J Biomed Sci, 2001 Sep, 8(5), 421 - 9 E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements; Rankin CA et al.; The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP) . Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP . We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins . We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay) . Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements . Since the activity assays were conducted with recombinant proteins purified from E . coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity . Plant Cell, 2001 Sep, 13(9), 2005 - 20 Secretory bulk flow of soluble proteins is efficient and COPII dependent; Phillipson BA et al.; COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway . In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved . In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate . To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway . First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation . A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity . A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley alpha-amylase and a modified version carrying an ER retention motif . Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously . We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay . The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed. Am J Respir Crit Care Med, 2001 Sep 1, 164(5), 873 - 8 High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice; Sadikot RT et al.; We examined the effects of dexamethasone treatment on nuclear factor (NF)-kappa B activation and lung inflammation in transgenic reporter mice expressing photinus luciferase under the control of an NF-kappa B-dependent promoter (HLL mice) . In vitro studies with bone marrow and peritoneal macrophages derived from these mice showed that treatment with dexamethasone blocked luciferase induction after treatment with Escherichia coli lipopolysaccharide (LPS); however, treatment of mice with intraperitoneal injection of dexamethasone at doses of 0.3 microg/g and 1 microg/g failed to inhibit NF-kappa B-dependent luciferase activity in the lungs . Furthermore, intraperitoneal treatment with 10 microg/g of dexamethasone prior to LPS paradoxically resulted in augmented luciferase activity as compared with that of mice treated with LPS alone . NF-kappa B-dependent luciferase expression in the lungs was detected by bioluminescence imaging and by measurement of luciferase activity in homogenized lung tissue . In these studies, there was an excellent correlation between indirect measurement of luciferase activity by bioluminescence in living mice and direct measurement of luciferase activity in lung tissue . Dexamethasone treatment did not affect LPS-induced neutrophilic influx or the concentration of macrophage inflammatory protein-2 in lung lavage fluid . These findings emphasize the potential error of extrapolating in vitro findings to complex in vivo events such as regulation of inflammation. Bioorg Med Chem Lett, 2001 Sep 17, 11(18), 2485 - 8 A selective inhibitor of Escherichia coli prephenate dehydratase; Husain A et al.; To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction . Screening experiments identified 18 as a promising new PDT inhibitor. Biochem Biophys Res Commun, 2001 Sep 14, 287(1), 153 - 9 Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model; Acharya AS et al.; Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI . The mutation was achieved using the megaprimer approach for PCR . The mutant genes were cloned and characterized by sequencing . The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates . All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate . The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein . The melting curves of the mutant proteins overlapped that of the wild-type enzyme . Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism . Microbios, 2001, 106 Suppl 1, 7 - 19 Identification, cloning and expression of sodC from an alkaline phosphatase gene fusion library of Mycobacterium avium subspecies paratuberculosis; Dupont C et al.; The phoA gene technology was used to investigate secreted proteins of the intracellular pathogen Mycobacteriumn avium subspecies paratuberculosis . This led to the identification of sodC, a gene which codes for a copper and zinc cofactored superoxide dismutase (Cu,ZnSOD) which has been implicated as a virulence factor for some pathogens . The predicted protein possessed a 76% identity with Cu,ZnSOD of Mycobacterium tuberculosis . To characterize Cu,ZnSOD from M . avium subspecies paratuberculosis, the gene was cloned and overexpressed in Escherichia coli . The renatured, affinity-purified recombinant protein possessed enzymatic activity that was inhibited by the presence of cyanide, which is characteristic of a Cu,ZnSOD. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 508 - 12 The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures; O'Brien M et al.; Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems . In the case of Escherichia coli, interference involves the Na+(Li+)/H+ antiporter transport system . The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions . The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance . In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs . The ultimate fate of the LiCl was not determined. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 465 - 73 Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the alpha-amylase genes; Uotsu-Tomita R et al.; A region downstream of the gene for pullulan-hydrolyzing alpha-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found . The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues . The TGA gene was subcloned and expressed in Escherichia coli . Enzymatic analyses showed that, like other glucoamylases, TGA produced beta-D-glucose from its substrate . However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides . The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low . The enzymatic properties of recombinant MGA produced in E . coli cells were similar to those of TGA . These findings indicate that TGA and MGA are novel glucoamy-lase-type enzymes with oligosaccaharide-metabolizing activity. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 425 - 30 Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene; Wang CL et al.; The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall . When grown in a defined salts medium supplemented with cadmium and cysteine, E . coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h . A control strain produced significantly less sulfide and removed significantly less cadmium . Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide . Without supplemental cysteine, both the E . coli producing cysteine desulfhydrase and the control E . coli demonstrated minimal cadmium removal. Semin Thromb Hemost, 2001 Aug, 27(4), 325 - 36 The production of improved tissue-type plasminogen activator in Escherichia coli; Mattes R; Tissue-type plasminogen activator (t-PA) is a valuable thrombolytic agent because of its high affinity to fibrin . When produced in mammalian cell lines, it is glycosylated, a modification that is believed to promote its rapid clearance from the circulation . Bacteria such as Escherichia coli have been tested as alternative expression systems but were not able to express the cDNA of t-PA effectively . The coding sequence for t-PA revealed a significant proportion of AGA and AGG codons, which are rarely used in the coding sequences of E . coli . The argU and argW gene products of E . coli proved to be minor tRNA(arg) species, respectively decoding the very rare triplets AGA/AGG and AGG for arginine . Analysis of genomic fragments from E . coli for both tRNA(arg) genes revealed the presence of defective, cryptic prophages integrated within the impaired tRNA genes . Cloning and supplementation of the limiting tRNA genes argU and argW on helper plasmids improved the translation of the rare AGA and AGG codons . This augmentation improved bacterial growth and enhanced t-PA production in the form of inactive inclusion bodies . This dependence on augmentation of tRNA(arg4) or tRNA(arg5) for improved cell growth and expression was also observed for other genes with a high content of these rare arginine codons . Construction and production of nonglycosylated t-PA in inclusion bodies in E . coli along with improvement of the subsequent renaturation and purification procedures resulted in material comparable to that derived from CHO cells . Deletion of domain-encoding segments yielded various "muteins" of t-PA (e.g., reteplase {rPA}) that could be produced in and activated from the purified inclusion bodies analogously . Furthermore, it was shown that rPA has an extended half-life in the circulation because of its lack of glycosylation and impaired receptor binding capability . rPA was successfully used in various clinical studies . It is a new-generation thrombolytic agent with a longer half-life and can thus be administered more conveniently as a double bolus. J Membr Biol, 2001 Sep 1, 183(1), 33 - 8 Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli; Ding PZ et al.; The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H(+), Na(+), or Li(+) . In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier . We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier . The resulting melibiose carriers retained 22-100% of their ability to transport melibiose . We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants . We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Crit Care Med, 2001 Sep, 29(9), 1761 - 6 Protective effect of tumor necrosis factor-alpha against subsequent endotoxemia in mice is mediated, in part, by interleukin-10; Murphey ED et al.; OBJECTIVE: Tumor necrosis factor (TNF)-alpha administration in large amounts can induce a state of shock similar to that observed in patients suffering from septic shock . Small doses of TNF-alpha induce only mild, transient hemodynamic alterations and can confer protection against subsequent inflammatory stimuli . The objective of this study was to determine whether this protective mechanism could be attributed to activity of the anti-inflammatory cytokine interleukin (IL)-10 . DESIGN: Prospective, randomized, controlled study . SETTING: Investigative intensive care unit at a medical university . SUBJECTS: Female BALB-c mice, 10-12 wks of age (approximately 20 g) . INTERVENTIONS: All mice were subjected to intraperitoneal (ip) injection of lipopolysaccharide (LPS; Escherichia coli 0111:B4, 125 microg) . Mice were randomly assigned to the following groups: TNF-alpha pretreated (100 microg ip 24 hrs before LPS); control (TNF vehicle alone 24 hrs before LPS); TNF/anti-IL-10 pretreated (TNF pretreatment as above and a neutralizing anti-IL-10 antibody); TNF/anti-IL-10 control (TNF pretreatment as above and an isotype-matched control antibody with no IL-10 activity); IL-10 (100 microg ip 1 hr before LPS); and IL-10 control (IL-10 vehicle 1 hr before LPS) . MEASUREMENTS AND MAIN RESULTS: Mice were observed for a 48-hr period after endotoxin administration . Mortality in each group was recorded . Separate groups of mice were pretreated with TNF (or vehicle) and killed at 0, 2, or 4 hrs after LPS injection for collection of serum and peritoneal lavage samples that were used to assay IL-10 concentrations . A small dose of TNF-alpha attenuated mortality in mice that were subsequently injected with a highly lethal dose of endotoxin and observed for 48 hrs . Peritoneal lavage fluid concentrations of IL-10 were consistently higher in TNF-pretreated mice after endotoxin administration . The TNF-alpha protective effect was reversed by administration of a neutralizing antibody directed against murine IL-10 . CONCLUSIONS: These findings indicate that administration of a low dose of TNF-alpha can induce cross-tolerance to endotoxin by induction of endogenous anti-inflammatory mechanisms. Crit Care Med, 2001 Sep, 29(9), 1666 - 9 Effect of mono-dose intraperitoneal cecropins in experimental septic shock; Giacometti A et al.; OBJECTIVE: To investigate the efficacy of three cecropins, cecropin A, cecropin B, and cecropin P1, in preventing lethality in a rat model of septic shock . DESIGN: Prospective, randomized, controlled animal study . SETTING: Research laboratory in a university hospital . SUBJECTS: Adult male Wistar rats . INTERVENTIONS: Rats were given an intraperitoneal injection of 2 x 10(10) colony forming units of Escherichia coli, with the exception of the uninfected control group (C0) . Animals were randomized to receive, immediately after bacterial challenge, intraperitoneally isotonic sodium chloride solution (untreated control group C1), 1 mg/kg cecropin A (group 2), 1 mg/kg cecropin B (group 3), 1 mg/kg cecropin P1 (group 4), 20 mg/kg imipenem (group 5), or 60 mg/kg piperacillin (group 6) . Each group included 15 animals . MEASUREMENTS AND MAIN RESULTS: We measured bacterial growth (quantitative agar culture) in abdominal exudate and plasma, endotoxin and tumor necrosis factor-alpha concentration in plasma, and mortality . Results were evaluated at 48 hrs after inoculation . Cecropins, piperacillin, and imipenem significantly reduced the lethality and the number of E . coli in abdominal fluid compared with saline treatment . In addition, cecropin B significantly decreased the lethality compared with piperacillin treatment . Finally, only cecropins significantly reduced plasma endotoxin concentration . CONCLUSIONS: Mono-dose cecropin treatment prevents bacterial growth, endotoxemia, and mortality in rats with septic shock . Cecropin B was the most effective compound in reducing all variables measured. J Biol Chem, 2001 Dec 7, 276(49), 45622 - 7 Epub 2001 Aug 23. Interaction of the periplasmic peptidylprolyl cis-trans isomerase SurA with model peptides . The N-terminal region of SurA id essential and sufficient for peptide binding; Webb HM et al.; One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases . To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E . coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts . The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides . From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA . This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli . Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains. J Biol Chem, 2001 Oct 26, 276(43), 39926 - 37 Epub 2001 Aug 23. De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase; Ackermann M et al.; Replication of positive strand flaviviruses is mediated by the viral RNA-dependent RNA polymerases (RdRP) . To study replication of dengue virus (DEN), a flavivirus family member, an in vitro RdRP assay was established using cytoplasmic extracts of DEN-infected mosquito cells and viral subgenomic RNA templates containing 5'- and 3'-terminal regions (TRs) . Evidence supported that an interaction between the TRs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements is required for RNA synthesis . Two RNA products, a template size and a hairpin, twice that of the template, were formed . To isolate the function of the viral RdRP (NS5) from that of other host or viral factors present in the cytoplasmic extracts, the NS5 protein was expressed and purified from Escherichia coli . In this study, we show that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo initiation . Furthermore, the incubation temperature during initiation, but not elongation phase of RNA synthesis modulates the relative amounts of the hairpin and de novo RNA products . A model is proposed that a specific conformation of the viral polymerase and/or structure at the 3' end of the template RNA is required for de novo initiation. J Biol Chem, 2001 Oct 26, 276(43), 40274 - 81 Epub 2001 Aug 23. Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR; Xiao B et al.; The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191 . The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints . The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded . Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis . A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A . The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile . Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP . The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle. J Biol Chem, 2001 Oct 26, 276(43), 39919 - 25 Epub 2001 Aug 23. Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase; Kozaki A et al.; Fatty acid synthesis in pea chloroplasts is regulated by light/dark . The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation . Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT) . To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation . We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state . Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides . Similar results were confirmed in the full-length CT . These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits . Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo . A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light. J Biol Chem, 2001 Nov 16, 276(46), 43374 - 82 Epub 2001 Aug 23. Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop; Jankova L et al.; The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution . The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet . Clear electron density is seen for all residues in the peptide . The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition . The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A . A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains . The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition . We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles . This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2. J Biol Chem, 2001 Nov 2, 276(44), 40680 - 6 Epub 2001 Aug 23. Vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins; Wright CF et al.; Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late . Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter . VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity . Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation . Overexpression and purification of these proteins from Escherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions . These studies identify two host cell factors potentially contributing to poxvirus replication in vivo. Mol Cell, 2001 Aug, 8(2), 455 - 63 Atomic structure of the clamp loader small subunit from Pyrococcus furiosus; Oyama T et al.; In eukaryotic DNA replication, replication factor-C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands at replication forks . The eukaryotic RFC is a complex consisting of one large and four small subunits . We have determined the crystal structure of the clamp loader small subunit (RFCS) from Pyrococcus furiosus . The six subunits, of which four bind ADP in their canonical nucleotide binding clefts, assemble into a dimer of semicircular trimers . The crescent-like architecture of each subunit formed by the three domains resembles that of the delta' subunit of the E . coli clamp loader . The trimeric architecture of archaeal RFCS, with its mobile N-terminal domains, involves intersubunit interactions that may be conserved in eukaryotic functional complexes. Mol Cell, 2001 Aug, 8(2), 449 - 54 ClpX-mediated remodeling of mu transpososomes: selective unfolding of subunits destabilizes the entire complex; Burton BM et al.; E . coli ClpX, a member of the Clp/Hsp100 family of ATPases, remodels multicomponent complexes and facilitates ATP-dependent degradation . Here, we analyze the mechanism by which ClpX destabilizes the exceedingly stable Mu transpososome, a natural substrate for remodeling rather than degradation . We find that ClpX has the capacity to globally unfold transposase monomers, the building blocks of the transpososome . A biochemical probe for protein unfolding reveals that ClpX also unfolds MuA subunits during remodeling reactions, but that not all subunits have their structure extensively modified . In fact, direct recognition and unfolding of a single transposase subunit are sufficient for ClpX to destabilize the entire transpososome . Thus, the ability of ClpX to unfold proteins is sufficient to explain its role in both complex destabilization and ATP-dependent proteolysis. Mol Cell, 2001 Aug, 8(2), 427 - 37 Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain; Zhou BL et al.; The UmuC/DinB family of bypass polymerases is responsible for translesion DNA synthesis and includes the human polymerases eta, iota, and kappa . We determined the 2.3 A resolution crystal structure of a catalytic fragment of the DinB homolog (Dbh) polymerase from Sulfolobus solfataricus and show that it is nonprocessive and can bypass an abasic site . The structure of the catalytic domain is nearly identical to those of most other polymerase families . Homology modeling suggests that there is minimal contact between protein and DNA, that the nascent base pair binding pocket is quite accessible, and that the enzyme is already in a closed conformation characteristic of ternary