Int J Parasitol, 2001 May 15, 31(7), 687 - 96 Differential gene expression in haemocytes of the snail Biomphalaria glabrata: effects of Schistosoma mansoni infection; Miller AN et al.; Parasite encapsulation and destruction in Biomphalaria glabrata has been shown to involve the cellular component of the snail's internal defence system, the haemocytes . To identify genes involved in the immunobiology of these cells, we used the method of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) to investigate differential gene regulation in haemocytes isolated from Schistosoma mansoni exposed and unexposed snails . RNA isolated from circulating haemocytes from resistant snails (BS-90 stock), previously exposed to S . mansoni, was analysed using 12 different arbitrary primers in conjunction with an anchored Oligo d(T(11)CG) primer . Transcription profiles between haemocytes of parasite exposed and unexposed snails were compared and a total of 87 differentially regulated bands were identified and isolated . Of these, 65 bands were cloned and used as probes in Southern blots to show the presence of corresponding sequences in the snail genome . RT-PCR was performed to verify the regulation of these transcripts . DNA sequence analysis showed that the majority of the cloned sequences were novel, although a few showed a high degree of sequence similarity to other sequences in the DNA and protein databases . One of these included a differentially expressed transcript that showed a significant degree of sequence identity to E . coli transposase Tn5, an enzyme whose activity is normally associated with generating mobility and instability in the genome. Biochem J, 2001 May 15, 356(Pt 1), 223 - 32 Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis; Bearne SL et al.; Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen . When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis . Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography . Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde . Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis . Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0 . The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde . Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition . When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx . 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis. Virology, 2001 May 10, 283(2), 197 - 206 The long repeat region is dispensable for fowl adenovirus replication in vitro; Ojkic D et al.; Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome . The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown . An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses . The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2 . Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro . The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR . Can J Anaesth, 2001 Apr, 48(4 Suppl), S32 - 40 Oxygen therapeutics--current concepts; Hill SE; PURPOSE: In an effort to develop agents that enhance the oxygen-delivery capability of blood without the risks associated with allogeneic blood transfusions, several products are undergoing development and clinical trials . These oxygen transport agents can be divided into two main groups, perfluorocarbon (PFC) emulsions and modified hemoglobin solutions . SOURCE: Articles from the literature on the development and clinical trials of oxygen therapeutic agents were reviewed . PRINCIPAL FINDING: PFCs are synthetic fluorinated hydrocarbons that increase dissolved oxygen in the fluid phase of the blood without binding the oxygen molecule . They enhance oxygen delivery significantly and may be used to augment the technique of intraoperative autologous donation . Two PFC products have been tested in Phase III clinical trials . Hemoglobin-based oxygen carriers (HBOCs) are either cross-linked or microencapsulated hemoglobin molecules . Modification of the human hemoglobin molecule with intra- and inter-molecular cross-linking eliminates renal toxicity and improves the oxygen dissociation characteristics of the molecule . These modifications are necessary because stroma-free hemoglobin (Hb) does not release oxygen in the physiologic range and dissociates into dimers which can be rapidly filtered by the kidney, leading to renal toxicity . In addition to human Hb, bovine hemoglobin is another source of raw material for HBOC products . Recombinant human Hb has also been produced, using an E . coli expression system, for HBOC manufacturing . Four cross-linked hemoglobin products have been tested in Phase III clinical trials . CONCLUSION: While no product has yet been approved for clinical use, preliminary studies with oxygen therapeutics show promising results, with effective oxygen carrying capacity and acceptable side effect profiles . In the future, the formation of a hybrid product which combines the best features from several of the products currently undergoing development may yield the ideal oxygen therapeutic agent. Shock, 2001 May, 15(5), 386 - 91 Endotoxin impairs agonist-stimulated intracellular free calcium (Ca(i)) responses in freshly dispersed aortic endothelial cells; Jones JJ et al.; Impairment in endothelial cell intracellular free calcium (Ca(i)) mobilization mechanisms may contribute to decreased nitric oxide (NO) biosynthesis and impaired vasorelaxation responses of endotoxemic guinea pigs to endothelium-dependent vasodilators . We tested this hypothesis using fura-2 microfluorometry to compare agonist-stimulated Ca(i) responses of aortic endothelial cells freshly dispersed from guinea pigs 16 h after intraperitoneal injection of Escherichia coli endotoxin (lipopolysaccharide, LPS; 4 mg/kg) or saline (CON) . In the presence of normal extracellular Ca2+ (2 mmol/L), basal (non-stimulated) endothelial Ca(i) (340/380 nm fluorescence ratio, R) was not different between CON and LPS cells (1.1 +/- 0.03 and 1.1 +/- 0.03, respectively) . However, exposure to ADP (10 micromol/L) produced a biphasic increase in Ca(i) that was markedly decreased in cells from LPS-treated animals (P < 0.0001) . Peak ADP-stimulated Ca(i) responses averaged 2.2 +/- 0.21 in CON cells and 1.5 +/- 0.11 (P < 0.01) in cells dispersed from LPS-treated animals . Exposure to acetylcholine (ACh; 10 micromol/L) produced sustained increases in Ca(i) (R = 1.4 +/- 0.13) in CON cells; however, LPS abolished Ca(i) responses to ACh . Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Ca(i) that was not different between groups . In the absence of extracellular Ca2+ (plus 10 micromol/L EGTA), exposure to ADP (10 micromol/L) produced transient increases in Ca(i) (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals . Exposure to ACh in zero Ca2+ (10 micromol/L) produced smaller increases in Ca(i) (peak R = 1.3 +/- 0.12) in CON cells (when compared to ADP); however, Ca(i) responses to ACh remained absent in cells from LPS-treated animals . Re-exposure to Ca2+ produced sustained ACh-induced Ca(i) responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P< 0.05) ADP-induced sustained Ca(i) responses . Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Ca(i) responses primarily involving impaired Ca2+ influx mechanisms . Known dependence of endothelial agonist-stimulated NO synthesis on Ca(i) suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation. Shock, 2001 May, 15(5), 378 - 85 Effects of nucleoside transport inhibition on hepatosplanchnic perfusion, oxygen extraction capabilities, and TNF release during acute endotoxic shock; Zhang H et al.; We explored the effects of the nucleoside transport inhibitor draflazine on regional blood flow, O2 extraction capabilities, and tumor necrosis factor (TNF) release in acute endotoxic shock . Fourteen anesthetized and mechanically ventilated dogs received 2 mg/kg of Escherichia coli endotoxin and were divided into two groups . Seven dogs received 0.1 mg/kg of draflazine 30 min before endotoxin, and 7 dogs served as a control group . Draflazine decreased arterial pressure without influencing cardiac index . Mesenteric and portal blood flow and ileum mucosal perfusion increased, but renal blood flow dramatically decreased . After endotoxemia, the draflazine-treated dogs had a lesser fall in cardiac index, filling pressures, and left ventricular stroke work index, and a lesser increase in pulmonary vascular resistance . After fluid resuscitation, they had a consistently lower renal blood flow and ileum mucosal perfusion, but a higher mixed venous and hepatic oxygen saturation and arterial pH than the control group . When cardiac index was reduced by tamponade to study the O2 extraction capabilities, renal blood flow and ileum mucosal perfusion remained lower in the draflazine group . Draflazine did not influence whole-body O2 extraction capabilities, but it delayed the occurrence of liver O2 supply dependency as indicated by a significantly lower liver DO2crit (27.7 +/- 3.9 vs . 43.3 +/- 10.8 mL/min) and a higher O2ERcrit (62.7 +/- 9.5 vs . 42.5 +/- 7.1%) than controls (both P< 0.05) . On the other hand, draflazine increased intestinal DO2crit (42.4 +/- 15.4 vs . 