J Clin Microbiol, 2004 Sep, 42(9), 4092 - 100 Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli; Reischl U et al.; To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness . Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format . A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib . LC-PCR findings from the testing of 161 E . coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis . The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes . The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation . Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay . Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib . The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC. J Cell Biol, 2004 Sep 13, 166(6), 769 - 74 YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins; Dalbey RE et al.; Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins . Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein-conducting channel . YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway. J Biol Chem, 2004 Nov 19, 279(47), 49259 - 67 Epub 2004 Sep 13. Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2; Zhao M et al.; Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis . Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation . However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis . In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase . The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation . ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation . The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI . These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation. J Biol Chem, 2004 Nov 19, 279(47), 49214 - 21 Epub 2004 Sep 13. Sugar recognition by the lactose permease of Escherichia coli; Vazquez-Ibar JL et al.; Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity . The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site . We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269 . Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates . Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N . It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit. J Biol Chem, 2004 Nov 19, 279(47), 48976 - 82 Epub 2004 Sep 10. Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites; Fukatsu K et al.; Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) plays an important role in neuronal functions; however, the lateral diffusion of IP3R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown . We expressed green fluorescent protein-tagged IP3R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique . IP3R1 showed lateral diffusion with an effective diffusion constant of approximately 0.3 microm2/s . Depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments . We also found that protein 4.1N, which binds to IP3R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP3R1 diffusion constant . Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP3R1 diffusion constant . The diffusion of IP3R type 3 (IP3R3), one of the isoforms of IP3Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence . These data suggest that 4.1N serves as a linker protein between IP3R1 and actin filaments . This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling. J Biol Chem, 2004 Nov 19, 279(47), 48876 - 82 Epub 2004 Sep 10. 4-cyanopyridine, a versatile spectroscopic probe for cytochrome P450 BM3; Ost TW et al.; The nitrogenous pi -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (Kd=1.8 microm for wild type P450 BM3) via its pyridine ring nitrogen . The reduced P450-4CNPy adduct displays unusual spectral properties that provide a useful spectroscopic handle to probe particular aspects of this P450 . 4CNPy is competitively displaced upon substrate binding, allowing a convenient route to the determination of substrate dissociation constants for ferrous P450 highlighting an increase in P450 substrate affinity on heme reduction . For wild type P450 BM3, Kd(red)(laurate)=82.4 microm (cf . Kd(ox)=364 microm) . In addition, an unusual spectral feature in the red region of the absorption spectrum of the reduced P450-4CNPy adduct is observed that can be assigned as a metal-to-ligand charge transfer (MLCT) . It was discovered that the energy of this MLCT varies linearly with respect to the P450 heme reduction potential . By studying the energy of this MLCT for a series of BM3 active site mutants with differing reduction potential (Em), the relationship EMLCT + (3.53 x = Em 17,005 cm)(-1) was derived . The use of this ligand thus provides a quick and accurate method for predicting the heme reduction potentials of a series of P450 BM3 mutations using visible spectroscopy, without the requirement for redox potentiometry. J Biol Chem, 2004 Nov 5, 279(45), 46787 - 93 Epub 2004 Sep 09. Solution structure of a ubiquitin-like domain from tubulin-binding cofactor B; Lytle BL et al.; Proper folding and assembly of tubulin alphabeta-heterodimers involves a stepwise progression mediated by a group of protein cofactors A through E . Upon release of the tubulin monomers from the chaperonin CCT, they are acted upon by each cofactor in the folding pathway through a unique combination of protein interaction domains . Three-dimensional structures have previously been reported for cofactor A and the C-terminal CAP-Gly domain of cofactor B (CoB) . Here we report the NMR structure of the N-terminal domain of Caenorhabditis elegans CoB and show that it closely resembles ubiquitin as was recently postulated on the basis of bioinformatic analysis (Grynberg, M., Jaroszewski, L., and Godzik, A . (2003) BMC Bioinformatics 4, 46) . CoB binds partially folded alpha-tubulin monomers, and a putative tubulin-binding motif within the N-terminal domain is identified from sequence and structure comparisons . Based on modeling of the homologous cofactor E ubiquitin-like domain, we hypothesize that cofactors B and E may associate via their beta-grasp domains in a