polymerase complexes . These observations afford insights into the sources of low fidelity and low processivity of the UmuC/DinB polymerases. Prostaglandins Leukot Essent Fatty Acids, 2001 Aug, 65(2), 59 - 65 Ibuprofen attenuates early lung injury in endotoxemic, neutropenic rats; Daphtary KM et al.; The objective of our study was to determine the role of ibuprofen in protecting neutropenic rats from cardiopulmonary injury due to endotoxemia . We hypothesized that ibuprofen would offer pulmonary protection by altering cytokine production . Neutropenic rats received E . coli lipopolysaccharide (LPS) alone or ibuprofen and LPS . After 4 h, arterial blood gases, heart rate and blood pressure were measured . Blood and bronchoalveolar lavage fluid (BALF) were collected for TNF- alpha and MIP-2 concentrations . Lung tissue for iNOS mRNA and myeloperoxidase were obtained . The ibuprofen group had decreased heart rate and better oxygenation . Ibuprofen suppressed TNF- alpha and MIP-2 production in blood and MIP-2 concentrations in BALF . Lung mRNA for iNOS was higher in the ibuprofen group . Neutrophil infiltration in the lung was similar in both groups . Ibuprofen attenuated cardiopulmonary dysfunction by decreasing the early cytokine response . The balance of vasodilator to vasoconstrictor production in the lung may favor vasodilation as shown by increased iNOS mRNA and suppression of thromboxane . J Mol Biol, 2001 Sep 7, 312(1), 221 - 8 Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain; Arndt KM et al.; Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback . To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization . We present an alternative format for stabilizing antibody Fv fragments . The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli . This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format . Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment . The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric . The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration) . Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem . J Mol Biol, 2001 Sep 7, 312(1), 79 - 93 Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies; Bach H et al.; Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential . However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disulfide bonds that are essential for correct folding and functionality of these antibody fragments . Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for efficient functional expression in the cell cytoplasm in a soluble, active form . The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP) . We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins . We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E . coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm . In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB(-) but not in trxB(+) E . coli cells . We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more efficiently in vivo as cytoplasmic intrabodies in E . coli . Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it . J Mol Biol, 2001 Sep 7, 312(1), 69 - 77 Helix packing in the lactose permease of Escherichia coli: localization of helix VI; Guan L et al.; Plasmids encoding "split" lactose permease constructs with discontinuities in either the periplasmic loop between helices V and VI (N(5)/C(7)) or between helices VI and VII (N(6)/C(6)) were used to localize helix VI within the tertiary structure by site-directed thiol cross-linking . A total of 57 double-Cys pairs, with one Cys residue in helix VI and another in helix V or VIII, were studied with homobifunctional cross-linking agents . Significant cross-linking is observed between the periplasmic ends of helices V (position 158 or 161) and VI (position 170) with rigid 6 or 10 A reagents . Furthermore, the Cys residue at position 170 (helix VI) also cross-links to a Cys residue at either position 264 or 265 (helix VIII) with a 21 A cross-linking agent . The data indicate that helices V, VI and VIII are in close proximity at the periplasmic face of the membrane, with helix VI significantly closer to helix V . In addition, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside induces a significant increase in cross-linking efficiency between helices VI and VIII and between helices V and VIII, with no significant change in cross-linking between helices V and VI . J Mol Biol, 2001 Sep 7, 312(1), 27 - 44 RNA folding pathway functional intermediates: their prediction and analysis; Shapiro BA et al.; The massively parallel genetic algorithm (GA) for RNA structure prediction uses the concepts of mutation, recombination, and survival of the fittest to evolve a population of thousands of possible RNA structures toward a solution structure . As described below, the properties of the algorithm are ideally suited to use in the prediction of possible folding pathways and functional intermediates of RNA molecules given their sequences . Utilizing Stem Trace, an interactive visualization tool for RNA structure comparison, analysis of not only the solution ensembles developed by the algorithm, but also the stages of development of each of these solutions, can give strong insight into these folding pathways . The GA allows the incorporation of information from biological experiments, making it possible to test the influence of particular interactions between structural elements on the dynamics of the folding pathway . These methods are used to reveal the folding pathways of the potato spindle tuber viroid (PSTVd) and the host killing mechanism of Escherichia coli plasmid R1, both of which are successfully explored through the combination of the GA and Stem Trace . We also present novel intermediate folds of each molecule, which appear to be phylogenetically supported, as determined by use of the methods described below. Annu Rev Microbiol, 2001, 55, 591 - 624 Periplasmic stress and ECF sigma factors; Raivio TL et al.; Envelope stress responses play important physiological roles in a variety of processes, including protein folding, cell wall biosynthesis, and pathogenesis . Many of these responses are controlled by extracytoplasmic function (ECF) sigma factors that respond to external signals by means of a membrane-localized anti-sigma factor . One of the best-characterized, ECF-regulated responses is the sigma(E) envelope stress response of Escherichia coli . The sigma(E) pathway ensures proper assembly of outer-membrane proteins (OMP) by controlling expression of genes involved in OMP folding and degradation in response to envelope stresses that disrupt these processes . Prevailing evidence suggests that, in E . coli, a second envelope stress response controlled by the Cpx two-component system ensures proper pilus assembly . The sensor kinase CpxA recognizes misfolded periplasmic proteins, such as those generated during pilus assembly, and transduces this signal to the response regulator CpxR through conserved phosphotransfer reactions . Phosphorylated CpxR activates transcription of periplasmic factors necessary for pilus assembly. J Bacteriol, 2001 Oct, 183(19), 5778 - 81 Regulation of potassium-dependent Kdp-ATPase expression in the nitrogen-fixing cyanobacterium Anabaena torulosa; Alahari A et al.; The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+) . In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation. J Bacteriol, 2001 Oct, 183(19), 5768 - 71 The phosphate-binding protein of Escherichia coli is not essential for P(i)-regulated expression of the pho regulon; Hoffer SM et al.; Disruption of pstS encoding the P(i)-binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon . We demonstrate that P(i)-controlled expression is restored when the activity of the P(i) transporter PitA or PitB is increased . Apparently, PstS is not an essential component of the signal transduction pathway. J Bacteriol, 2001 Oct, 183(19), 5747 - 50 The djlA gene acts synergistically with dnaJ in promoting Escherichia coli growth; Genevaux P et al.; The DnaK chaperone of Escherichia coli is known to interact with the J domains of DnaJ, CbpA, and DjlA . By constructing multiple mutants, we found that the djlA gene was essential for bacterial growth above 37 degrees C in the absence of dnaJ . This essentiality depended upon the J domain of DjlA but not upon the normal membrane location of DjlA. J Bacteriol, 2001 Oct, 183(19), 5599 - 608 Binding specificity of the Porphyromonas gingivalis heme and hemoglobin receptor HmuR, gingipain K, and gingipain R1 for heme, porphyrins, and metalloporphyrins; Olczak T et al.; Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases . In this study we report on the binding specificity of the recombinant P . gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay . Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains . Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M . Recombinant E . coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring . Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains . Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring . We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins . Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR . Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed. J Bacteriol, 2001 Oct, 183(19), 5523 - 8 Overexpression of yccL (gnsA) and ydfY (gnsB) increases levels of unsaturated fatty acids and suppresses both the temperature-sensitive fabA6 mutation and cold-sensitive secG null mutation of Escherichia coli; Sugai R et al.; A multicopy suppressor of the cold-sensitive secG null mutation was isolated . The suppressor contained sfa and yccL, the former of which has been reported to be a multicopy suppressor of the fabA6 mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph . Subcloning of the suppressor gene revealed that yccL, renamed gnsA (secG null mutant suppressor), was responsible for the suppression of both the secG null mutation and the fabA6 mutation . In contrast, the sfa gene did not suppress the fabA6 mutation . The ydfY (gnsB) gene, encoding a protein which is highly similar to GnsA, also suppressed both the secG null mutation and the fabA6 mutation . Although both gnsA and gnsB are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response . A gnsA-gnsB double null mutant grew normally under all conditions examined; thus, the in vivo functions of gnsA and gnsB remain unresolved . However, overexpression of gnsA and gnsB stimulated proOmpA translocation of the secG null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids . Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature. J Bacteriol, 2001 Oct, 183(19), 5496 - 505 LuxArray, a high-density, genomewide transcription analysis of Escherichia coli using bioluminescent reporter strains; Van Dyk TK et al.; A sequenced collection of plasmid-borne random fusions of Escherichia coli DNA to a Photorhabdus luminescens luxCDABE reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions . This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in E . coli . High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression . The cellular response to nalidixic acid perturbation was analyzed using this format . As expected, fusions to promoters of LexA-controlled SOS-responsive genes dinG, dinB, uvrA, and ydjM were found to be upregulated in the presence of nalidixic acid . In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated . The responses of two of these, fusions to oraA and yigN, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon . The responses of the other four were neither induced by mitomycin C nor dependent on lexA function . Thus, the promoters of ycgH, intG, rihC, and a putative operon consisting of lpxA, lpxB, rnhB, and dnaE were not generally DNA damage responsive and represent a more specific response to nalidixic acid . These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses. J Bacteriol, 2001 Oct, 183(19), 5482 - 90 ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release; Barthel TK et al.; We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK . Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP . The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant . The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK . The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished . All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP . The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP . ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein . The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK . The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell. Genome Res, 2001 Sep, 11(9), 1503 - 10 Functional versatility and molecular diversity of the metabolic map of Escherichia coli; Tsoka S et al.; We have analyzed the known metabolic enzymes of Escherichia coli in relation to their biochemical reaction properties and their involvement in biochemical pathways . All enzymes involved in small-molecule metabolism and their corresponding protein sequences have been extracted from the EcoCyc database . These 548 metabolic enzymes are clustered into 405 protein families according to sequence similarity . In this study, we examine the functional versatility within enzyme families in terms of their reaction capabilities and pathway participation . In addition, we examine the molecular diversity of reactions and pathways according to their presence across enzyme families . These complex, many-to-many relationships between protein sequence and biochemical function reveal a significant degree of correlation between enzyme families and reactions . Pathways, however, appear to require more than one enzyme type to perform their complex biochemical transformations . Finally, the distribution of enzyme family members across different pathways provides support for the "recruitment" hypothesis of biochemical pathway evolution. Genes Dev, 2001 Sep 1, 15(17), 2295 - 306 Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift; Bregeon D et al.; Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells . In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins . This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site . Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift . The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function. Genes Dev, 2001 Sep 1, 15(17), 2273 - 81 Recruitment of HU by piggyback: a special role of GalR in repressosome assembly; Kar S et al.; In Gal repressosome assembly, a DNA loop is formed by the interaction of two GalR, bound to two distal operators, and the binding of the histone-like protein, HU, to an architecturally critical position on DNA to facilitate the GalR-GalR interaction . We show that GalR piggybacks HU to the critical position on the DNA through a specific GalR-HU interaction . This is the first example of HU making a specific contact with another protein . The GalR-HU contact that results in cooperative binding of the two proteins to DNA may be transient and absent in the final repressosome structure . A sequence-independent DNA-binding protein being recruited to an architectural site on DNA through a specific association with a regulatory protein may be a common mode for assembly of complex nucleoprotein structures. Trends Plant Sci, 2001 Sep, 6(9), 407 - 13 Caffeine: a well known but little mentioned compound in plant science; Ashihara H et al.; Caffeine, a purine alkaloid, is a key component of many popular drinks, most notably tea and coffee, yet most plant scientists know little about its biochemistry and molecular biology . A gene from tea leaves encoding caffeine synthase, an N-methyltransferase that catalyses the last two steps of caffeine biosynthesis, has been cloned and the recombinant enzyme produced in E . coli . Similar genes have been isolated from coffee leaves but the recombinant protein has a different substrate specificity to the tea enzyme . The cloning of caffeine biosynthesis genes opens up the possibility of using genetic engineering to produce naturally decaffeinated tea and coffee. J Med Chem, 2001 Sep 13, 44(19), 3166 - 74 Properties and interaction of heterologously expressed glutamate decarboxylase isoenzymes GAD(65kDa) and GAD(67kDa) from human brain with ginkgotoxin and its 5'-phosphate; Buss K et al.; Two isoforms of glutamate decarboxylase (GAD(65kDa) and GAD(67kDa)) from human brain, which had previously been overexpressed in Escherichia coli as fusion proteins containing a glutathione-S-transferase domain, were purified by affinity chromatography on glutathione Sepharose 4B . Both isoforms were also expressed in Saccharomyces cerevisiae . After modification of a HPLC based assay, the enzymes were characterized with respect to their biochemical properties . Comparison of kinetic data, pH, and temperature optima as well as of the mode of interaction with pyridoxal phosphate as a cofactor revealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features . Investigation of the influence of 4'-O-methylpyridoxine (ginkgotoxin) (1), a neurotoxin occurring in Ginkgo biloba L., on the different isoenzymes, indicates that the phosphorylated form of the toxin, 4'-O-methylpyridoxine-5'-phosphate (2), decreases GAD(65kDa) activity, although in unphysiologically high concentrations, whereas GAD(67kDa) activity seems to be hardly affected. J Med Chem, 2001 Sep 13, 44(19), 3083 - 91 Design and synthesis of potent C(2)-symmetric diol-based HIV-1 protease inhibitors: effects of fluoro substitution; Pyring D et al.; Implementation of derivatized carbohydrates as C(2)-symmetric HIV-1 protease inhibitors has previously been reported . With the objective of improving the anti-HIV activity of such compounds, we synthesized a series of fluoro substituted P1/P1' analogues . These compounds were evaluated for antiviral activity toward both wild type and mutant virus . The potency of the analogues in blocking HIV-1 protease was moderate, with K(i) values ranging from 1 to 7 nM . Nonetheless, compared to the parent nonfluorous inhibitors, a majority of the compounds exhibited improved antiviral activity, for example the 3-fluorobenzyl derivative 9b, which had a K(i) value of 7.13 nM and displayed one of the most powerful antiviral activities in the cellular assay of the series . Our results strongly suggest that fluoro substitution can substantially improve antiviral activity . The X-ray crystal structures of two of the fluoro substituted inhibitors (9a and 9f) cocrystallized with HIV-1 protease are discussed. Virology, 2001 Sep 15, 288(1), 89 - 95 Enzymatic depalmitoylation of viral glycoproteins with acyl-protein thioesterase 1 in vitro; Veit M et al.; Many glycoproteins of enveloped viruses as well as cellular proteins are covalently modified with fatty acids . Palmitoylation is often reversible, but the enzymology of this hydrophobic protein modification is not understood . Recently a cytosolic enzyme designated acyl-protein thioesterase 1 (APT1) was purified, which depalmitoylates several cellular proteins . Since hitherto no transmembrane proteins have been tested as substrates for APT1 we have investigated whether palmitoylated viral membrane glycoproteins can be deacylated by use of this enzyme . Recombinant APT1 was purified from Escherichia coli, and depalmitoylation of {3H}palmitate-labeled glycoproteins present in virus particles was measured by SDS-PAGE, fluorography, and scanning densitometry . We find that APT1 causes rapid and almost complete cleavage of fatty acids from the G-protein of vesicular stomatitis virus, hemagglutinin proteins of influenza A and C virus, and E2 of Semliki Forest virus (SFV) . In contrast, E1 of SFV is largely resistant against APT1 activity . This substrate specificity of APT1 was also observed using microsomes prepared from SFV-infected cells . Our data emphasize the potential of APT1 as a tool for functional analysis of protein-bound fatty acids . Genomics, 2001 Sep, 77(1-2), 27 - 34 Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast; Zeng C et al.; We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere) . The cassette allows transferring of BACs into yeast for their further modification . Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones . Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation . Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule . Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T . brucei cloned in BACs . We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity. Acta Astronaut, 1985 Feb, 12(2), 131 - 4 Preliminary results of Cytos 2 experiment; Tixador R et al.; Cytos 2 experiment, carried out during the French-Soviet manned flight (July 1982), has studied the antibiotics sensitivity of bacteria cultivated in vitro during the orbital flight . The results show an increase of the antibiotics resistance and a larger thickness of the cellular envelope for the inflight cells . The increase of antibiotics resistance can be related to a stimulating effect of space on the cell growth rate or to changes of the cellular envelope structure. Adv Space Res, 1983, 3(8), 65 - 9 W-reactivation and W-mutagenesis in phage phi X 174; Yatagai F et al.; W-reactivation (WR) and W-mutagenesis (WM) were observed after irradiation of phage chi X 174 with He and N ions (4.6 MeV/amu) . The relative amount of phage RFI DNA (RARFI) extracted from the infected cells was determined by agarose gel electrophoresis . It was found that RARFI was increased by prior UV-irradiation of host cells and this increase corresponded to the efficiency of WR. Acta Astronaut, 1988 Feb, 17(2), 267 - 70 Microgravity and the organisms: results of the Spacelab mission D1; Volkmann D; During the Spacelab mission D1 different organisms were investigated at the unicellular and multicellular level respectively . Microgravity affects growth and development of the organisms in a different manner, some processes are enhanced, others are inhibited . On the other hand, there are a lot of parameters . e.g . circadian rhythm or cell and organ polarity, which seem to be exclusively under genetical control. Acta Astronaut, 1998 Aug-Sep, 43(3-6), 329 - 48 Bioconversion systems for food and water on long term space missions; Benjaminson MA et al.; An imperative for prolonged Space flight missions is the conservation of resources . Extensive resupply could pose technological and logistical challenges for those responsible for the management and successful completion of the mission . Therefore, the biological waste water reclamation system (BWWR) which requires little or no expendable supplies and the waste cellulose to edible mushroom conversion system (CMCS) which is conceived as a low energy crop waste recycling system are prototype instruments which have been conceived as solutions to the mission resupply problem . Out tests, conducted with relatively crude devices based on the original concepts, indicate that further research on the basic principles underlying the systems and refinement of the engineering designs will lead to hardware with the potential to satisfy the requirement for minimal re-supply while providing recycled water and edible mushrooms. Nucleic Acids Symp Ser, 1997, 36, 125 - 8 Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase; Martinis SA et al.; Recombinant E . coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine . {32P}-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging . Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent . The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively . Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism. J Gen Appl Microbiol, 1997 Jun, 43(3), 175 - 7 Relationship between LET and RBE values for Escherichia coli determined using carbon ion beams from the TIARA cyclotron and HIMAC synchrotron; Imamura M et al.; NASA: Researchers studied the effects of ion beams on cell lethality in two strains of Escherichia coli . Experiments were conducted on the wild-type strain and a DNA repair-deficient mutant strain that lacks the ability to repair DNA damage . A final aspect of the study was to examine the relationship between the linear energy transfer and relative biological effectiveness values for E . coli cell lethality and dose-response for decreasing the survival fraction to 10 percent . Acta Astronaut, 1995 Oct-Dec, 36(8-12), 545 - 58 Chronic orthostatic and antiorthostatic restraint induce neuroendocrine, immune and neurophysiologial disorders in rats; Assenmacher I et al.; The tail-cast suspension rat model has been developed in ground laboratories interested in space physiology for extensive study of mechanisms causing the pathophysiological syndrome associated with space flights . We used individually-caged male rats to explore the effects of acute and chronic (7d) orthostatic restraint (OR) and head-down anti-orthostatic restraint (AOR) on a series of physiological variables . The acute restraint study showed that (1) the installation of the OR device induced an acute reaction for 2 days, with a substantial rise in ACTH (x2) and CORT (x6), and that (2) the head-down tilt from OR to AOR induced (i) within 10 min and lasting 60 min a 2-fold rise in the intra-cerebro-ventricular pressure (Picv) monitored with an icv telemetric recording system, which receded to normal between 60 and 120 min; and (ii) within 30 min a short-lived 4-fold rise in plasma ACTH and CORT levels . Chronic OR induced (1) the suppression of the diurnal ACTH/CORT rhythm, with increased mean levels, especially for ACTH, (2) a degraded circadian locomotor activity rhythm manifested by a significant reduction in the spectral power of the 24h periodicity and a concomitant emergence of shorter (ultradian) periodicities, (3) an associated, but less pronounced alteration of the diurnal rhythm in body temperature; and (4) a marked increase in baseline plasma levels of IL-1 beta and an increased reactivity in cytokine release following an E . coli endotoxin (LPS) challenge . AOR induced (1) a similar obliteration of the circadian ACTH/CORT rhythm, (2) the loss of close correlation between ACTH and CORT, (3) a generalized increase in baseline plasma IL-1 beta levels and (4) more extensive degradation of the circadian periodicity for both locomotor activity and, to a lesser extent, body temperature, replaced by dominant spectral powers for ultradian periodicities (3 to 10h) . In conclusion, both experimental paradigms--but AOR more than OR--caused a blockade of the circadian rhythmicity of major physiological variables, the loss of normal correlations between ACTH and CORT, and inflammatory-immune hyperreactivity . These pathophysiological disorders may all be parts of a complex chronic stress syndrome. Am Zool, 1994, 34(3), 343 - 52 Genetic control of early embryogenesis in the red flour beetle, Tribolium castaneum; Brown SJ et al.; The power of genetic analysis possible with the fruit fly, Drosophila melanogaster, has yielded a detailed understanding of pattern formation controlled by homeotic and segmentation genes in early embryogenesis . We are studying the genetic regulation of embryogenesis in the red flour beetle, Tribolium castaneum . The dynamic process of germ rudiment formation and sequential segmentation exhibited by Tribolium provides a context different than Drosophila within which to assess the function of homeotic and segmentation gene homologs . Our analyses of the genes in the HOM-C suggest many similarities in structure and function with the well-characterized Drosophila genes . Abdominal resembles its Drosophila homolog abdominal-A in functioning to establish segmental identities in the abdomen, such that in each case mutations result in homeotic transformations to PS6 . Although the anterior functional boundary of abdominal-A homologs is precisely conserved, the domain within which Abdominal is important extends more posterior than that of abdominal-A . The final expression pattern of the segmentation gene engrailed in Tribolium is identical to Drosophila, suggesting that these homologs are involved in a conserved developmental process . However, as expected the development of that pattern is different; engrailed stripes anticipate the formation of each new segment as they appear sequentially in the elongating germ band . Although the grasshopper even-skipped and fushi tarazu homologs are not apparently important in segmentation, the expression patterns of the Tribolium homologs strongly suggest that they have gained a role in segmentation in the lineage leading to beetles and flies . Nevertheless, differences between Tribolium and Drosophila in the dynamics of even-skipped expression and the fushi tarazu mutant phenotype indicate divergence in the regulation and roles of these genes. Physica A, 1995, 221, 180 - 92 Statistical properties of DNA sequences; Peng CK et al.; We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated . We do not find such a long-range correlation in the coding regions of the gene . We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA) . We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database . Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages . Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts . These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information. Nucl Instrum Methods Phys Res B, 1996 Feb, 107(1-4), 287 - 91 The calculation of radial dose from heavy ions: predictions of biological action cross sections; Katz R et al.; The track structure model of heavy ion cross sections was developed by Katz and co-workers in the 1960s . In this model the action cross section is evaluated by mapping the dose-response of a detector to gamma rays (modeled from biological target theory) onto the radial dose distribution from delta rays about the path of the ion . This is taken to yield the radial distribution of probability for a "hit" (an interaction leading to an observable end-point) . Radial integration of the probability yields the cross section . When different response from ions of different Z having the same stopping power is observed this model may be indicated . Since the 1960s there have been several developments in the computation of the radial dose distribution, in the measurement of these distributions, and in new radiobiological data against which to test the model . The earliest model, by Butts and Katz made use of simplified delta ray distribution functions, of simplified electron range-energy relations, and neglected angular distributions . Nevertheless it made possible the calculation of cross sections for the inactivation of enzymes and viruses, and allowed extension to tracks in nuclear emulsions and other detectors and to biological cells . It set the pattern for models of observable effects in the matter through which the ion passed . Here we outline subsequent calculations of radial dose which make use of improved knowledge of the electron emission spectrum, the electron range-energy relation, the angular distribution, and some considerations of molecular excitation, of particular interest both close to the path of the ion and the outer limits of electron penetration . These are applied to the modeling of action cross sections for the inactivation of several strains of E-coli and B . subtilis spores where extensive measurements in the "thin-down" region have been made with heavy ion beams . Such calculations serve to test the radial dose calculations at the outer limit of electron penetration . We lack data from which to test these calculations in regions close to the path of the ion aside from our earliest work on latent tracks in plastics, though it appears that the criterion then suggested for the threshold of track formation, of a minimal dose at a minimal distance (of about 20 angstroms, in plastics), remains valid. Adv Space Res, 1994 Oct, 14(10), 277 - 84 Influence of thiols and oxygen on the survival of gamma-irradiated plasmid DNA and cells; Schulte-Frohlinde D et al.; Some of the recent progress made in the understanding of the quantitative aspects of the oxygen effect in radiation biology by several groups is summarized . Examples are: the importance of unrepairable damage for the quantitative description of the oxygen effect; proof that protein thiols hardly contribute to protection in cells in the absence of oxygen; the proposal that protection by thiols in concentration ranges where all DNA radicals react with oxygen is due to the formation of hydroperoxides which can be repaired enzymatically by glutathione peroxydase; the finding that unscavengeable damage in plasmid DNA is mainly due to spur-induced clustered damages, but that the precursors of the scavengeable and the unscavengeable damage are comparably well repaired by thiols; the result that E . coli repair wild type strains are better protected by addition of thiols than strains with deficiencies in enzymatic repair capacities. Adv Space Res, 1995 Mar, 15(3), 203 - 10 Biochemical constraints for survival under Martian conditions; Dose K et al.; A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments . Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins . These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate . If an organism has no chance to repair the accumulated DNA damage during intermittent periods of active life, survival will not exceed a few decades . The restriction of survival by dryness-induced DNA lesions is corroborated by new data on conidia of Aspergillus and the free plasmid pBR 322 . Our results will be discussed with respect to the chance of finding dormant life or biochemical fossils on the surface of Mars. Planta, 1991, 186, 115 - 21 Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii; Huppe HC et al.; The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii . The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system . Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins . In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P . Decottignies et al., 1991, Eur . J . Biochem . 198, 505-512) . The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast . The NTR was purified from Chlamydomonas using thioredoxin h from the same source . Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx . 36 kDa and was specific for NADPH . Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin . Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin . Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f. Proteins, 2001 Oct 1, 45(1), 55 - 61 Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease; Dominguez MA Jr et al.; Incorporation of fluorine into proteins has long served as a means of probing structure and function, yet there are few studies that examine the impact of fluorine substitution, particularly at locations distant from the active sites of enzymes . The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and conformation . The unnatural amino acids o-, m-, and p-fluorophenylalanines were incorporated biosynthetically into the representative PvuII restriction endonuclease . Interestingly, m-fluoro-Phe-PvuII endonuclease exhibits very similar conformational stability to that of the native enzyme, but it exhibits a reproducible, 2-fold higher average specific activity . Given the level of incorporation and the distribution of species, the species of modified enzyme responsible for this increase in specific activity is most likely even faster . Further, moving the fluorine atom from the meta- to the para-position of Phe results in a 4-fold decrease in specific activity and a decrease in conformational stability of 1.5 kcal/mol . Since none of the Phe residues in PvuII endonuclease lies near the DNA recognition or catalytic sites, this differential behavior alludes to the impact of subtle changes in enzyme conformation on endonuclease activity and suggests novel ways to influence catalytic behavior . Bioelectromagnetics, 2001 Sep, 22(6), 449 - 55 Influence of intestinal myoelectrical activity on the growth of Escherichia coli; Grzesiuk E et al.; Intestinal bacteria, particularly those adhering to intestinal epithelial cells, are exposed to electric fields and currents generated by the muscular activity of the small intestine . This activity displays a regular pattern known as the myoelectrical migrating complex (MMC) . In order to explore the possibility that these endogenous electric fields could affect bacterial growth, a digitised duodenal signal obtained via serosal electrodes from a healthy calf was recorded and then applied via platinum electrodes to Escherichia coli cultures . The culture tubes were placed within a Faraday shield, incubated at 37 degrees C with shaking, and stimulated by the electric current for 5 or 8 h . The growth of E . coli stimulated by the electric current was significantly altered compared to those of non-stimulated controls: after a period of intensive growth, inhibition of cell division was observed . This was not the case when the bacteria with lon mutation were used . Moreover, synchronic bacterial culture could not be achieved in the presence of the MMC-related electric field . These results suggest that the myoelectrical activity of the duodenum, through action on cell membrane, can affect cell division of intestinal bacteria . Biotechnol Bioeng, 2001 Oct 5, 75(1), 120 - 9 Stochastic kinetic analysis of the Escherichia coli stress circuit using sigma(32)-targeted antisense; Srivastava R et al.; A stochastic Petri net model was developed for simulating the sigma(32) stress circuit in E . coli . Transcription factor sigma(32) is the principal regulator of the response of E . coli to heat shock . Stochastic Petri net (SPN) models are well suited for kinetics characterization of fluxes in biochemical pathways . Notably, there exists a one-to-one mapping of model tokens and places to molecules of particular species . Our model was validated against experiments in which ethanol (inducer of heat shock response) and sigma(32)-targeted antisense (downward regulator) were used to perturb the sigma(32) regulatory pathway . The model was also extended to simulate the effects of recombinant protein production . Results show that the stress response depends heavily on the partitioning of sigma(32) within the cell; that is, sigma(32) becomes immediately available to mediate a stress response because it exists primarily in a sequestered, inactive form, complexed with chaperones DnaK, DnaJ, and GrpE . Recombinant proteins, however, also compete for chaperone proteins, particularly when folded improperly . Our simulations indicate that when the expression of recombinant protein has a low requirement for DnaK, DnaJ, and GrpE, the overall sigma(32) levels may drop, but the level of heat shock proteins will increase . Conversely, when the overexpressed recombinant protein has a strong requirement for the chaperones, a severe response is predicted . Interestingly, both cases were observed experimentally . Biotechnol Bioeng, 2001 Oct 5, 75(1), 100 - 3 Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions; Wu CF et al.; It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000) . In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)) . Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH) . Importantly, the fusion protein was still fluorescent as determined via microscopy . In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone . Med Sci Monit, 2001 Sep-Oct, 7(5), 861 - 8 Alteration of DNA base excision repair enzymes hMYH and hOGG1 in hydrogen peroxide resistant transformed human breast cells; Gu Y et al.; BACKGROUND: Oxidative stress is a major causative agent of carcinogenesis, aging, and a number of diseases . 8-oxoG is the most stable and deleterious lesion of oxidative DNA damage . The 8-oxoG lesions can be eliminated by human repair systems consisting of three enzymes hMTH1, hOGG1, and hMYH homologous to E . coli MutT, MutM, and MutY proteins, respectively . MATERIAL AND METHODS: Human cells (P1, P2, and P3) resistant to H(2)O(2) were derived from the non-tumorigenic human breast cell line MCF10A by sequential treatment of the cells with H(2)O(2) . The protein expression levels of DNA repair enzymes were analyzed by Western blotting . The DNA binding and glycosylase activities of hMYH and hOGG1 were measured in the extracts of the H(2)O(2) resistant cells . RESULTS: The H(2)O(2) resistant cells displayed tremendously greater anchorage-independent growth capability and higher expression of the anti-apoptotic protein BCL-2 than the parental cells . H(2)O(2) detoxification ability was elevated in P1 and P2 cells, but not in P3 cells, suggesting P3 cells might employ a different defense mechanism from P1 and P2 cells . In P3 cells, both hOGG1 and hMYH glycosylase activities were reduced but their protein levels increased . Two A/8-oxoG binding complexes were detected with cell extracts: the fast-migrating complex (bottom form) was dominated in MCF10A cells, and was greatly reduced in P3 cells . Interesting, the P3 cells showing the least amount of bottom form had the weakest hMYH glycosylase activity . CONCLUSIONS: Our results demonstrated, for the first time, that alteration of base excision repair pathways is correlated to cell resistance to oxidative stress. Microbiology, 2001 Sep, 147(Pt 9), 2493 - 504 Chemotaxis in the human gastric pathogen Helicobacter pylori: different roles for CheW and the three CheV paralogues, and evidence for CheV2 phosphorylation; Pittman MS et al.; The complete genome sequence of Helicobacter pylori has revealed the presence of a novel set of chemotaxis genes including three cheV paralogues . CheV is a bi-functional protein, the N-terminal domain being homologous to the signalling-complex linker protein CheW, while the C-terminal domain is homologous to the response-regulator CheY, but its precise function in chemotaxis is unknown . In this study, each of the three cheV paralogues were insertionally inactivated in strain 26695 to determine their importance in the chemotactic signal-transduction pathway of H . pylori . Mutation of HP0019 (cheV1) had a severe inhibitory effect on chemotaxis, as determined by a swarm-plate assay . In contrast, strains carrying single mutations in either cheV2 (HP0616) or cheV3 (HP0393) displayed wild-type swarming behaviour, as did a cheV2/cheV3 double mutant . However, expression of the cheV2 or cheV3 genes in Escherichia coli resulted in an inhibition of chemotaxis in a wild-type strain, indicating their role in chemotaxis, although these genes were unable to complement isogenic E . coli cheW or cheY mutants . The product of cheV2/HP0616 was overexpressed in E . coli and purified to homogeneity . Protein fluorescence quenching experiments showed that CheV2 was capable of binding acetyl phosphate, a small-molecule phosphodonor . The measured K(m) for acetyl phosphate was 21 mM . It is concluded that in the absence of a cheZ gene, the CheV proteins could act as phosphate sinks to control the cellular level of phospho-CheY in H . pylori . However, only CheV1 was critical for chemotaxis, indicating a specific role distinct from the other paralogues in the signal-transduction pathway . Significantly, none of the CheV proteins could substitute for the loss of CheW, as an H . pylori cheW null mutant was non-chemotactic. Microbiology, 2001 Sep, 147(Pt 9), 2399 - 408 Identification of the heat-shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti; Oke V et al.; Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors . The first gene, rpoH1, complements an Escherichia coli rpoH mutation . Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa . A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth . The second gene, designated rpoH2, is 42% identical to the S . meliloti rpoH1 gene . Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa . An rpoH2-gusA fusion increases in expression during the stationary phase of growth . The presence of two rpoH-like sequences in S . meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes. Blood, 2001 Sep 15, 98(6), 1828 - 35 Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress; Morigi M et al.; Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of postdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to renal and other organ microvascular thrombosis . Why thrombi form only on arterioles and capillaries is not known . This study investigated whether VT-1 directly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress . Human umbilical vein endothelial cells (HUVECs) were used for comparison as a large-vessel endothelium . HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blood prelabeled with mepacrine . Results showed that VT-1 significantly increased platelet adhesion and thrombus formation on HMEC-1 in comparison with unstimulated control cells . An increase in thrombus formation was also observed on HUVECs exposed to VT-1, but to a remarkably lower extent . The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as documented by FACS analysis . The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of the same dermal origin (HDMECs) . The adhesive molecules involved in VT-induced thrombus formation were also studied . Blocking the binding of von Willebrand factor to platelet glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of platelet alpha(IIb)beta(3)-integrin by chimeric 7E3 Fab resulted in a significant reduction of VT-1-induced thrombus formation, suggesting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon . Functional blockade of endothelial beta(3)-integrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specific antibodies was associated with a significant decrease of the endothelial area covered by thrombi . Confocal microscopy studies revealed that VT-1 increased the expression of vitronectin receptor and P-selectin and redistributed PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet adhesion and thrombus formation in microvascular endothelium . These results might help to explain why thrombi in HUS localize in microvessels rather than in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS. Vaccine, 2001 Sep 14, 19(32), 4816 - 23 Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: identification of a T cell determinant; Sinnathamby G et al.; Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease 'peste des petits ruminants' in goats and sheep . This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants . PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins . The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity . In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant . We report that the immunized goats develop both humoral and cell-mediated immune responses . Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro . Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123-137) and a C-terminal domain (amino acids 242-609) harboring potential T cell determinant(s) in goats. J Comput Biol, 2001, 8(3), 283 - 303 Tsukuba BB: a branch and bound algorithm for local multiple alignment of DNA and protein sequences; Horton P; In this paper we present a branch and bound algorithm for local gapless multiple sequence alignment (motif alignment) and its implementation . The algorithm uses both score-based bounding and a novel bounding technique based on the "consistency" of the alignment . A sequence order independent search tree is used in conjunction with a technique for avoiding redundant calculations inherent in the structure of the tree . This is the first program to exploit the fact that the motif alignment problem is easier for short motifs . Indeed, for a short fixed motif width, the running time of the algorithm is asymptotically linear in the size of the input . We tested the performance of the program on a dataset of 300 E . coli promoter sequences and a dataset of 85 lipocalin protein sequences . For a motif width of 4, the optimal alignment of the entire set of sequences can be found . For the more natural motif width of 6, the program can align 21 sequences of length 100, more than twice the number of sequences which can be aligned by the best previous exact algorithm . The algorithm can relax the constraint of requiring each sequence to be aligned, and align 105 of the 300 promoter sequences with a motif width of 6 . For the lipocalin dataset, we introduce a technique for reducing the effective alphabet size with a minimal loss of useful information . With this technique, we show that the program can find meaningful motifs in a reasonable amount of time by optimizing the score over three motif positions. Hum Gene Ther, 2001 Sep 1, 12(13), 1639 - 49 Adenovirus gene transfer vectors inhibit growth of lymphatic tumor metastases independent of a therapeutic transgene; Korst RJ et al.; Adenovirus (Ad) gene transfer vectors traffic to regional lymph nodes (RLNs) after footpad injections in mice, resulting in localized production of interferon gamma (IFN-gamma) . With this background, we evaluated the hypothesis that Ad vector administration may inhibit RLN tumor metastasis independent of the transgene in the expression cassette . Tumors of MM48, a cell line with a propensity toward lymphogenous metastasis, were established in the footpads of syngeneic C3H mice, and E1(-)E3(-) Ad vectors encoding no transgene (AdNull) or encoding an irrelevant transgene (AdCD; Escherichia coli cytosine deaminase with no 5-fluorocytosine administration) were administered (10(10) particles) in a peritumoral location . Both vectors suppressed the growth of tumor in the regional (popliteal) lymph node . This effect was localized to the regional, but not distant, lymph nodes (p < 0.05) . Heat inactivation of the vector or decreasing the dose of the vector to 10(9) particles did not suppress RLN growth of the tumor when compared with 10(10) particles of active AdNull (p < 0.05 and p < 0.01, respectively) . The ability of an E1(-)E4(-) vector expressing beta-galactosidase (AdRSVbetagal.11) to suppress RLN tumor growth showed that the E4 region of the Ad vector was not responsible for the effect . Blocking either IFN-gamma or natural killer (NK) cells with systemic antibody treatment in immunocompetent mice allowed rapid growth of RLN metastases despite Ad vector administration, and Ad vector injection into the footpads of tumor-free mice induced the accumulation of NK cells in the RLN . These data demonstrate that, in a metastatic murine tumor model, a low dose (10(10) particles) of replication-deficient Ad vectors inhibits RLN metastases independent of a therapeutic transgene, an effect that is mediated, at least in part, by IFN-gamma and NK cells. Biochem J, 2001 Sep 15, 358(Pt 3), 783 - 90 Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity; Park HS et al.; The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O(-)(2) at the expense of NADPH . The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli . During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane . Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms . The leucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphorylation sites . In the present study, we report that CK2 is able to catalyse the phosphorylation of p47(phox) in vitro . Phosphoamino acid analysis of phosphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on serine residues . CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47(phox) . Dependence of phosphorylation on the conformation of p47(phox) is supported by the finding that p47(phox) undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47(phox) . In addition, 5,6-dichloro-1-beta-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells . Taken together, we propose that CK2 is the p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase. Biochem J, 2001 Sep 15, 358(Pt 3), 757 - 61 Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins; Jiang DW et al.; As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli . The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography . Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length . TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0) . TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0) . In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. J Am Chem Soc, 2001 Sep 12, 123(36), 8750 - 9 Total synthesis of cyclic ADP-carbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic ADP-ribose; Shuto S et al.; The synthesis of cyclic ADP-carbocyclic-ribose (cADPcR, 4) designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, was achieved using as the key step a condensation reaction with the phenylthiophosphate-type substrate 14 to form an intramolecular pyrophosphate linkage . The N-1-carbocyclic-ribosyladenosine derivative 16 was prepared via the condensation between the imidazole nucleoside derivative 17, prepared from AICA-riboside (19), and the readily available optically active carbocyclic amine 18 . Compound 16 was then converted to the corresponding 5' '-phosphoryl-5'-phenylthiophosphate derivatives 14 . Treatment of 14 with AgNO3 in the presence of molecular sieves (3 A) in pyridine at room temperature gave the desired cyclization product 32 in 93% yield, and subsequent acidic treatment provided the target cADPcR (4) . This represents a general method for synthesizing biologically important cyclic nucleotides of this type . 