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 transc