manner analogous to the PB1 and caspase-activated deoxyribonuclease superfamily of protein interaction domains. Chest, 2004 Sep, 126(3), 860 - 7 Experimental human endotoxemia is associated with depression of load-independent contractility indices: prevention by the lipid a analogue E5531; Kumar A et al.; OBJECTIVE: To evaluate the efficacy of a novel lipopolysaccharide (LPS) antagonist, E5531, in blocking LPS-induced cardiac responses including myocardial depression (as assessed by relatively load-independent echocardiographic indices of contractility) in a human model of experimental endotoxemia . DESIGN: Randomized, prospective, placebo-controlled, double-blind trial . SETTING: ICU procedure room . PARTICIPANTS: Thirty-two healthy, male volunteers . INTERVENTIONS: Administration of LPS (4 ng/kg) and either a placebo or one of four sequential doses of E5531 (100 microg, 250 microg, 500 microg, or 1,000 microg) followed by volumetric echocardiography before and during 4-L saline solution infusion (3 L over 3 h, followed by 1 L over 2 h) . RESULTS: In addition to the generation of a hyperdynamic circulation throughout the study period, administration of LPS resulted in a biphasic contractility response . Ejection fraction (EF), rate-corrected mean velocity of circumferential fiber shortening (Vcfc), peak systolic BP (SBP)/end-systolic volume index (ESVI) ratio, and end-systolic pressure (Pes)/ESVI ratio increased at the 3-h post-LPS assessment, compared to a control group of subjects receiving only similar amounts of saline solution (minimum p < 0.001) . End-systolic myocardial wall stress (sigmaes)/ESVI ratio, one of the most load independent of the contractility indices, was unchanged . At 5 h after endotoxin, EF, Vcfc, SBP/ESVI, Pes/ESVI, and sigmaes/ESVI were all decreased (minimum p < 0.01), indicating myocardial depression . When present, early (3 h after LPS), apparent enhancement of myocardial contractility and later (5 h after LPS) myocardial depression were substantially blunted by administration of E5531 (minimum p < 0.025), typically in a concentration-dependent manner . CONCLUSIONS: Endotoxin generates significant myocardial depression when measured using highly load-independent indices of cardiac contractility . E5531 is a potent inhibitor of the early hyperdynamic cardiovascular and later myocardial depression response seen in experimental human endotoxemia. J Mol Biol, 2004 Oct 1, 342(5), 1547 - 58 Inter-domain motions of the N-domain of the KdpFABC complex, a P-type ATPase, are not driven by ATP-induced conformational changes; Haupt M et al.; P-type ATPases are involved in the active transport of ions across biological membranes . The KdpFABC complex (P-type ATPase) of Escherichia coli is a high-affinity K+ uptake system that operates only when the cell experiences osmotic stress or K+ limitation . Here, we present the solution structure of the nucleotide binding domain of KdpB (backbone RMSD 0.17 A) and a model of the AMP-PNP binding mode based on intermolecular distance restraints . The calculated AMP-PNP binding mode shows the purine ring of the nucleotide to be "clipped" into the binding pocket via a pi-pi-interaction to F377 on one side and a cation-pi-interaction to K395 on the other . This binding mechanism seems to be conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB) . Thus, we conclude that the Kdp-ATPase (currently type IA) is misgrouped and has more similarities to type III ATPases . The KdpB N-domain is the smallest and simplest known for a P-type ATPase, and represents a minimal example of this functional unit . No evidence of significant conformational changes was observed within the N-domain upon nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the reaction cycle. J Mol Biol, 2004 Oct 1, 342(5), 1471 - 85 Crystal structures of Escherichia coli RecA in a compressed helical filament; Xing X et al.; The X-ray crystal structure of uncomplexed Escherichia coli RecA protein has been determined in three new crystal forms at resolutions of 1.9 A, 2.0 A, and 2.6 A . The RecA protein used for this study contains the extra residues Gly-Ser-His-Met at the N terminus, but retains normal ssDNA-dependent ATPase and coprotease activities . In all three crystals, RecA is packed in a right-handed helical filament with a pitch of approximately 74 A . These RecA filaments are compressed relative to the original crystal structure of RecA, which has a helical pitch of 82.7 A . In the structures of the compressed RecA filament, the monomer-monomer interface and the core domain are essentially the same as in the RecA structure with the 83 A pitch . The change in helical pitch is accommodated by a small movement of the N-terminal domain, which is reoriented to preserve the contacts it makes at the monomer-monomer interface . The new crystal structures show significant variation in the orientation and conformation of the C-terminal domain, as well as in the inter-filament packing interactions . In crystal form 2, a calcium ion is bound closely to a beta-hairpin of the C-terminal domain and to Asp38 of a neighboring filament, and residues 329-331 of the C-terminal tail become ordered to contact a neighboring filament . In crystal forms 3 and 4, a sulfate ion or a phosphate anion is bound to the same site on RecA as the beta-phosphate group of ADP, causing an opening of the P-loop . Altogether, the structures show the conformational variability of RecA protein in the crystalline state, providing insight into many aspects of RecA function. J Mol Biol, 2004 Oct 1, 342(5), 1379 - 90 Statistical analysis of the spatial distribution of operons in the transcriptional regulation network of Escherichia coli; Warren PB et al.; We have performed a statistical analysis of the spatial distribution of operons along the DNA in the transcriptional regulation network of Escherichia coli . The analysis reveals that pairs of operons that regulate each other