27.7 +/- 6.5 mL/min, P < 0.05) compared to the control group . TNF levels remained higher in the draflazine group than in the control group, particularly 3 and 4 h after endotoxin administration . We conclude that nucleoside transport inhibition with draflazine does not alter global and hepatosplanchnic hemodynamics but may decrease gut mucosal perfusion and renal blood flow . However, this intervention can improve liver O2 extraction capabilities in acute endotoxic shock. J Biol Chem, 2001 Jul 20, 276(29), 27555 - 61 Epub 2001 May 02. The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus; Horer S et al.; Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature . In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor . Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance . Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose . Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli . Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme . The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3% . It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes . The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173). FEBS Lett, 2001 Apr 27, 495(3), 167 - 71 Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3; Grill S et al.; Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria . However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions . Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site . We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes . Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery. Int Microbiol, 2000 Dec, 3(4), 239 - 45 The purified colicin S8 is a multimeric protein; Concepcion Curbelo JL et al.; Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc . Colicins are commonly inducible and extracellular . Their molecular masses range from 30 to 90 kDa . Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure . In our hands, purified colicin S8 was an aggregation of extremely related polypeptides . Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa . Behavior on molecular filtration indicated a molecular weight higher than 200 kDa . Similar results were obtained when purification was carried out through FPLC . Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide . We conclude that more than one form of colicin S8 exists . The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides . Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components. Antisense Nucleic Acid Drug Dev, 2001 Apr, 11(2), 77 - 85 Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers; Vorobjev PE et al.; The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated . These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present . It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli . The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers . Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers . Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H . As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy. Nature, 2001 May 3, 411(6833), 110 - 4 An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes; Fabrega C et al.; Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs . There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each . These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs . Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret . Here we describe a different cysteinyl-tRNA synthetase from M . jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo . This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin . The existence of this protein contrasts with proposals that aminoacylation with cysteine in M . jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase. J Virol, 2001 Jun, 75(11), 5197 - 204 Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner; Gewurz BE et al.; The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome . We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus . Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules . The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry . These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules . Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2 . The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E . We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM . The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules. J Virol, 2001 Jun, 75(11), 5141 - 50 Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection; Tumpey TM et al.; Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses . We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I . Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone . In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge . Mice that were i.n . administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge . The immune mediators of Het-I were investigated . The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively . beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge . Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice . Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5 . These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential. J Mass Spectrom, 2001 Apr, 36(4), 384 - 91 Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9; Havlicek V et al.; The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp) . The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain . This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP . The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC . However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT) . In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9 . This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323 . Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found . In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence . We also discuss these results in comparison with those from recombinant ACT expressed in E . coli strain K12 XL1-Blue . The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . J Neurobiol, 2001 Jun 5, 47(3), 183 - 93 Conditional ablation of neurones in transgenic mice; Isles AR et al.; Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo . This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation . Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo . As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter . We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost . The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic . Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo . Plant Cell Physiol, 2001 Apr, 42(4), 349 - 57 Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds; Mizuno K et al.; cDNA clones encoding an isoform of starch branching enzyme, RBE4, have been identified from a developing rice seed cDNA library, using a synthetic oligonucleotide probe corresponding to the N-terminal amino acid sequence of RBE4 . The cDNA-derived amino acid sequence indicated that RBE4 is initially produced as a precursor protein of 841 amino acids, including a 53-residue transit peptide at the N-terminus . The mature form of RBE4 shared a high degree of sequence identity (80%) with mature RBE3, and possessed an N-terminal extra sequence, as found in RBE3 . Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds . The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development . To examine enzymatic functions of RBE4, recombinant proteins were produced in Escherichia coli cells, and purified by two chromatographic separations . The branched alpha-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer . The peak of major branches of the products by RBE3 or RBE4 was 6 glucose units, whereas the peaks of major branches of the products by RBE1 were 6 and 11 glucose units . The similar property between RBE3 and RBE4 is supported by high similarity of the amino acid sequences between them. J Biol Chem, 2001 Jul 6, 276(27), 24790 - 6 Epub 2001 May 01. CYP83b1 is the oxime-metabolizing enzyme in the glucosinolate pathway in Arabidopsis; Hansen CH et al.; CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates . Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-l-cysteine, the next proposed intermediate in the glucosinolate pathway . The production was shown to be dependent on a cytochrome P450 monooxygenase . We searched the genome of A . thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides . By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli . The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a "P450mox" that converted oximes into toxic compounds that the plant detoxified into glucosinolates. J Biol Chem, 2001 Jul 6, 276(27), 25399 - 403 Epub 2001 May 01. HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding; Macdonald JR et al.; HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens . Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K(half)) of 1.4 x 10(-7) m, and a Hill coefficient of 4.3 was observed . Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli . Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker . Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed . Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix . Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant . Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix. J Biol Chem, 2001 Jul 20, 276(29), 27345 - 53 Epub 2001 May 01. Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase; Ludlam AV et al.; Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein . To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement . The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB . Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to {alpha-(32)P}ATP . These alleles also failed to complement a dnaC28 mutant . Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation . Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M . W., Miyajima, A., Kobori, J., and Arai, K . (1987) J . Biol . Chem . 262, 10475-10480). J Biol Chem, 2001 Jul 6, 276(27), 24498 - 505 Epub 2001 May 01. Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components; Ebisu K et al.; Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-associated flavocytochrome b(558) . Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J . T., Uhlinger, D . J., and Lambeth, J . D . (1992) J . Biol . Chem . 267, 7529-7538) . To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed . In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K(m) for NADPH . O(2) generating activity was remarkably stabilized by using p67N-p47N . The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors . In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components . Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b(558) relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures . From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system . The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex . Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b(558) in the active complex. J Biol Chem, 2001 Jul 6, 276(27), 25014 - 21 Epub 2001 May 01. The Escherichia coli heat labile toxin binds to Golgi membranes and alters Golgi and cell morphologies using ADP-ribosylation factor-dependent processes; Zhu X et al.; The fate of the catalytic subunit of the Escherichia coli heat labile toxin (LTA(1)) was studied after expression in mammalian cells to assess the requirement for ADP-ribosylation factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors . A progression in LTA(1) localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA(1) expression . At the highest levels of LTA(1) expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A . Thus, LTA(1) binds to the Golgi and can alter its morphology in two distinct ways . However, point mutants of LTA(1) that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology . Co-expression of mutants of ARF3 that regained binding to these same mutant LTA(1) proteins restored the localization and activities of the toxin . Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes . A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels . These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-ribosylation activity but are the least toxic LTA(1) mutants yet described, making them the best candidates for development as mucosal adjuvants. Genetics, 2001 May, 158(1), 41 - 64 Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli; Courcelle J et al.; The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor . Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E . coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control . We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E . coli chromosome . We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold . In addition, several transcripts were either downregulated or degraded following UV irradiation . These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation. Genetics, 2001 May, 158(1), 29 - 39 Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli; Onda M et al.; We studied the role of DNA ligase in illegitimate recombination in Escherichia coli . A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation . Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type . Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation . In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp) . However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp) . Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation . These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism. Electrophoresis, 2001 Mar, 22(5), 966 - 9 Reliable quantification of in vitro synthesized green fluorescent protein: comparison of fluorescence activity and total protein levels; Nemetz C et al.; At any time in vitro or in vivo expressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples . Quantification via protein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms . Here, wild-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coli lysates . Fluorescent activity was determined in dependence of oxygen and compared to total protein levels . In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable via determination of fluorescence activity . A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining is described. Electrophoresis, 2001 Mar, 22(5), 933 - 45 Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels; Walker AK et al.; We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs) . The use of this technique to visualize proteins from IPGs was explored in this study . Whole cell Escherichia coli extracts of various loadings were separated on IPGs . These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel . Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel . This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE) . These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels . Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa . In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features. Nat Rev Mol Cell Biol, 2001 May, 2(5), 350 - 6 Protein targeting by the twin-arginine translocation pathway; Robinson C et al.; The twin-arginine translocation pathway operates in the thylakoid membrane of chloroplasts and in the plasma membrane of most free-living bacteria . Its main function is to transport fully folded proteins across the membrane . Three important tat genes have been identified and the sequences of the encoded proteins, together with the unusual properties of the pathway, indicate that the Tat system is completely different from other protein translocases. Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5521 - 5 Epub 2001 May 01. The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase; Ribbe MW et al.; It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion . Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially ( approximately 45%) FeMoco deficient . The "free" FeMoco that is not inserted accumulates in the cell . We were able to insert this "free" FeMoco into the partially pure FeMoco-deficient MoFe protein . This insertion reaction required crude extract of the DeltanifHDK A . vinelandii strain CA12, Fe protein and MgATP . We used this as an assay to purify a required "insertion" protein . The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E . coli GroEL, and its NH(2)-terminal polypeptide sequence . The NH(2)-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms . Purified GroEL of A . vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing . By using GroEL-containing extracts from a DeltanifHDK strain of A . vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein . The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes. J Biol Chem, 2001 Jul 20, 276(29), 27449 - 54 Epub 2001 Apr 30. Structure of Ala(20) --> Pro/Pro(64) --> Ala substituted subunit c of Escherichia coli ATP synthase in which the essential proline is switched between transmembrane helices; Dmitriev OY et al.; The structure of the A20P/P64A mutated subunit c of Escherichia coli ATP synthase, in which the essential proline has been switched from residue 64 of the second transmembrane helix (TMH) to residue 20 of the first TMH, has been solved by (15)N,(1)H NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture . The cA20P/P64A mutant grows as well as wild type, and the F(0)F(1) complex is fully functional in ATPase-coupled H(+) pumping . Residues 20 and 64 lie directly opposite to each other in the hairpin-like structure of wild type subunit c, and the prolinyl 64 residue is thought to induce a slight bend in TMH-2 such that it wraps around a more straightened TMH-1 . In solution, the A20P/P64A substituted subunit c also forms a hairpin of two alpha-helices, with residues 41-45 forming a connecting loop as in the case of the wild type protein, but, in this case, Pro(20) induces a bend in TMH-1, which then packs against a more straightened TMH-2 . The essential prolinyl residue, whether at position 64 or 20, lies close to the aspartyl 61 H(+) binding site . The prolinyl residue may introduce structural flexibility in this region of the protein, which may be necessary for the proposed movement of the alpha-helical segments during the course of the H(+) pumping catalytic cycle. J Biol Chem, 2001 Jun 29, 276(26), 24051 - 8 Epub 2001 Apr 30. Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*; Ghosh I et al.; Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp . PCC6803 . First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion . The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid . We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway . Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves . N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves . The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association . This is the first evidence showing that the conserved residues in block F are required for protein splicing. Bioinformatics, 2001 May, 17(5), 445 - 54 The utility of different representations of protein sequence for predicting functional class; King RD et al.; MOTIVATION: Data Mining Prediction (DMP) is a novel approach to predicting protein functional class from sequence . DMP works even in the absence of a homologous protein of known function . We investigate the utility of different ways of representing protein sequence in DMP (residue frequencies, phylogeny, predicted structure) using the Escherichia coli genome as a model . RESULTS: Using the different representations DMP learnt prediction rules that were more accurate than default at every level of function using every type of representation . The most effective way to represent sequence was using phylogeny (75% accuracy and 13% coverage of unassigned ORFs at the most general level of function: 69% accuracy and 7% coverage at the most detailed) . We tested different methods for combining predictions from the different types of representation . These improved both the accuracy and coverage of predictions, e.g . 40% of all unassigned ORFs could be predicted at an estimated accuracy of 60% and 5% of unassigned ORFs could be predicted at an estimated accuracy of 86%. Bioinformatics, 2001 May, 17(5), 429 - 37 Analysis of genomic sequences by Chaos Game Representation; Almeida JS et al.; MOTIVATION: Chaos Game Representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to find the coordinates for their position in a continuous space . This distribution of positions has two properties: it is unique, and the source sequence can be recovered from the coordinates such that distance between positions measures similarity between the corresponding sequences . The possibility of using the latter property to identify succession schemes have been entirely overlooked in previous studies which raises the possibility that CGR may be upgraded from a mere representation technique to a sequence modeling tool . RESULTS: The distribution of positions in the CGR plane were shown to be a generalization of Markov chain probability tables that accommodates non-integer orders . Therefore, Markov models are particular cases of CGR models rather than the reverse, as currently accepted . In addition, the CGR generalization has both practical (computational efficiency) and fundamental (scale independence) advantages . These results are illustrated by using Escherichia coli K-12 as a test data-set, in particular, the genes thrA, thrB and thrC of the threonine operon. Biochemistry, 2001 May 8, 40(18), 5548 - 55 Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase; Rong J et al.; The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain . We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units {Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M . (2000) Biochem . J . 346, 463-468} . In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST . Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho{(35)S}sulfate . Incubations with O-desulfated heparin, predominantly composed of {(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-}(n)(), resulted in 2-O-sulfation of iduronic acid . When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure {(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-}(n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid . Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid . In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM). Biochemistry, 2001 May 8, 40(18), 5506 - 10 Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli; Ding PZ et al.; The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+) . This membrane transport protein consists of 12 transmembrane helices . Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I . The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS) . Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C) . PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C) . We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment . Second site revertants were isolated from K18C and Y31C . The revertants were found to have mutations in helices I, IV, and VII. Biochemistry, 2001 May 8, 40(18), 5488 - 95 Stability and global fold of the mouse prohormone convertase 1 pro-domain; Tangrea MA et al.; We have purified the mouse prohormone convertase 1 (PC1) pro-domain expressed in Escherichia coli cells and demonstrated, using a number of biophysical methods, that this domain is an independent folding unit with a T(m) of 39 degrees C at a protein concentration of 20 microM and pH 7.0 . This differs significantly from similar pro-domains in bacteria and human furin, which are unfolded at 25 degrees C and require the catalytic domain in order to be structured {Bryan et al . (1995) Biochemistry 34, 10310-10318; Bhattacharjya et al . (2000) J . Biomol . NMR 16, 275-276} . Using heteronuclear NMR spectroscopy, we have determined the backbone (1)H, (13)C, and (15)N assignments for the pro-domain of PC1 . On the basis of (1)H/(13)C chemical shift indices, NOE analysis, and hydrogen exchange measurements, the pro-domain is shown to consist of a four-stranded beta-sheet and two alpha-helices . The results presented here show that both the bacterial pro-domain in complex with subtilisin and the uncomplexed mouse PC1 pro-domain have very similar overall folds despite a lack of sequence homology . The structural data help to explain the location of the secondary processing sites in the pro-domains of the PC family, and a consensus sequence for binding to the catalytic domain is proposed. Biochemistry, 2001 May 8, 40(18), 5376 - 81 A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu; Mursinna RS et al.; Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA . Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection . These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation . We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site . Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA . Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site . This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site. Appl Microbiol Biotechnol, 2001 Mar, 55(2), 187 - 91 High-level production of heme-containing holoproteins in Escherichia coli; Jung Y et al.; The expression of recombinant protein is essential for the investigation of the functions and properties of heme-containing protein as an electron carrier . For the expression of fully active recombinant protein, conversion of the expressed apoprotein into holoprotein is the most important and difficult problem . In this study, a system was developed for the production of heme-containing protein in a pure, recombinant holoprotein form, using the bovine cytochrome b5 tryptic fragment and Escherichia coli bacterioferritin as heterologous and homologous heme-containing model proteins, respectively . This system is based on the slow synthesis of recombinant apoprotein, which can maintain the balanced consumption of amino acids between protein synthesis and heme synthesis, so that the synthesized apoprotein continues to act as a heme sink . From a 1-1 culture, 15 mg of cytochrome b5 and 40 mg of bacterioferritin were purified as pure holoprotein forms . Our expression system provides a rapid and simple method for obtaining large quantities of the active holo-form of heme-containing proteins. Biosci Biotechnol Biochem, 2001 Mar, 65(3), 694 - 7 Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting; Takai T et al.; A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli . The cells were solubilized and run on SDS-PAGE under reducing conditions . Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis. Ultramicroscopy, 2001 Apr, 87(3), 155 - 64 4Pi-confocal microscopy of live cells; Bahlmann K et al.; By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy . So far this improvement was possible only in glycerol-mounted, fixed cells . Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells . Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope . The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria . The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 98 - 100 {Cloning and expression of the genes of glutathione synthetases}; Shen LX et al.; The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively . The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh . E . coli BL21 was transformed by pTrc-gsh for expression of the related enzymes . Analysis of SDS-PAGE showed that the expected products were expressed . E . coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5 . The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG . The expressed products were up to 25% of the total protein of the bacteria . Acetone-treated cells of the engineered strain could synthesize GSH efficiently. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 90 - 3 {A study on the expression of human leptin in the mammary glands of transgenic mice}; Liu JZ et al.; Human leptin expressed by E . coli had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas . In this study, human leptin gene about 1.0 kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2 kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3 kb were used to construct a expression vector . Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172 bp) and parts of the third exon(including part of the encoding sequences and part of the 3' untranslated region) . The final expression vector was digested with NotI and a fragment of 7.5 kb was collected and dissolved in TE(10 mmol/L Tris.Cl, pH7.4; 0.1 mmol/L EDTA) for later microinjection . The concentration of DNA was about 2 micrograms/mL, the copy number in 1 mL was about 2.4 x 10(11), every 1 to 2 pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage . After standard microinjection procedures, 48 live mice were obtained . The tails of the mice were cut(about 0.1 g) at four weeks of age, genomic DNA was extracted and digested completely with EcoRI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48) . The two female transgenic mice(2# and C3) were mated with nontransgenic male mice . The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage . SDS-PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of non-transgenic mouse at fifth day after parturition as control . SDS-PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin . The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1-2 mg/mL. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 78 - 83 {Cloning, expression and preliminary application of a alpha-hydroxynitrile lyase from cassave}; Cheng SH et al.; alpha-Hydroxynitrile lyase (ME-HNLs, E.C . 4.1.2.3.37) from the cyanogenic crop cassava(Manihot esculentz, Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)-cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals . The cDNA of a ME-HNL were obtained by RT-PCR and cloned . The sequencing result for the cDNA showed that the sequence encoded for the ME-HNL was inconsistent with all those which are published, such as hnl10, hnl24, hnl4 . The full sequence analysis demonstrated that the cloned cDNA was about 75.2%, 79.8%, 99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142, 337, 476, 634 and 636, respectively . The two base substitutions lead to change the amino acid sequence, i.e., Ser113-->Gly113, Phe158-->Tyr158 . To construct the recombinant plasmid pET30a-hnl, the cDNA was inserted into an expression vector pET30a . After transformation of pET30a-hnl and induction with IPTG, the ME-HNL was efficiently expressed in E . coli . BL21 (DE3) and reached over 2100 units/L of culture with the specific activity 8.5 u/mg protein . By one simple treatment, incubating 10 minutes at 70 degrees C, the recombinant ME-HNL may be used as an catalyst for production of (S)-mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 7 - 10 {Site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin (chymosin)}; Cheng HJ et al.; During the work of site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin, it was found that the corresponding template sequence had the potential to form a loop-stem structure with free energy of -16.1 kcal/mol, which prevent the template from pairing with primer and, in turn, the synthesis of the mutated DNA strand . Rapid annealing can overcome this difficulty . Five expression plasmids of prochymosin muants with deletion of Cys206-Cys210 (C206A, C210A, C206A/C210A, C210S and C206S/C210S) were constructed . Except for C206A they were expressed at high level in E . coli amounting to 50% of the total cellular proteins . Renaturation of the mutant prochymosin indicated that Cys206-Cys210 is dispensable for correct refolding of prochymosin . However, the amino acid residues at Cys206 and/or Cys 210 play a critical role in determining the renaturation . Among the five mutants the reactivation efficiency of C206A/C210A were about 4.5-fold, 20-fold and 30-fold higher than that of C206S/C210S, C210A and C210S respectively . C206A can not correctly refold at all . CD spectra in the far UV region indicate that C206A/C210A and C206S/C210S chymosin analogs have a secondary structure almost identical to that of the wild-type chymosin . Fluorescence spectroscopic analysis revealed that mutant chymosins have the same emission maximum at 333 nm as the wild-type chymosin but their fluorescence intensities at 333 nm are much higher than that of the wild-type chymosin . Considering that the mutants and the wild-type chymosin exhibit almost the same specific activity, it is reasonable to conclude that the mutant proteins assume a native active information with a perturbance around some tryptophan residues. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 68 - 72 {Cloning and expression of urate oxidase and its application in serum uric acid analysis}; Zhu XJ et al.; Anurate oxidase (uricase, EC 1.7.3.3) gene from Candida utilis AS2.117 was cloned by PCR amplification with primers derived from conserved regions of published uicase DNA sequence . The DNA sequence of cloned uricase gene was determined and a high homology compared to the reported gene was found . The cloned gene was inserted into Bam H I and Nde I sites of pET21a to create the recombinant plasmid pURO . In Escherichia coli BL21(DE3) host, the expression lever of uricase reached to about 40% of total soluble proteins of the cell . The western blot analysis confirmed the result of expression . Properties of the enzyme protein produced by E . coli BL21(DE3)/pURO were determined and similar with those of original protein from Candida utilis AS2.117 . Furthermore, the thermostability of the expressed protein was enhanced . The purified recombinant uricase was used in serum uric acid analysis. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 59 - 63 {High cell density culture of phosphotransacetylase mutants of Escherichia coli BL21(DE3)}; Zhang WC et al.; Cell culture, organic acid production and foreign protein (TNF) expression of E . coli BL21(DE3) and its pta mutant were investigated . Under shaking conditions, TNF expression in pta mutant increased by 23% . During the fed-batch culture without limitation of specific growth rates, the mutant reached a cell density as high as 32.5 g(DCW)/L and total TNF expression at 2.8 g/L, while the parental strain only obtained 19.5 g(DCW)/L and 0.84 g/L . The results indicate that utility of pta mutant as a host is advantageous in foreign protein expression and high cell density culture . Meanwhile, the analysis data of organic acids accumulated during fed-batch culture showed that as the decrease of acetate production(42% of the parental strain), the accumulation of other organic acids(mainly pyruvate, lactate and succinate) obviously increased . As a result, the amount of total organic acids increased by 123% over its parent . The lactate production may be the main obstacle in further growth of the cells. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 50 - 4 {A three-domain antibody fragment VH/L specific to tumor blood vessels}; Wu XP et al.; AA98 is a specificaally anti-angiogenic antibody generated in our lab . The heavy chain variable region (VH) attached with mutagenized 36 nucleotides sequence derived from the heavy chain constant region1 (CH1) was amplified VH and light chain (L) were inserted into high-level expression vector pET21a successively, thus pET21a-VH/L was constructed . VH/L was expressed in E . coli BL21 (DE3) after induction with IPTG . The expression of VH/L was 20% of the total bacterial proteins . The refolding of VH/L was conducted by dilution and gel filtration chromatography . The refolded VH/L could bind to HUVEC specifically . Its affinity to the antigen is similar to that of recombinant AA98Fab, but lower than that of the parent antibody AA98. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 46 - 9 {Gene chimeric fusion and expression of nucleocapsid NS3 regions and NS4 regions of hepatitis C virus genome}; Shen XC et al.; Genes encoding HCV core and NS4 antigen epitopes and C33c antigen were cloned from HCV genome by PCR, respectively . Two fused genes were constructed . One contained these three genes, another contained genes encoding C33c antigen and NS4 antigen epitopes . These fused genes were cloned into expression plasmid pET-24(a)+ and pET-22(b)+ under T7 promoter and transformed into E . coli BL21 (DE3) respectively . SDS-PAGE analysis revealed that these fused antigens CCN, CN were highly expressed after the induction by 1 mmol/L IPTG . These Expression products were detected by western blotting with anti-HCV serum. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 16 - 9 {Study on the expression of pig zona pellucida-3 beta excluding N-terminus signal peptide and C-terminus transmembrance-like domain in Escherichia coli}; Xu WX et al.; Pig oocytes are surrounded by an acellular translucent envelope-zona pellucida(pZP), which is comprised of three biochemically and immunologically distinct glycoproteins(pZP1, pZP3 alpha and pZP3 beta) . Due to the cross-reactivity of anti-pZP3 beta antibodies with human ZP in vitro, pZP3 beta has been considered as potential one of immunogens for developing human contraceptive vaccine . In order to express pZP3 beta directly in E . coli; we amplified the core fragment of pZP3 beta cDNA by PCR that was deleted 5'- and 3'-terminal sequences of coding for signal peptide and transmembrane-like domain . The DNA sequenced EcoRI and SalI restriced core fragment was cloned in a frame downstream of PRPL promoter in the pBV221 vector . SDS-PAGE analysis showed that pZP3 beta protein was expressed especially in E . coli after thermal induction, which the expression band displayed the molecular weight of about 38 kD matching with its deduced molecular weight(36.5 kD) . In addition, the specially expressed protein band on SDS-PAGE gel showed specific immunological reaction with anti-pig ZP IgGs of rabbit in Western blot. Nippon Ganka Gakkai Zasshi, 2001 Apr, 105(4), 230 - 6 {Effects of topical prostaglandin analogues on the aqueous flare intensity in rabbit eyes at an early phase of endotoxin-induced uveitis}; Kiuchi Y et al.; PURPOSE: We examined the effects of prostaglandin analogues on the blood-aqueous barrier(BAB) permeability