacteria (Escherichia coli, or Staphylococous aureus), or LPS and cytokines were measured both by ELISA and the membrane protein array . Of the 16 cytokines measured via ELISA, only IFN-gamma was below detection level . The other 15 cytokines were present in concentrations up to several thousand picograms/ml . Of the 20 cytokines measured via membrane protein array, only 3 could be detected (IL-6, IL-8 and MIP-1beta) . Additionally, the membrane protein array did not detect TNF-alpha from the LPS-stimulated blood . These results indicate that the low-cost membrane protein array may lack sufficient sensitivity to adequately detect cytokines levels in complex biological fluids such as human plasma. Can J Microbiol, 2003 Nov, 49(11), 723 - 6 Expression and characterization of the 42 kDa chitinase of the biocontrol fungus Metarhizium anisopliae in Escherichia coli; Baratto CM et al.; Albeit Metarhizium anisopliae is the best-characterized entomopathogenic fungus, the role of some hydrolytic enzymes during host cuticle penetration has not yet been established . Three chitinase genes (chit1, chi2, chi3) from Metarhizium have already been isolated . To characterize the chitinase coded by the chit1 gene, we expressed the active protein (CHIT42) in Escherichia coli using a T7-based promoter expression vector . The recombinant protein, CHIT42, is active against glycol chitin and synthetic N-acetylglucosamine (GlcNAc) dimer and tetramer substrates . These activities suggest that the recombinant CHIT42 acts as an endochitinase. Mol Biotechnol, 2004 Jan, 26(1), 35 - 8 Cloning genes from a library using a clustering strategy and PCR; Diaz Prado S et al.; A new polymerase chain reaction (PCR)-based method is described for the isolation of clones of interest from a library when only part of a sequence is available . In actuality, this occurs with many genomes that have been partially sequenced using a random strategy . The method presented here, discriminating clusters by PCR (DCbyPCR), is a nonradioactive and improved alternative to colony hybridization. Mol Biotechnol, 2004 Jan, 26(1), 27 - 34 A novel simple and rapid PCR-based site-directed mutagenesis method; Rabhi I et al.; Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed . Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector . In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest . The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified . The PCR is carried out using Pfu DNA polymerase . The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli . Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing . This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively. Mol Biotechnol, 2004 Jan, 26(1), 1 - 6 Establishment of stable melanoma cell line expressing a novel gene, jpk, using a tetracycline-controlled gene expression system; Kim BG et al.; Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed . To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells . Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations . The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin . Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical . Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future. J Immunol, 2004 Feb 1, 172(3), 1916 - 25 Lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs; Lemke CD et al.; Amid growing evidence that numerous viral infections can produce immunopathology, including nonspecific polyclonal lymphocyte activation, the need to test the direct impact of an infecting virus on the immune system of the host is crucial . This can best be tested in the isolator piglet model in which maternal and other extrinsic influences can be excluded . Therefore, neonatal isolator piglets were colonized with a benign Escherichia coli, or kept germfree, and then inoculated with wild-type porcine reproductive and respiratory syndrome virus (PRRSV) or sham medium . Two weeks after inoculation, serum IgM, IgG, and IgA levels were 30- to 50-, 20- to 80-, and 10- to 20-fold higher, respectively, in animals receiving virus vs sham controls, although <1% was virus specific . PRRSV-infected piglets also had bronchial tree-associated lymph nodes and submandibular lymph nodes that were 5-10 times larger than colonized, sham-inoculated animals . Size-exclusion fast performance liquid chromatography revealed that PRRSV-infected sera contained high-molecular-mass fractions that contained IgG, suggesting the presence of immune complexes . Lesions, inflammatory cell infiltration, glomerular deposits of IgG, IgM, and IgA, and Abs of all three isotypes to basement membrane and vascular endothelium were observed in the kidneys of PRRSV-infected piglets . Furthermore, autoantibodies specific for Golgi Ags and dsDNA could be detected 3-4 wk after viral inoculation . These data demonstrate that PRRSV induces B cell hyperplasia in isolator piglets that leads to immunologic injury and suggests that the isolator piglet model could serve as a useful model to determine the mechanisms of virus-induced immunopathology in this species. J Immunol, 2004 Feb 1, 172(3), 1727 - 34 Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses; Kikuchi T et al.; Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia . In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified . In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L . pneumophila . Heat- and formalin-killed L . pneumophila, but not live L . pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns . The mechanisms of the DC maturation by heat- or formalin-killed L . pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively . After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L . pneumophila . The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L . pneumophila . Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L . pneumophila, as evidenced by the up-regulation of MHC class II . Finally, DCs, but not macrophages, exhibited a proliferative response to live L . pneumophila that was consistent with their cell cycle progression . These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection. J Nucl Med, 2004 Jan, 45(1), 30 - 9 Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates; Griffiths GL et al.; The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol . METHODS: We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates . The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit . The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies . A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system . Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer . RESULTS: A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work . We synthesized the bivalent peptide termed IMP 241 {DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)} and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling . When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody . For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide . The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins . High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241 . When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively . CONCLUSION: The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application. FASEB J, 2004 Mar, 18(3), 534 - 6 Epub 2004 Jan 20. Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice; Anders HJ et al.; How bacterial or viral infections trigger flares of autoimmunity is poorly understood . As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E . coli) on the course of nephritis in MRL(lpr/lpr) mice . In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates . After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls . Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro . In vivo both E . coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice . This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria . CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment . In contrast control GpC-ODN had no effect . We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis. J Biol Chem, 2004 Apr 23, 279(17), 17562 - 9 Epub 2004 Jan 20. Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition; Lee MH et al.; Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families . There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets . TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-alpha-converting enzyme, ADAM-17) . The molecular basis for such inactivity is unknown . Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L) . Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP . Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE . The binding affinity of the mutant (V4S/TIMP-3-AB-loop/V69L/T98L) (K (app)(i) 0.14 nm) surpassed that of TIMP-3 (K (app)(i) 0.22 nm), the only natural TIMP inhibitor of the enzyme . The requirement for leucine is absolute for the transformation in inhibitory pattern . On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1 . Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity. J Biol Chem, 2004 Apr 16, 279(16), 16368 - 76 Epub 2004 Jan 20. Stopped-flow fluorescence analysis of the conformational changes in the GroEL apical domain: relationships between movements in the apical domain and the quaternary structure of GroEL; Taniguchi M et al.; GroEL undergoes numerous conformational alterations in the course of facilitating the folding of various proteins, and the specific movements of the GroEL apical domain are of particular importance in the molecular mechanism . In order to monitor in detail the numerous movements of the GroEL apical domain, we have constructed a mutant chaperonin (GroEL R231W) with wild type-like function and a fluorescent probe introduced into the apical domain . By monitoring the tryptophan fluorescence changes of GroEL R231W upon ATP addition in the presence and absence of the co-chaperonin GroES, we detected a total of four distinct kinetic phases that corresponded to conformational changes of the apical domain and GroES binding . By introducing this mutation into a single ring variant of GroEL (GroEL SR-1), we determined the extent of inter-ring cooperation that was involved in apical domain movements . Surprisingly, we found that the apical domain movements of GroEL were affected only slightly by the change in quaternary structure . Our experiments provide a number of novel insights regarding the dynamic movements of this protein. J Biol Chem, 2004 Apr 2, 279(14), 14464 - 71 Epub 2004 Jan 20. Differential specificity of human and Escherichia coli endonuclease III and VIII homologues for oxidative base lesions; Katafuchi A et al.; In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2) . In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates . hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1 . This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII . Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates . In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal . hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues . The profiles of the excision of the Tg isomers with HeLa and E . coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells . However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells. Bioinformatics, 2004 Jan 22, 20(2), 226 - 34 Minimal cut sets in biochemical reaction networks; Klamt S et al.; MOTIVATION: Structural studies of metabolic networks yield deeper insight into topology, functionality and capabilities of the metabolisms of different organisms . Here, we address the analysis of potential failure modes in metabolic networks whose occurrence will render the network structurally incapable of performing certain functions . Such studies will help to identify crucial parts in the network structure and to find suitable targets for repressing undesired metabolic functions . RESULTS: We introduce the concept of minimal cut sets for biochemical networks . A minimal cut set (MCS) is a minimal (irreducible) set of reactions in the network whose inactivation will definitely lead to a failure in certain network functions . We present an algorithm which enables the computation of the MCSs in a given network related to user-defined objective reactions . This algorithm operates on elementary modes . A number of potential applications are outlined, including network verifications, phenotype predictions, assessing structural robustness and fragility, metabolic flux analysis and target identification in drug discovery . Applications are illustrated by the MCSs in the central metabolism of Escherichia coli for growth on different substrates . AVAILABILITY: Computation and analysis of MCSs is an additional feature of the FluxAnalyzer (freely available for academic users upon request, special contracts for industrial companies; see web page below) . Supplementary information: http://www.mpi-magdeburg.mpg.de/projects/fluxanalyzer FEMS Microbiol Lett, 2004 Jan 15, 230(1), 9 - 12 Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli; Burdychova R et al.; This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies . The gene coding Apx I toxin was amplified from the A . pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter . The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E . coli cells . The gene coding A . pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin . Expression of the Apx I coding sequence in E . coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol . The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 646 - 53 Eotaxin and monocyte chemotactic protein-3 use different modes of action; Chung IY et al.; Eotaxin selectively binds CC chemokine receptor (CCR) 3, whereas monocyte chemotactic protein (MCP)-3 binds CCR1, CCR2, and CCR3 . To identify the functional determinants of the chemokines, we generated four reciprocal chimeric chemokines-M10E9, M22E21, E8M11, and E20M23-by shuffling the N-terminus and N-loop of eotaxin and MCP-3 . M22E21 and E8M11, which shared the N-loop from MCP-3, bound to monocytes with high affinity, and activated monocytes . In contrast, M10E9 and E20M23, which lacked the N-loop, failed to bind and transduce monocyte responses, identifying the N-loop of MCP-3 as the selectivity determinant for CCR1/CCR2 . A BIAcore assay with an N-terminal peptide of CCR3 (residues 1-35) revealed that all chimeras except E20M23 exhibited varying degrees of binding affinity with commensurate chemotaxis activity of eosinophils . Surprisingly, E20M23 could neither bind the CCR3 peptide nor activate eosinophils, despite having both N-terminal motifs from eotaxin . These results suggest that the two N-terminal motifs of eotaxin must cooperate with other regions to successfully bind and activate CCR3. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 622 - 30 Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein; Yang L et al.; We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli . Huang et al . The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs . To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein . RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed . Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA . More interestingly, the C-terminal domain was a target of protein kinase C (PKC) . Phosphorylation of the C-terminal domain of p68 abolished its RNA binding . Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68 . The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 555 - 60 Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system; Landino LM et al.; Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases . We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH . Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot . Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent . Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition . In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC . In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 459 - 67 Analysis of type I signal peptidase affinity and specificity for preprotein substrates; Geukens N et al.; Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins . Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance . The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis . In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M) . The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity . The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening . In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 434 - 9 Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex; Yamamoto T et al.; We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L . cv . Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex . Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly . The relationship between the two is unexpected, but of great interest . The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa . Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent . The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2) . The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme. J Endotoxin Res, 2003, 9(6), 361 - 6 Structural basis for endotoxic and antagonistic activities: investigation with novel synthetic lipid A analogs; Kusumoto S et al.; Our early work using homogeneous synthetic preparations demonstrated the presence of a lipid A analog which antagonizes endotoxic activities of LPS and lipid A . The first example was a tetraacylated biosynthetic precursor, now known as precursor Ia or lipid IVa, that contains four 3-hydroxytetradecanoyl moieties linked to the bisphosphorylated disaccharide backbone common to the endotoxic hexa-acyl Escherichia coli lipid A . Various compounds with both endotoxic and antagonistic activities have subsequently been reported from either natural or synthetic sources, but little is known about the factors determining the type of the activities of the respective compounds . To approach this issue, we have synthesized a series of lipid A analogs with various numbers and chain lengths of acyl groups on the backbone . Some were prepared by the aid of a novel affinity separation procedure . The phosphate moieties were also synthetically replaced . Biological tests showed that at least three acyl groups are required for antagonistic activity but one or even both of the phosphates can be replaced with other acidic moieties without losing the activity . The effect of Kdo residues linked to lipid A is also briefly discussed . Molecular dynamics calculations reasonably explain possible conformations required for the biological activity. Bioconjug Chem, 2004 Jan-Feb, 15(1), 16 - 26 Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand; Albrecht H et al.; ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs . In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv . The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain . Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics . Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared . In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys . These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues . In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates . ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2 . ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC. Lipids, 2003 Nov, 38(11), 1167 - 72 Kinetics of barley FA hydroperoxide lyase are modulated by salts and detergents; Koeduka T et al.; The cDNA from barley coding FA hydroperoxide lyase (HPL) was cloned . A recombinant protein derived from the cDNA was expressed in Escherichia coli as an active enzyme . Thus far, there have been no reports on HPL in monocotyledonous plants . The recombinant protein was shown to be most active to linolenic acid 13-hydroperoxide, followed by linoleic acid 13-hydroperoxide . 9-Hydroperoxides of the FA could not be substrates for the recombinant HPL . The activity was dramatically enhanced in the presence of a detergent and/or a salt in the reaction mixture . At the same time, the kinetics of the reaction, including inactivation and the Vmax value of the HPL, were also greatly modulated, depending on the concentration of a monovalent cation and/or a detergent in the reaction mixture . These results suggest that these effectors induced a conformational change in barley HPL, resulting in an improvement in substrate binding and in enzyme activity. Lipids, 2003 Nov, 38(11), 1149 - 56 Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay; Cho KH et al.; The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7alpha-hydroxylase, which in turn regulates bile acid production and cholesterol excretion . To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library . Three human FXR cDNA, designated FXR20, FXR33, and FXR53 cDNA, were subcloned and ligated into a pET28a expression vector . Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography . Approximately 5 mg of FXR33 (1-182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR53 (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity . The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N1-labeled anti-His antibody . From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR33 and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 microM) . The ligand-binding domain of FXR (FXR33) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR53) or the DNA-binding domain of FXR (FXR20). Acta Biochim Biophys Sin (Shanghai), 2004 Jan, 36(1), 37 - 41 DNA vaccine of SARS-Cov S gene induces antibody response in mice; Zhao P et al.; The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of the most important antigen candidates for vaccine design . In the present study, three fragments of the truncated S protein were expressed in E.coli, and analyzed with pooled sera of convalescence phase of SARS patients . The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice . The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E . coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen . The results showed that all the three fragments of S protein expressed by E.coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization . These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines. Acta Biochim Biophys Sin (Shanghai), 2004 Jan, 36(1), 27 - 32 Purification, gene cloning and expression of an acidic phospholipase A2 from Agkistrodon shedaoensis Zhao; Jin Q et al.; A protein with the activity of phospholipase A(2) named asAPLA(2) was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S, and Mono Q FPLC . Its molecular weight was estimated to be 19 kD by SDS-PAGE, and its pI was about 3.5 by IEF analysis . It inhibited the platelet aggregation that was induced by 1 micromol/ L ADP, and the IC(50) was determined to be 6 micromol/L . Degenerating primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA(2) . Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland . Its molecular weight and the pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar software according to the deduced amino acid sequence . Then the gene was cloned into the expression plasmid pET-40b(+) and expressed in E . coli BL21(DE3) . Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme. J Biol Chem, 2004 Apr 2, 279(14), 13607 - 15 Epub 2004 Jan 19. Identification and characterization of structural domains of human ERp57: association with calreticulin requires several domains; Silvennoinen L et al.; The amino acid sequence of ERp57, which functions in the endoplasmic reticulum together with the lectins calreticulin and calnexin to achieve folding of newly synthesized glycoproteins, is highly similar to that of protein disulfide isomerase (PDI), but they have their own distinct roles in protein folding . We have characterized the domain structure of ERp57 by limited proteolysis and N-terminal sequencing and have found it to be similar but not identical to that of PDI . ERp57 had three major protease-sensitive regions, the first of which was located between residues 120 and 150, the second between 201 and 215, and the third between 313 and 341, the data thus being consistent with a four-domain structure abb'a' . Recombinant expression in Escherichia coli was used to verify the domain boundaries . Each single domain and a b'a' double domain could be produced in the form of soluble, folded polypeptides, as verified by circular dichroism spectra and urea gradient gel electrophoresis . When the ability of ERp57 and its a and a' domains to fold denatured RNase A was studied by electrospray mass analyses, ERp57 markedly enhanced the folding rate at early time points, although less effectively than PDI, but was an ineffective catalyst of the overall process . The a and a' domains produced only minor, if any, increases in the folding rate at the early stages and no increase at the late stages . Interaction of the soluble ERp57 domains with the P domain of calreticulin was studied by chemical cross-linking in vitro . None of the single ERp57 domains nor the b'a' double domain could be cross-linked to the P domain, whereas cross-linking was obtained with a hybrid ERpabb'PDIa'c polypeptide but not with ERpabPDIb'a'c, indicating that multiple domains are involved in this protein-protein interaction and that the b' domain of ERp57 cannot be replaced by that of PDI. Nitric Oxide, 2003 Nov, 9(3), 153 - 64 Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis; Secco DD et al.; There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration . The aim of the present investigation was to clarify this situation . Treatment of rats with non-selective, NG-nitro-L-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS) . The neutrophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also enhanced by nitro or amino treatments . The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L-arginine, suggesting an involvement of the L-arginine/NOS pathway in the process . The administration of Cg in iNOS deficient (iNOS(-/-)) mice also enhanced the neutrophil migration compared with wild type mice . This enhancement was markedly potentiated by treatment of iNOS(-/-) mice with nitro . Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils . Similar results were observed in iNOS(-/-) mice, in which these mechanisms were potentiated and reverted by nitro and L-arginine treatments, respectively . In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration . This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils. Trends Cell Biol, 1995 Oct, 5(10), 380 - 3 Directionality in protein translocation across membranes: the N-tail phenomenon; Dalbey RE et al.; Protein translocation normally starts from an N-terminal signal peptide and proceeds in an N-to-C-terminal direction . However, in certain integral membrane proteins an N-terminal tail is translocated even though it is not preceded by a signal peptide . In eukaryotic cells this process involves the normal Sec-machinery . In contrast, recent studies in Escherichia coli show that translocation of such N-terminal tails occurs by a mechanism that does not appear to involve the Sec proteins and is most efficient for short tails lacking positively charged residues . These novel observations suggest that the Sec-machinery has an inherent N-to-C-terminal directionality and cannot work 'in reverse'. Trends Cell Biol, 1991 Nov, 1(5), 110 - 2 Recombination and RNA processing: a common strand? Kearsey S, Kipling D. Genetic recombination is a basic cellular process required for altering genome structure . The RecA protein of Escherichia coli has a central role in homologous recombination, and a eukaryotic protein with similar properties has been discovered in the yeast Saccharomyces cerevisiae . Unexpectedly, this RecA-like protein has additional biochemical activities, and its function may not be restricted to recombination. Trends Cell Biol, 1991 Nov, 1(5), 107 - 9 A turnstile for initiation of DNA replication; Boye E; The progress of a cell through its growth cycle is a multifaceted process; so far we have seen only a glimpse of the complex interplay between the macromolecules performing and regulating the different steps involved . In most organisms, control mechanisms ensure that all chromosomal DNA sequences are replicated once, and only once, between two cell divisions . This enables each division to produce two daughter cells with a genetic content identical to that of their mother . Although the biochemical synthetic processes involved in replicating DNA have been described in detail, our knowledge of the regulatory mechanisms of DNA replication remains scant . In recent experiments with Escherichia coli, new light has been shed on these elusive control mechanisms, and evidence has emerged that may signal an end to our ignorance about this important biological problem. Mol Microbiol, 2004 Feb, 51(3), 873 - 85 Deletion analyses of the peptidoglycan-associated lipoprotein Pal reveals three independent binding sequences including a TolA box; Cascales E et al.; The Tol-Pal system of the Escherichia coli cell envelope is composed of five proteins . TolQ, TolR and TolA form a complex in the inner membrane, whereas TolB is a periplasmic protein interacting with Pal, the peptidoglycan-associated lipoprotein anchored to the outer membrane . This system is required for outer membrane integrity and has been shown to form a trans-envelope bridge linking inner and outer membranes . The TolA-Pal interaction plays an important role in the function of this system and has been found to depend on the proton motive force and the TolQ and TolR proteins . The Pal lipoprotein interacts with many components, such as TolA, TolB, OmpA, the major lipoprotein and the murein layer . In this study, six pal deletions were constructed . The analyses of the resulting Pal protein functions and interactions defined an N-terminal region of 40 residues, which can be deleted without any cell-damaging effect, and three independent regions required for its interaction with TolA, OmpA and TolB or the peptidoglycan . The analyses of the integrity of the cells producing the various Pal lipoproteins revealed strong outer membrane destabilization only when binding regions were deleted . Furthermore, a conserved polypeptide sequence located downstream of the peptidoglycan binding motif of Pal was required for the TolA-Pal interaction and for the maintenance of outer membrane stability. Mol Microbiol, 2004 Feb, 51(3), 849 - 59 Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions; Mujacic M et al.; Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps) . To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones . Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature . The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity . Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells . On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions . This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding. Mol Microbiol, 2004 Feb, 51(3), 813 - 26 Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N-acetylgalactosamine metabolism in Escherichia coli K-12; Ray WK et al.; The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N-acetylgalactosamine and galactosamine in Escherichia coli . In some strains of E . coli, particularly the common K-12 strain, a portion of this cluster is missing because of a site-specific recombination event that occurred between sites in agaW and agaA . Strains that have undergone this recombination event have lost the ability to utilize either N-acetylgalactosamine or galactosamine as sole sources of carbon . Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators . Promoters upstream of agaR, agaZ and agaS were characterized . All three promoters had elevated activity in the presence of N-acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA-binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting . In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP-CRP . Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA-binding or response to inducer . These residues of AgaR were targeted for site-directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non-inducible . The apparent ability to produce negatively dominant and non-inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function. Mol Microbiol, 2004 Feb, 51(3), 799 - 811 Multiple stress signal integration in the regulation of the complex sigma S-dependent csiD-ygaF-gabDTP operon in Escherichia coli; Metzner M et al.; The csiD-ygaF-gabDTP region in the Escherichia coli genome represents a cluster of sigma S-controlled genes . Here, we investigated promoter structures, sigma factor dependencies, potential co-regulation and environmental regulatory patterns for all of these genes . We find that this region constitutes a complex operon with expression being controlled by three differentially regulated promoters: (i) csiDp, which affects the expression of all five genes, is cAMP-CRP/sigma S-dependent and activated exclusively upon carbon starvation and stationary phase; (ii) gabDp1, which is sigma S-dependent and exhibits multiple stress induction like sigma S itself; and (iii) gabDp2{previously suggested by Schneider, B.L., Ruback, S., Kiupakis, A.K., Kasbarian, H., Pybus, C., and Reitzer, L . (2002) J . Bacteriol . 184: 6976-6986}, which appears to be Nac/sigma 70-controlled and to respond to poor nitrogen sources . In addition, we identify a novel repressor, CsiR, which modulates csiDp activity in a temporal manner during early stationary phase . Finally, we propose a physiological role for sigma S-controlled GabT/D-mediated gamma-aminobutyrate (GABA) catabolism and glutamate accumulation in general stress adaptation . This physiological role is reflected by the activation of the operon-internal gabDp1 promoter under the different conditions that also induce sigma S, which include shifts to acidic pH or high osmolarity as well as starvation or stationary phase. Mol Microbiol, 2004 Feb, 51(3), 777 - 90 The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers; Khemici V et al.; In Escherichia coli, REP-stabilizers are structural elements in polycistronic messages that protect 5'-proximal cistrons from 3'-->5' exonucleolytic degradation . The stabilization of a protected cistron can be an important determinant in the level of gene expression . Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP-stabilized mRNA . However, subsequent degradation of mRNA fragments containing a REP-stabilizer poses a special challenge to the mRNA degradation machinery . Two enzymes, the DEAD-box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP-stabilizers by polynucleotide phosphorylase (PNPase) . This is the first in vivo evidence that these enzymes are required for the degradation of REP-stabilizers . Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments. Mol Microbiol, 2004 Feb, 51(3), 645 - 57 R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division; Koppelman CM et al.; We investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles . Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter . This hydrophobic loop contains an arginine residue at position 174 . To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP) . FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ . FtsZ-R174D could also localize in an FtsZ depletion strain . However, in contrast to wild-type FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation . In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2 . This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize . The mutant is able to form protofilaments in vitro but fails to bundle . It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division. Biochemistry, 2004 Jan 27, 43(3), 799 - 807 Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site; Elliott SJ et al.; The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode . Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors . At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative . As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations . This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force . However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out . This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate . Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential . The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site . The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E . coli. Biochemistry, 2004 Jan 27, 43(3), 791 - 8 Superoxide destroys the {2Fe-2S}2+ cluster of FNR from Escherichia coli; Sutton VR et al.; The oxygen sensing ability of the transcription factor FNR depends on the presence of a {4Fe-4S}2+ cluster . In the presence of O2, conversion of the {4Fe-4S}2+ cluster to a {2Fe-2S}2+ cluster inactivates FNR, but the fate of the {2Fe-2S}2+ cluster in cells grown under aerobic conditions is unknown . The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the {2Fe-2S}2+ cluster, like the {4Fe-4S}2+ cluster, is not stable under these conditions . By quantifying the amount of {2Fe-2S}2+ cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the {2Fe-2S}2+ cluster . Mossbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the {2Fe-2S}2+ cluster . We conclude that the {2Fe-2S}2+ cluster of FNR is labile to superoxide both in vitro and in vivo . This lability may explain the absence of the {2Fe-2S}2+ cluster form of FNR under aerobic growth conditions. Biochemistry, 2004 Jan 27, 43(3), 773 - 81 A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity; Kervinen M et al.; The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices . We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis . The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined . Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed . The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E . coli cells on malate as the main carbon source . The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes . Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant. Biochemistry, 2004 Jan 27, 43(3), 651 - 62 A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry; Chepanoske CL et al.; MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA . MutY contains a {4Fe-4S}2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif . This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily . Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY . The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY . The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry . This analysis revealed Arg 143 as the site of modification in MutY . Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily . Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs . In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G . Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone . The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the {4Fe-4S}2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate. Electrophoresis, 2004 Jan, 25(1), 14 - 9 Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2; Suck R et al.; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts . Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2 . Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity . Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties . After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC) . The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform . Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts. Planta, 2004 Apr, 218(6), 989 - 98 Epub 2004 Jan 17. sll1722, an unassigned open reading frame of Synechocystis PCC 6803, codes for L-myo-inositol 1-phosphate synthase; Chatterjee A et al.; L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to L-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms . Two forms of the enzyme have been characterized from higher plants, viz . cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g . Spirulina sp.), the presumed chloroplast progenitors . The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp . PCC 6803 . Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx . 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools . The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS . The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl beta-D-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification . Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others. Nat Struct Mol Biol, 2004 Feb, 11(2), 179 - 86 Epub 2004 Jan 11. Mapping structural differences between 30S ribosomal subunit assembly intermediates; Holmes KL et al.; Under appropriate conditions, functional Escherichia coli 30S ribosomal subunits assemble in vitro from purified components . However, at low temperatures, assembly stalls, producing an intermediate (RI) that sediments at 21S and is composed of 16S ribosomal RNA (rRNA) and a subset of ribosomal proteins (r-proteins) . Incubation of RI at elevated temperatures produces a particle, RI*, of similar composition but different sedimentation coefficient (26S) . Once formed, RI* rapidly associates with the remaining r-proteins to produce mature 30S subunits . To understand the nature of this transition from RI to RI*, changes in the reactivity of 16S rRNA between these two states were monitored by chemical modification and primer extension analysis . Evaluation of this data using structural and biochemical information reveals that many changes are r-protein-dependent and some are clustered in functional regions, suggesting that this transition is an important step in functional 30S subunit formation. Nat Struct Mol Biol, 2004 Feb, 11(2), 135 - 41 Epub 2004 Jan 18. Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase; Diez M et al.; Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell . F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation . We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis . The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences . The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis . The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis. J Biomed Sci, 2004 Jan-Feb, 11(1), 117 - 26 Early detection of antibodies against various structural proteins of the SARS-associated coronavirus in SARS patients; Wu HS et al.; Severe acute respiratory syndrome (SARS), a new disease with symptoms similar to those of atypical pneumonia, raised a global alert in March 2003 . Because of its relatively high transmissibility and mortality upon infection, probable SARS patients were quarantined and treated with special and intensive care . Therefore, instant and accurate laboratory confirmation of SARS-associated coronavirus (SARS-CoV) infection has become a worldwide interest . For this need, we purified recombinant proteins including the nucleocapsid (N), envelope (E), membrane (M), and truncated forms of the spike protein (S1-S7) of SARS-CoV in Escherichia coli . The six proteins N, E, M, S2, S5, and S6 were used for Western blotting (WB) to detect various immunoglobulin classes in 90 serum samples from 54 probable SARS patients . The results indicated that N was recognized in most of the sera . In some cases, S6 could be recognized as early as 2 or 3 days after illness onset, while S5 was recognized at a later stage . Furthermore, the result of recombinant-protein-based WB showed a 90% agreement with that of the whole-virus-based immunofluorescence assay . Combining WB with existing RT-PCR, the laboratory confirmation for SARS-CoV infection was greatly enhanced by 24.1%, from 48.1% (RT-PCR alone) to 72.2% . Finally, our results show that IgA antibodies against SARS-CoV can be detected within 1 week after illness onset in a few SARS patients . Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2648 - 51 Coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans; Tonouchi N et al.; The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated . By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G . oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities . It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G . oxydans. Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2524 - 32 Transaldolase/glucose-6-phosphate isomerase bifunctional enzyme and ribulokinase as factors to increase xylitol production from D-arabitol in Gluconobacter oxydans; Sugiyama M et al.; Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated . Two proteins in the soluble fraction were found to have the ability to increase xylitol production . Both of these xylitol-increasing factors were purified, and on the basis of their NH(2)-terminal amino acid sequences the genes encoding both of the factors were cloned . Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase . Using membrane and soluble fractions of G . oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors. RNA, 2004 Feb, 10(2), 265 - 76 Preferential translation of cold-shock mRNAs during cold adaptation; Giuliodori AM et al.; Upon temperature downshift below the lower threshold of balanced growth (approximately 20 degrees C), the Escherichia coli translational apparatus undergoes modifications allowing the selective translation of the transcripts of cold shock-induced genes, while bulk protein synthesis is drastically reduced . Here we were able to reproduce this translational bias in E . coli cell-free extracts prepared at various times during cold adaptation which were found to display different capacities to translate different types of mRNAs as a function of temperature . Several causes were found to contribute to the cold-shock translational bias: Cold-shock mRNAs contain cis-elements, making them intrinsically more prone to being translated in the cold, and they are selective targets for trans-acting factors present in increased amounts in the translational apparatus of cold-shocked cells . CspA was found to be among these trans-acting factors . In addition to inducing a higher level of CspA, cold shock was found to cause a strong (two- to threefold) stoichiometric imbalance of the ratio between initiation factors (IF1, IF2, IF3) and ribosomes without altering the stoichiometric ratio between the factors themselves . The most important sources of cold-shock translational bias is IF3, which strongly and selectively favors translation of cold-shock mRNAs in the cold . IF1 and the RNA chaperone CspA, which stimulate translation preferentially in the cold without mRNA selectivity, can also contribute to the translational bias . Finally, in contrast to a previous claim, translation of cold-shock cspA mRNA in the cold was found to be as sensitive as that of a non-cold-shock mRNA to both chloramphenicol and kanamycin inhibition. RNA, 2004 Feb, 10(2), 231 - 9 Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli; del Campo M et al.; Escherichia coli pseudouridine synthase RluD makes pseudouridines 1911, 1915, and 1917 in the loop of helix 69 in 23S RNA . These are the most highly conserved ribosomal pseudouridines known . Of 11 pseudouridine synthases in E . coli, only cells lacking RluD have severe growth defects and abnormal ribosomes . We have determined the 2.0 A structure of the catalytic domain of RluD (residues 77-326), the first structure of an RluA family member . The catalytic domain folds into a mainly antiparallel beta-sheet flanked by several loops and helices . A positively charged cleft that presumably binds RNA leads to the conserved Asp 139 . The RluD N-terminal S4 domain, connected by a flexible linker, is disordered in our structure . RluD is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RsuA, TruB, and TruA . We identify five sequence motifs, two of which are novel, in the RluA, RsuA, TruB, and TruA families, uniting them as one superfamily . These results strongly suggest that four of the five families of pseudouridine synthases arose by divergent evolution . The RluD structure also provides insight into its multisite specificity. RNA, 2004 Feb, 10(2), 192 - 9 Not all pseudouridine synthases are potently inhibited by RNA containing 5-fluorouridine; Spedaliere CJ et al.; RNA containing 5-fluorouridine has been assumed to inhibit strongly or irreversibly the pseudouridine synthases that act on the RNA . RNA transcripts containing 5-fluorouridine in place of uridine have, therefore, been added to reconstituted systems in order to investigate the importance of particular pseudouridine residues in a given RNA by inactivating the pseudouridine synthase responsible for their generation . In sharp contradiction to the assumption of universal inhibition of pseudouridine synthases by RNA containing 5-fluorouridine, the Escherichia coli pseudouridine synthase TruB, which has physiologically critical eukaryotic homologs, is not inhibited by such RNA . Instead, the RNA containing 5-fluorouridine was handled as a substrate by TruB . The E . coli pseudouridine synthase RluA, on the other hand, forms a covalent complex and is inhibited stoichiometrically by RNA containing 5-fluorouridine . We offer a hypothesis for this disparate behavior and urge caution in interpreting results from reconstitution experiments in which RNA containing 5-fluorouridine is assumed to inhibit a pseudouridine synthase, as normal function may result from a failure to inactivate the targeted enzyme rather than from the absence of nonessential pseudouridine residues. J Med Microbiol, 2004 Feb, 53(Pt 2), 97 - 102 Protective effect of ethyl-3-(3-dimethyl aminopropyl)urea dihydrochloride (EDU) against LPS-induced death in mice; Matsumoto T et al.; Evaluation of anti-adhesive gels and bioresorbable films in animal models of intra-abdominal infection has shown that a product of the cross-linking reaction between hyaluronic acid (HA) and CM-cellulose, 1-ethyl-3-(3-dimethyl aminopropyl)urea dihydrochloride (EDU), has immunomodulatory properties . The effects of EDU were evaluated by using an endotoxin-induced shock mouse model . Pre-treatment of mice with EDU (50 mg kg(-1)) in DMSO resulted in a significant reduction in mortality following injection of LPS, compared to vehicle (DMSO) pre-treatment alone . Serum levels of TNF-alpha, IL1beta and IFN-gamma in EDU-treated mice were significantly lower than those in vehicle-treated mice . Nitric oxide (NO) concentrations in the sera of mice after inoculation with LPS were significantly lower in the EDU-treated group than in the vehicle-treated group at various time-points . In contrast, EDU pre-treatment was associated with an enhanced IL10 response after LPS injection, compared to vehicle pre-treatment alone . In vitro studies revealed that IL10 production by RAW 264.7 macrophages, elicited by LPS, was increased significantly when EDU was added to the culture medium . These results suggest that the protective effect of EDU during LPS-induced shock in mice is the result of inhibition of proinflammatory cytokines and NO production and an enhanced IL10 response. Plant Cell, 2004 Feb, 16(2), 544 - 54 Epub 2004 Jan 16. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis; Nair RB et al.; Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants . The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants . Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids . Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity . These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis . When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid . These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde . This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes. J Bacteriol, 2004 Feb, 186(3), 880 - 4 Inhibiting cell division in Escherichia coli has little if any effect on gene expression; Arends SJ et al.; DNA microarrays were used to compare gene expression in dividing and nondividing (filamentous) cultures of Escherichia coli . Although cells from these cultures differed profoundly in morphology, their gene expression profiles were nearly identical . These results extend previous evidence that there is no division checkpoint in E . coli, and progression through the cell cycle is not regulated by the transcription of different genes during different parts of the cell cycle. J Bacteriol, 2004 Feb, 186(3), 785 - 93 A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli; Schmidt KL et al.; FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria . Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial . Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX . RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop . We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl . We also found that in wild-type E . coli both FtsE and FtsX localize to the division site . Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI . Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not . Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent . We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media. J Bacteriol, 2004 Feb, 186(3), 654 - 60 Genetic analysis of disulfide isomerization in Escherichia coli: expression of DsbC is modulated by RNase E-dependent mRNA processing; Zhan X et al.; We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity . The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell . Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain . Following chemical mutagenesis, a total of eight E . coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated . This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC . In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product . Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci . The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37 degrees C in wild-type cells but was two- to threefold longer in some of the stronger mutants . These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression. J Biol Chem, 2004 Apr 2, 279(14), 14165 - 70 Epub 2004 Jan 16. Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli; Kramer G et al.; The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli . TF unifies two functions in one to promote proper protein folding in vitro . First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding . Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding . Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center . Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro . In contrast, all other activities of TF F198A tested were comparable with wild type TF . TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in vitro, and associated with nascent polypeptides in an in vitro transcription/translation system . Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of DeltatigDeltadnaK cells and prevented global protein misfolding at temperatures between 20 and 34 degrees C in these cells . We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins. J Mol Biol, 2004 Jan 30, 335(5), 1289 - 97 Structure formation in the C terminus of type III collagen guides disulfide cross-linking; Boudko SP et al.; In type III collagen the main triple-helical domain is followed by a disulfide knot and the C-terminal propeptide, which are both essential for nucleation, stabilization and registration of the triple helix . We demonstrate that oxidative inter-chain disulfide bridging does not occur between the knot sequences GlyProCysCysGly of dissociated randomly coiled chains . N-terminal fusion of the obligatory trimeric domain of mini-fibritin is able to direct this process efficiently, demonstrating a folded precursor mechanism in which the thiol groups have to be properly placed for the formation of native disulfide bonds . The natural C-propeptide domain may act in a similar way as the mini-fibritin domain . After disulfide linkage and triple-helix formation the catalyzing mini-fibritin domain was removed by thrombin cleavage . In this way a short but stable triple-helical collagen fragment was expressed in Escherichia coli for structural and functional studies. J Mol Biol, 2004 Jan 30, 335(5), 1251 - 64 Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum; Eaazhisai K et al.; In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival . Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs . Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy . We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution . The overall architecture of P . falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants . Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species . Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme . The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues . Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme. Biosystems, 2004 Jan, 73(1), 57 - 71 Time hierarchies in the Escherichia coli carbohydrate uptake and metabolism; Kremling A et al.; The analysis of metabolic pathways with mathematical models contributes to the better understanding of the behavior of metabolic processes . This paper presents the analysis of a mathematical model for carbohydrate uptake and metabolism in Escherichia coli . It is shown that the dynamic processes cover a broad time span from some milliseconds to several hours . Based on this analysis the fast processes could be described with steady-state characteristic curves . A subsequent robustness analysis of the model parameters shows that the fast part of the system may act as a filter for the slow part of the system; the sensitivities of the fast system are conserved . From these findings it is concluded that the slow part of the system shows some robustness against changes in parameters of the fast subsystem, i.e . if a parameter shows no sensitivity for the fast part of the system, it will also show no sensitivity for the slow part of the system. Gene, 2004 Feb 4, 326, 157 - 65 Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage; Li J et al.; A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres . EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro . Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls . Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules . Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces . The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection . Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated . Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease . Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible. Vet Parasitol, 2003 Dec 30, 118(3-4), 177 - 85 Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1; Chahan B et al.; Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs . The surface antigen 1 of N . caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis . In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) . The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N . caninum antibodies in cattle . The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle . In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii . Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA . Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N . caninum . Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens . These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N . caninum infection in cattle. Eur J Pharmacol, 2004 Jan 12, 483(2-3), 317 - 22 Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood; Marshall M et al.; Exposure of anti-coagulated human blood to Escherichia coli lipopolysaccharide (50 ng/ml) resulted in the time-dependent (maximum at 5 h) biosynthesis of interleukin-1beta and tumour necrosis factor-alpha (TNF-alpha) . Preincubation with nitroparacetamol or nitroflurbiprofen (but not paracetamol or flurbiprofen) caused dose-related inhibition of the formation of interleukin 1 beta (IC(50)s, 44.5 and 362 microM, n=12) and tumour necrosis factor-alpha (IC(50)s, 9.0 and 0.0009 microM, n=12) . The inhibitory effect of nitroparacetamol was completely reversed by (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide potassium (carboxy-PTIO, 100 microM; NO scavenging agent) . Neither the nuclear factor-kappaB transduction inhibitor, pyrrolidinedithiocarbamate (10-1000 microM) nor the nitric oxide donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5, 10-1000 microM), affected cytokine formation in these experiments.
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