and those that are co-regulated tend to lie much closer to one another than would be expected for a random network . Moreover, these pairs of operons tend to be transcribed in diverging directions . This spatial arrangement of operons allows the upstream regulatory domains to overlap and interfere with each other and our analysis also demonstrates the statistical significance of this motif of overlapping operons . Overlapping operons afford additional regulatory control, such as the correlated or anticorrelated expression of operons . We show by a mean-field analysis of a feed-forward loop that overlapping operons can drastically enhance the performance of gene regulatory networks . Our results suggest that regulatory control can provide a selective pressure that drives operons together in the course of evolution. FEMS Immunol Med Microbiol, 2004 Oct 1, 42(2), 167 - 72 Lethality in LPS-induced endotoxemia in C3H/HeCr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin; Zimecki M et al.; C3H/HeCr mice are more susceptible to infection compared with other strains . Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia . In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice . We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v . LPS injection than the control, CBA strain . 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level . IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice . That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs . 60% in control, PBS treated mice . In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant . These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain . We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response. Eur J Pharmacol, 2004 Sep 13, 498(1-3), 211 - 7 Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats; Buyukafsar K et al.; Effects of bacterial lipopolysaccharide (Escherichia coli serotype, 055:B5, 20 mg kg(-1), i.p., for 6 h) and a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, Y-27632 (10(-9)-10(-5) M) were investigated on the contractile responses of the rat mesenteric artery to phenylephrine (10(-9)-3 x 10(-5) M), angiotensin-2 (10(-10)-10(-6) M) and endothelin-1 (10(-10)-10(-7) M) . Moreover, alteration in the level of Rho-kinase (ROCK-2) expression was examined in the superior mesenteric artery obtained from saline- and lipopolysaccharide-treated rats by Western blotting . Endotoxemic rat mesenteric rings exhibited no different contractions to phenylephrine and angiotensin-2 but augmented contractile activity to endothelin-1 . In the mesenteric artery obtained from the endotoxemic rats, acetylcholine-induced vasorelaxation did not differ; pD2 value for acetylcholine was 7.85+/-0.12 in the endotoxemic rings; however, it was 7.81+/-0.15 in the control rings (P>0.05) . Y-27632 induced relaxation, which was the same in the control arteries as in endotoxemic ones when contracting agent was phenylephrine . However, when endothelin-1 was used to precontract the rings, Y-27632 produced enhanced relaxation in endotoxemic vessels . pD2 values for Y-27632 were, respectively, 7.69+/-0.12 and 8.20+/-0.10 in control and endotoxemic rings precontracted by endothelin-1 (10(-8) M) (P<0.01) . Moreover, Y-27632 (10(-5) M) suppressed the contraction induced by angiotensin-2 (10(-10)-10(-6) M) . Western blot analysis revealed that Rho-kinase was upregulated significantly in the mesenteric artery obtained from the rats treated with LPS for 6 h . In addition, serum NO2-/NO3- level, which was detected by Griess method, was 10.0+/-1.4 microM in endotoxemic rats; however, it was 6.6+/-0.5 microM in control (P<0.05) . Taken together, these results show that the expression of the contractile protein Rho-kinase could be upregulated in endotoxemic mesenteric artery and this upregulation may be coincided with an enhanced contraction to endothelin-1 but not phenylephrine and angiotensin-2. Exp Parasitol, 2004 Jul-Aug, 107(3-4), 163 - 72 Echinococcus multilocularis: identification and molecular characterization of a Ral-like small GTP-binding protein; Spiliotis M et al.; In mammals, Ral (Ras-like) GTPases have been implicated in the regulation of several cellular key processes such as oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics . Here we provide, for the first time, molecular data on a Ral homologue from a parasitic helminth . We have cloned and characterized the complete cDNA molecule and the chromosomal locus encoding a novel GTP binding protein, EmRal, of the human parasite Echinococcus multilocularis . The encoded protein contained all highly conserved amino acid residues of the protein family at corresponding positions and shared significant sequence homologies with human RalA (53% identity) and RalB (54%) . Upon heterologous expression of EmRal in Escherichia coli, the recombinant protein was able to bind GTP, thus indicating functionality of the Echinococcus factor . Using an in vitro prenylation assay, the purified protein was shown to be geranylgernylated, but not farnesylated, in both rabbit reticulocyte and Echinococcus cell extracts . The EmRal mRNA was found to be processed via trans-splicing and, using RT-PCR and virtual Northern blot experiments, expression of the factor could be demonstrated for the larval stages metacestode and protoscolex during an infection of the intermediate host . The data presented herein provide a solid basis for further investigations on Ras-Ral signaling mechanisms in Echinococcus. Anal Chem, 2004 Sep 15, 76(18), 5423 - 30 Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins; Stapels MD et al.; Proteins from Escherichia coli were isolated based on their ability to bind DNA and digested in-solution with trypsin; the resulting peptides were separated using HPLC and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments . Various properties of the peptides observed with the two ionization techniques were compared taking into account