1H NMR analysis of cADPcR suggested that its conformation in aqueous medium is similar to that of cADPR . cADPcR, unlike cADPR, was stable under neutral and acidic conditions, where under basic conditions, it formed the Dimroth-rearranged N6-cyclized product 34 . cADPcR was also stable in rat brain membrane homogenate which has cADPR degradation activity . Furthermore, cADPcR was resistant to the hydrolysis by CD38 cADPR hydrolase, while cADPR was rapidly hydrolyzed under the same conditions . When cADPcR was injected into sea urchin eggs, it caused a significant release of Ca2+ in the cells, an effect considerably stronger than that of cADPR . Thus, cADPcR was identified as a stable mimic of cADPR. Biochemistry, 2001 Sep 11, 40(36), 10901 - 10 The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation; Palackal NT et al.; Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1 . Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones . We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones . cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized . AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR . AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz{a}anthracene-3,4-diol, 7-methylbenz{a}anthracene-3,4-diol, and 7,12-dimethylbenz{a}anthracene-3,4-diol . AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols . The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested . The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry . AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo{a}pyrene-7(R),8(R)-dihydrodiol . AKR1A1 also preferred (-)-benz{a}anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz{a}anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz{a}anthracene-3(R),4(R)-dihydrodiol . The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo{a}pyrene-7,8-dione by LC/MS . Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed . The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues. Biochemistry, 2001 Sep 11, 40(36), 10846 - 52 Arfophilin is a common target of both class II and class III ADP-ribosylation factors; Shin OH et al.; Arfophilin was first identified as a target protein for GTP-ARF5 . The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756) . This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner . Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin . Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind . In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding . Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating . Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector. Biochemistry, 2001 Sep 11, 40(36), 10825 - 31 Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase; Shi XB et al.; Ten truncated mutants of chloroplast ATP synthase epsilon subunit from spinach (Spinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PCR . These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF(1), and the ATPase activity and proton conductance of thylakoid membrane were examined . Deletions of as few as 3 amino acid residues from the N-terminus or 6 residues from the C-terminus of epsilon subunit significantly affected their ATPase-inhibitory activity in solution . Deletion of 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore proton impermeability . Considering the consequence of interaction of epsilon and gamma subunit in the enzyme functions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay . In addition, the structures of these mutants were modeled through the SWISS-MODEL Protein Modeling Server . These results suggested that in chloroplast ATP synthase, both the N-terminus and C-terminus of the epsilon subunit show importance in regulation of the ATPase activity . Furthermore, the N-terminus of the epsilon subunit is more important for its interaction with gamma and some CF(o) subunits, and crucial for the blocking of proton leakage . Compared with the epsilon subunit from E . coli {Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M . (1992) Arch . Biochem . Biophys . 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M . (1988) J . Biol . Chem . 263, 4335-4340}, the chloroplast epsilon subunit is more sensitive to N-terminal or C-terminal truncations. Biochemistry, 2001 Sep 11, 40(36), 10782 - 91 Structural comparison of monomeric variants of the chemokine MIP-1beta having differing ability to bind the receptor CCR5; Kim S et al.; MIP-1beta, a member of the chemokine family of proteins, tightly binds the receptor CCR5 as part of its natural function in the immune response, and in doing so also blocks the ability of many strains of HIV to enter the cell . The single most important MIP-1beta residue known to contribute to its interaction with the receptor is Phe13, which when mutated reduces the ability of MIP-1beta to bind to CCR5 by more than 1000-fold . To obtain a structural understanding of the dramatic effect of the absence of Phe13 in MIP-1beta, we used multidimensional heteronuclear NMR to determine the three-dimensional structure of the MIP-1beta F13A variant . We had previously shown that, unlike the wild-type protein which has been shown to be a tight dimer, the F13A mutant is monomeric even at high concentrations {Laurence, J . S., Blanpain, C., Burgner, J . W., Parmentier, M., and LiWang, P . J . (2000) Biochemistry 39, 3401-3409}, leading to significant changes in the NMR spectra of F13A and the wild-type protein . We have obtained a total of 940 structural restraints for MIP-1beta F13A, and have calculated a family of structures having a backbone rmsd from the average of 0.55 A (residues 12-67) . A structural comparison of the F13A mutant with a fully active monomeric variant, P8A, shows that despite some differences in the (1)H-(15)N HSQC spectra the two are nearly identical in NOE distance restraints and in backbone conformation . A comparison of F13A with the wild-type protein shows largely the same fold, although differences exist in the N-terminal and loop regions for which the loss of the dimer in F13A can mainly account . A dynamics comparison confirms greater flexibility in F13A than in the wild-type protein in regions of dimer contact in the wild-type protein . In an analysis to determine if the large functional effect resulting from the loss of Phe13 is due to the local side chain change or due to more global structural changes, we conclude that local effects predominate . This suggests that a strategy for designing tight binding anti-CCR5 therapeutics should include a Phe-like component. Biochemistry, 2001 Sep 11, 40(36), 10756 - 63 Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli; Zhu Z et al.; The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector . The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl) . The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl . The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct . Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size . Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD . Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence . These data confirm that the purified protein was the intact hAR LBD . AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD . The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for {(3)H}-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate. Plasmid, 2001 Jul, 46(1), 57 - 9 A yeast expression vector and leucine selection in Escherichia coli to aid in the identification of novel genes; Palmer EA et al.; The complete sequencing of the Saccharomyces cerevisiae genome provides a powerful tool for studying and elucidating essential cellular processes . To aid in the application of this resource to investigations into the molecular mechanisms of endoplasmic reticulum-associated protein degradation, a simple procedure was designed to generate a unique 2-microm LEU2-selectable yeast expression vector . Putative genes easily inserted into this vector are under control of the ADH1 promoter and transcription terminator sequences . Furthermore, a LEU2 selection in both yeast and Escherichia coli was used to allow the isolation of underrepresented plasmid from a pool of multiple vectors . Together, these advances in technology will be useful in the systematic analysis of novel yeast gene function . Plasmid, 2001 Jul, 46(1), 1 - 9 Nucleotide polymorphism in microcin V plasmids; Pinou T et al.; DNA sequence polymorphism was determined for the microcin V gene cluster encoded on the microcin V plasmids of 12 natural isolates of Escherichia coli . These microcin V gene clusters are similar in DNA sequence, with only 10 of the 683 bp polymorphic . Further, the levels and patterns of microcin V gene cluster polymorphism differ from those of a chromosomal region, trpORF2, sequenced from each of the host isolates . These contrasting levels and patterns of polymorphism suggest that the microcin V gene cluster has experienced an evolutionary history different from that of the host . Analyst, 2001 Aug, 126(8), 1279 - 84 Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy; LeCaptain DJ et al.; A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated . Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field . Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation . Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation . The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error . The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient . Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system . The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis. J Virol, 2001 Oct, 75(19), 9274 - 81 Evidence that Equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding; Warner S et al.; Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus . In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop . Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif . To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1) . GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV . An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro . Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors . This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction . A separate cell-binding assay also implicated GST-VP1 in receptor binding . Importantly, anti-GST-VP1 antibodies