in rabbit eyes at an early phase of endotoxin-induced uveitis(EIU) . SUBJECTS AND METHODS: One drop of 0.005% latanoprost or 0.12% unoprostone were applied to rabbit eyes . Escherichia coli lipopolysaccharides were injected to induce uveitis . The changes in flare intensity in normal eyes and EIU eyes after application of eye drops were evaluated . The effect of cyclooxygenase inhibitor on the flare intensity changes caused by the application of unoprostone was also examined . RESULTS: Flare intensity increased significantly after a single instillation of unoprostone, and the increase was not prevented by pretreatment with cyclooxygenase inhibitor . In eyes with EIU, unoprostone caused an additional increase of flare intensity to uveitis induced flare change . Latanoprost had no effects on BAB in eyes with normal and with uveitic conditions . CONCLUSION: Latanoprost and unoprostone did not cause an excessive inflammatory reaction in rabbit eyes at an early phase of EIU. J Radiat Res (Tokyo), 2000 Dec, 41(4), 355 - 66 Redox reactions of sanazole (AK-2123) in aqueous solutions: a pulse radiolysis study; Kapoor S et al.; The redox chemistry of sanazole, an efficient hypoxic cell radiosensitizer, generally referred to as AK-2123, was studied by pulse radiolysis with eaq-, CO2-., 2-propanol radicals and CH2OH radicals . AK-2123 reacts with eaq-, CO2- . and 2-propanol radicals at almost diffusion-controlled rates, producing a nitro radical anion (lambda max = 290 nm) within a few microseconds . The decay kinetics of the radical anion was independent of the pH . The radical anion reacts with oxygen with a rate constant of 3.4 x 10(6) dm3 mol-1 s-1 . An electron-transfer reaction was observed from the thymine radical anion to AK-2123 . From redox equilibria with methyl viologen, the one-electron reduction potential of AK-2123 in aqueous solution, determined by pulse radiolysis, was estimated to be -0.33 +/- 0.02 V vs . NHE . Depletion of intracellular nonprotein thiols did not mitigate the radiosensitizing affect of the hypoxic radiosensitizer, AK-2123. J Radiat Res (Tokyo), 2000 Dec, 41(4), 349 - 54 2-hydroxyadenine in DNA is a very poor substrate of the Escherichia coli MutY protein; Kamiya H et al.; To test the possibility that the Escherichia coli MutY or MutM protein acts as a 2-hydroxyadenine (2-OH-Ade) glycosylase, we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E . coli MutY or MutM protein in vitro . We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand . Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY . The cleavage of oligonucleotides with 2-OH-Ade by MutM was not observed . These results indicate that neither MutY nor MutM plays an important role in the removal of 2-OH-Ade from DNA. Clin Diagn Lab Immunol, 2001 May, 8(3), 652 - 7 Oral administration of influenza vaccine in combination with the adjuvants LT-K63 and LT-R72 induces potent immune responses comparable to or stronger than traditional intramuscular immunization; Barackman JD et al.; Mucosal immunization strategies are actively being pursued in the hopes of improving the efficacy of vaccines against the influenza virus . Our group investigated the oral immunization of mice via intragastric gavage with influenza hemagglutinin (HA) combined with mutant Escherichia coli heat-labile enterotoxins K63 (LT-K63) and R72 (LT-R72) . These oral immunizations resulted in potent serum antibody and HA inhibition titers, in some cases stronger than those obtained with traditional intramuscular administration, in addition to HA-specific immunoglobulin A in the saliva and nasal secretions . This study demonstrates that it may be possible to develop effective oral influenza vaccines. Clin Diagn Lab Immunol, 2001 May, 8(3), 637 - 40 Prevalence of known P-fimbrial G alleles in Escherichia coli and identification of a new adhesin class; Manning SD et al.; Screening a large Escherichia coli collection for P-fimbrial adhesin classes identified 20 unclassifiable strains . Cloning and sequencing of papG from an unclassifiable strain identified another G allele . The novel adhesin gene has 65% identity to the class I adhesin gene, 44% identity to the class II adhesin gene, and 43% identity to the class III adhesin gene. Biochemistry, 2001 Feb 20, 40(7), 2282 - 90 Effects of benzo{a}pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation; Alekseyev YO et al.; Benzo{a}pyrene diol epoxide (B{a}PDE) adducts are strong blocks of DNA replication in vitro, allowing the rare incorporation of a nucleotide across from the lesion and negligibly small extent of further bypass . To study the mechanistic details of this process, a gel-retardation assay was used to measure the dissociation constants for the binding of DNA polymerase I (Klenow fragment) (KF) to the primer-templates containing a (+)-trans- or (+)-cis-B{a}P-N(2)-dG adduct . When the primer was terminated one nucleotide before the adduct, the presence of a (+)-trans-B{a}P-N(2)-dG adduct did not affect the binding while a (+)-cis-B{a}P-N(2)-dG adduct caused a slight decrease in affinity . The presence of any dNTP decreased the affinity of KF to the modified primer-templates . (In contrast, a strong increase of the affinity to unmodified primer-templates was observed in the presence of the next correct dNTP.) Limited protease digestion experiments indicated that a closed ternary complex of KF with the modified primer-templates was not detectable in the presence of any dNTP, whereas it was clearly observed with unmodified template in the presence of the next correct nucleotide . These findings suggest that these adducts may interfere with the conformational change to the catalytically active closed ternary complex and/or cause significant destabilization of this complex . When the primers extended to the position across from the adduct, the affinity of KF was significantly decreased irrespective of the identity of the base across from the adduct, possibly explaining the low bypass of the lesion . Interestingly, the stability of these DNA-polymerase complexes correlated with nucleotide insertion kinetics for the unmodified and (+)-trans-B{a}PDE-modified templates. Biochemistry, 2001 Feb 20, 40(7), 2276 - 81 Escherichia coli transcription termination factor Rho binds and hydrolyzes ATP using a single class of three sites; Stitt BL; Escherichia coli transcription termination factor Rho uses the energy of ATP hydrolysis to travel 5' --> 3' along RNA . We previously showed that the hexameric Rho protein binds three molecules of ATP in active sites and that hydrolysis of the three bound ATP molecules upon RNA binding is sequential . Other models of Rho ATP hydrolysis activity have arisen from reports of additional ATP binding sites on Rho . Here we present further evidence from binding, isotope partitioning, and rapid mix/chemical quench experiments, in support of the presence of only three equivalent ATP binding sites on Rho that are catalytic sites and that fire sequentially . These results are incorporated into a proposed mechanism for directional Rho tracking along RNA. Biochemistry, 2001 Feb 20, 40(7), 2148 - 54 Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer's beta(25-35) amyloid fibril; Konno T; Amyloid-induced aggregation and precipitation of soluble proteins were investigated in vitro using the amyloid fibrils of the beta(25--35) peptide, a cytotoxic fragment of the Alzheimer's beta-peptide at positions 25--35 . The aggregation rate of firefly luciferase was found to be modulated by both a chaperone molecule DnaK and the beta(25--35) amyloid, but their effects were opposite in direction . The amyloid fibril drastically facilitated the luciferase aggregation, which may define a kind of anti-chaperone activity . The effect of the beta(25--35) amyloid to promote protein aggregation and precipitation was further demonstrated for a wide variety of target proteins . The amount of coprecipitation was well correlated with the predicted isoelectric point of the target proteins, indicating that the interaction between the beta(25--35) amyloid and the target was driven by an electrostatic force between them . This view was confirmed by the experiments using an electrically neutral mutant peptide, beta(25--35)KA . It was also found that clustering of the beta(25--35) peptide to form amyloid and the conformation of the target protein are additional factors that determine the strength of the amyloid-protein interaction . Spectroscopic and electron microscopic methods have revealed that the proteins coprecipitated with the beta(25--35) amyloid formed amorphous aggregates deposited together with the amyloid fibrils . The conformation of protein molecules left in the residual soluble fraction was also damaged in the amyloid-containing solution . As a summary, this study has proposed a scheme for events related to the nonspecific amyloid-protein interaction, which may play substantial roles in in vivo conditions. Biochemistry, 2001 Feb 20, 40(7), 2104 - 12 Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function; Hitchcock-DeGregori SE et al.; Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform . Periodic repeats in the sequence have been proposed to correspond to actin binding sites . To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5) . Recombinant Tms (unacetylated) were expressed in Escherichia coli . Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn) . dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition . Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding . dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1 . In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding . The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding . The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA . In the presence of Ca(2+), relief of inhibition by these Tms was incomplete . We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different. Biochemistry, 2001 Feb 20, 40(7), 1996 - 2003 Functional estimation of loop-helix boundaries in the lactose permease of Escherichia coli by single amino acid deletion analysis; Wolin CD et al.; Mutants with single amino acid deletions in the loops of lactose permease retain activity, while mutants with single deletions in transmembrane helices are inactive, and the loop--helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic deletion of single residues {Wolin, C . D., and Kaback, H . R . (1999) Biochemistry 38, 8590-8597} . The experimental approach is applied here to the remainder of the permease . Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions . In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted . Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues . Surprisingly, deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual deletion of only three residues in this region abolishes activity . Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts. Biochemistry, 2001 Feb 20, 40(7), 1984 - 95 The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: kinetic and structural effects of mutations of Phe-50; Czerwinski RM et al.; The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide . Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) {Czerwinski, R . M., Harris, T . K., Johnson, Jr., W . H., Legler, P . M., Stivers, J . T., Mildvan, A . S., and Whitman, C . P . (1999) Biochemistry 38, 12358-12366} . Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site . Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 A from Pro-1 and is one of 12 apolar residues within 9 A of Pro-1 . Replacing Phe-50 with Tyr does not significantly alter k(cat) or K(m) and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by (15)N NMR spectroscopy, comparable to that observed for wild type . (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme . In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by UV and (1)H NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-1 in this mutant . In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by (15)N NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K(m) rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6 . A loss of structure of the beta-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT . (1)H-(15)N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone (15)N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degrees C and their disappearance at 43 degrees C due to rapid exchange with solvent . These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1 . In addition, the F50A mutation decreased k(cat) 167-fold and increased K(m) 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1 . The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K(m) by 3.3- and 3.8-fold, respectively. Biochemistry, 2001 Feb 20, 40(7), 1956 - 63 Determination of the redox properties of human NADPH-cytochrome P450 reductase; Munro AW et al.; Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains . Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) {oxidized/semiquinone} = -286 +/- 6 mV, E(2) {semiquinone/reduced} = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) {FMN} = -66 +/- 8 mV, E(2) {FMN} = -269 +/- 10 mV; E(1) {FAD} = -283 +/- 5 mV, E(2) {FAD} = -382 +/- 8 mV) . The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains . Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples . Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain . In both cases, large amounts of the higher potential FMN are in the semiquinone form . The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase . However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes. Biochemistry, 2001 Feb 20, 40(7), 1913 - 21 Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites; Olsen LR et al.; N-Acetylglucosamine-1-PO(4) uridyltransferase (GlmU) is a trimeric bifunctional enzyme that catalyzes the last two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc . The X-ray crystal structure of Escherichia coli GlmU in complex with UDP-GlcNAc and CoA has been determined to 2.1 A resolution and reveals a two-domain architecture that is responsible for these two reactions . The C-terminal domain is responsible for the CoA-dependent acetylation of Glc-1-PO(4) to GlcNAc-1-PO(4) and displays the longest left-handed parallel beta-helix observed to date . The acetyltransferase active site defined by the binding site for CoA makes use of residues from all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO(4) acetyl acceptor . The N-terminal domain catalyzes uridyl transfer from UTP to GlcNAc-1-PO(4) to form the final products UDP-GlcNAc and pyrophosphate . This domain is composed of a central seven-stranded beta-sheet surrounded by six alpha-helices in a Rossmann fold-like topology . A Co(2+) ion binds to just one of the two independent pyrophosphorylase active sites present in the crystals studied here, each of which nonetheless binds UDP-GlcNAc . The conformational changes of the enzyme and sugar nucleotide that accompany metal binding may provide a window into the structural dynamics that accompany catalysis. Biochemistry, 2001 Feb 20, 40(7), 1897 - 902 Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug; Phan J et al.; Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries . The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution . In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits . This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme . The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A . The thiophene ring of the drug is disordered between two parallel positions. J Biol Chem, 2001 Jul 20, 276(29), 26852 - 9 Epub 2001 Apr 27. Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli; Assarsson M et al.; The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component . The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein . The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state . We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present . Paramagnetic intermediates, i.e . a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions . A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined . Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204 . A general scheme for the reconstitution reaction is presented based on EPR and structure results . This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site. DNA Seq, 2000, 11(5), 419 - 31 MglA and mglB of Treponema denticola; similarity to ABC transport and spa genes; Lepine G et al.; The mglA and mglB genes (td-mglA and td-mglB) of the oral spirochete Treponema denticola were sequenced . These two T . denticola genes are highly homologous to the E . coli and Treponema pallidum mglA and mglB genes which are part of the three gene beta-methylgalactoside transport operon, mglBAC . Both Td-mglA and td-mglB are also homologous to the high affinity ABC-type transporters for ribose and arabinose, and surface presentation antigens (spa) locus, part of the type III secretion systems in enteropathogens . Td-mglB and td-mglA are co-transcribed as a single mRNA in T . denticola as well as in E . coli cells as determined by reverse transcription PCR (RT-PCR) . Homology to td-mglB and its expressed protein was found in other oral spirochetes as determined by Southern and western blot analysis. DNA Seq, 2000, 11(5), 395 - 404 Identification and characterization of the gene encoding the mitochondrial elongation factor G in rice; Kato A et al.; A plant nuclear gene coding for a mitochondrial elongation factor G (mEF-G) was cloned from a cDNA library and genomic library of rice (Oryza sativa L.) . This DNA sequence predicts a 757-amino-acid protein exhibiting 79%, 55% and 49% homology to Arabidopsis thaliana, Saccharomyces cerevisiae and rat mEF-G respectively, 53% homology to the elongation factor G in Escherichia coli and 43% homology to soybean chloroplast elongation factor G . The deduced amino acid sequence contains the characteristic motifs shared by all GTP binding proteins . Comparison of the sequence of the genomic clone to that of the cDNA clone revealed that this gene is split nineteen times by introns, although the gene of Arabidopsis is split seventeen times by introns . Some of the introns found in the rice genome are relatively long and they result in a long gene with a size of approximately 15 kb. J Biochem (Tokyo), 2001 May, 129(5), 761 - 8 The amino acid residues affecting the activity and azole susceptibility of rat CYP51 (sterol 14-demethylase P450); Nitahara Y et al.; The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation . Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli . Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues . Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51 . H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species . A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole . Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51 . Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51. J Biochem (Tokyo), 2001 May, 129(5), 709 - 16 Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression; Yubisui T et al.; A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5) . The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs . The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5) . A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues . The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration . The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase . The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5) . Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5 . Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low . This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian. Br J Haematol, 2001 Apr, 113(1), 115 - 9 Drug monitoring of low-dose PEG-asparaginase (Oncaspar) in children with relapsed acute lymphoblastic leukaemia; Vieira Pinheiro JP et al.; Use of asparaginase (ASNase) in the treatment of relapsed childhood acute lymphoblastic leukaemia (ALL) is associated with a high rate of hypersensitive reactions . 'Silent' inactivation may additionally reduce treatment intensity . Therefore, PEG-ASNase (Oncaspar), a polyethylene glycol conjugate of the native Escherichia coli-ASNase, was introduced into the Berlin-Frankfurt-Munster (BFM) 96 treatment protocol for relapsed ALL under drug monitoring conditions . A single i.v . dose of 500 IU/m2 PEG-ASNase, substituted for the native ASNases, was administered to supply a plasma activity of 100 IU/l for 1 week . From November 1997 to March 2000, 35 patients from 23 BFM-associated hospitals, with or without a previous allergic reaction to one or both native preparations, underwent monitoring . After 82 applications, a total of 270 samples were submitted to be tested for ASNase activity . The ASNase activity on the day of the administration and the following day ranged between < 20 and 693 IU/l, with a median of 413 IU/l (53 samples) . The median on d 7 +/- 1 was 199 IU/l (range <20--421 IU/l; 41 samples) and on d 14 +/- 1, 105 IU/l (range <20--188 IU/l; 19 samples) . An ASNase activity of > 100 IU/l was seen on d 7 in 36 activity time courses of 52 interpretable applications (69%) . Intraindividual variability of activity time courses was low . However, a rapid decrease in ASNase activity after repeated applications was observed in 4 out of 20 children . Previously experienced allergic reactions to native ASNases did not influence PEG-ASNase pharmacokinetics . PEG-ASNase is a useful alternative to the native ASNases in children with relapsed ALL . Whenever possible, drug monitoring should be performed to identify patients with 'silent' inactivation. Biochemistry, 2001 Feb 27, 40(8), 2588 - 98 HU binding to DNA: evidence for multiple complex formation and DNA bending; Wojtuszewski K et al.; HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA . The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes . The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation . These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex . The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length . Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity . The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe . These results are suggestive of a local bending or unwinding of the DNA . On the basis of these results we propose a model in which bending of DNA accompanies HU binding . Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes . We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities. Biochemistry, 2001 Feb 27, 40(8), 2502 - 10 Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer; Mizuno H et al.; The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated . The recombinant DsRed expressed in E . coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm . Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm . Incubation of E . coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak . In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R) . Light-scattering analysis revealed that DsRed proteins expressed in E . coli and HeLa cells form a stable tetramer complex . DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm . The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm) . When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin . Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved . Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy . Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant. Biochemistry, 2001 Feb 13, 40(6), 1844 - 9 N-terminal intramolecularly conserved histidines of three domains in Gonyaulax luciferase are responsible for loss of activity in the alkaline region; Li L et al.; Gonyaulax luciferase is a single-chain ( approximately 137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each) . Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments ( approximately 35 kDa) of luciferase are active . The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8 . While the activity loss at higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual domains . The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves similar to that of the full-length enzyme . For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-activity profiles mimicked that of the proteolytic fragments reported earlier . Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at high pH . This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence. Biochemistry, 2001 Feb 13, 40(6), 1835 - 43 Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein; Ding H et al.; SecA ATPase is an essential component of the Sec-dependent protein translocation machinery . Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane . To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions . Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure . Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins . Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands . Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide . These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation. Biochemistry, 2001 Feb 13, 40(6), 1804 - 11 Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase; Johnson EA et al.; The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM) . The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1 . CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F) . CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322 . Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20% . RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45% . The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57 . These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer . This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1 . These new RET measurements also allow the alignment of the predicted epsilon subunit structure . The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1. Biochemistry, 2001 Feb 13, 40(6), 1796 - 803 Evidence in Escherichia coli that N3-methyladenine lesions induced by a minor groove binding methyl sulfonate ester can be processed by both base and nucleotide excision repair; Shah D et al.; It has been previously reported that a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (MeOSO(2)-lex) selectively affords N3-methyladenine lesions . To study the interaction of the neutral lex dipeptide with calf thymus DNA, we have prepared stable, nonmethylating sulfone analogues of MeOSO(2)-lex that are neutral and cationic . Thermodynamic studies show that both the neutral and monocationic sulfone compounds bind to DNA with K(b)'s of 10(5) in primarily entropy-driven reactions . To determine how the cytotoxic N3-methyladenine adduct generated from MeOSO(2)-lex is repaired in E . coli, MeOSO(2)-lex was tested for toxicity in wild-type E . coli and in mutant strains defective in base excision repair (tag and/or alkA glycosylases or apn endonuclease), nucleotide excision repair (uvrA), and both base and nucleotide excision repair (tag/alkA/uvrA) . The results clearly demonstrate the cellular toxicity of the N3-methyladenine lesion, and the protective role of base excision glycosylase proteins . A novel finding is that in the absence of functional base excision glycosylases, nucleotide excision repair can also protect cells from this cytotoxic minor groove lesion . Interaction between base and nucleotide excision repair systems is also seen in the protection of cells treated with cis-diamminedichloroplatinum(II) but not with anti-(+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene. Biochemistry, 2001 Feb 13, 40(6), 1764 - 73 Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p; Thual C et al.; The {URE3} factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2 . The molecular basis of the propagation of this phenotype is unknown . We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded . Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils {Thual et al . (1999) J . Biol . Chem . 274, 13666-13674} . To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions . We show here that Ure2p dimerizes through its C-terminal region . We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils . A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain . Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein . This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils . Our results contradict the intramole