the differences between the mass analyzers . This empirical analysis of a data set containing hundreds of peptides and thousands of individual amino acids supports some of the currently held notions regarding the complementary nature of the two ionization processes . Specifically, ESI tends to favor the identification of hydrophobic peptides whereas MALDI tends to lead to the identification of basic and aromatic species . Findings from the present study suggest that ESI and MALDI may be complementary due to the biases of the two ionization techniques for certain classes of amino acids . From a practical standpoint, these biases indicate that, for the present at least, analyses must be performed on both types of instruments in order to gain the most information possible out of a given set of samples in a proteomics study. Biochemistry, 2004 Sep 21, 43(37), 11862 - 72 Isomerase-independent chaperone function of cyclophilin ensures aggregation prevention of adenosine kinase both in vitro and under in vivo conditions; Chakraborty A et al.; Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers {Chakraborty et al . (2002) J . Biol . Chem . 277, 47451-47460} . Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state . To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed . Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme . In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme . Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state . Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions. Biochemistry, 2004 Sep 21, 43(37), 11836 - 41 Discrimination of cognate and noncognate substrates at the active site of class II lysyl-tRNA synthetase; Ataide SF et al.; Within the two unrelated aminoacyl-tRNA synthetase classes, lysyl-tRNA synthetase (LysRS) is the only example known to exist in both classes . To probe the role of the amino acids responsible for L-lysine binding in the active site of the class II LysRS (LysRS2), we studied the lysS-encoded Escherichia coli protein . On the basis of the structure of L-lysine complexed with E . coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination were systematically replaced . Steady-state kinetic parameters for these variants showed reductions in the catalytic efficiency (k(cat)/K(M)) of 1-3 orders of magnitude, allowing the assignment of specific roles for key residues in the active site of LysRS2 . To further investigate the role of each residue in discrimination against noncognate amino acids, steady-state kinetic parameters were determined for the nonprotein amino acid S-(2-aminoethyl)-L-cysteine, a potent inhibitor of LysRS2 . While a number of variants showed reductions of several hundred-fold in efficiency of S-(2-aminoethyl)-L-cysteine utilization, this was uniformly accompanied by similar reductions in the efficiency of lysine utilization . Thus, manipulation of the amino acid binding site only allowed up to a 4-fold improvement in S-(2-aminoethyl)-L-cysteine discrimination . This is in contrast to the highly effective discrimination against S-(2-aminoethyl)-L-cysteine by class I LysRS and correlates with the fundamentally different roles of conserved aromatic residues in the two LysRS active sites . This now provides a mechanistic basis for the proposal that differences in amino acid discrimination have been pivotal in the evolution of two unrelated LysRSs. Biochemistry, 2004 Sep 21, 43(37), 11770 - 81 Escherichia coli lipoyl synthase binds two distinct {4Fe-4S} clusters per polypeptide; Cicchillo RM et al.; Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)) . In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor . Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported {Cicchillo, R . M., Iwig, D . F., Jones, A . D., Nesbitt, N . M., Baleanu-Gogonea, C., Souder, M . G., Tu, L., and Booker, S . J . (2004) Biochemistry 43, 6378-6386} . However, the exact identity of the sulfur donor remains unknown . We report herein that LS from Escherichia coli can accommodate two {4Fe-4S} clusters per polypeptide and that this form of the enzyme is relevant to turnover . One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases . When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide . The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide . Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group . Mossbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration {4Fe-4S}(2+) . When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group . Mossbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of {4Fe-4S}(2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 {4Fe-4S}(2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species . Although the Mossbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs. Biochemistry, 2004 Sep 21, 43(37), 11630 - 6 Association of spin-labeled lipids with beta-barrel proteins from the outer membrane of Escherichia coli; Ramakrishnan M et al.; The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins . The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol . The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids . The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio . It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein . Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins . The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA. J Mol Recognit, 2004 Sep-Oct, 17(5), 488 - 96 Large changes in cytoplasmic biopolymer concentration with osmolality indicate that macromolecular crowding may regulate protein-DNA interactions and growth rate in osmotically stressed Escherichia coli K-12; Cayley S et al.; From determination of amounts and concentrations of biopolymers and solutes in the cytoplasm of Escherichia coli, we are obtaining information needed to assess the effect of macromolecular crowding on cytoplasmic properties and processes of osmotically stressed bacteria . We observe that growth rate, and the amount of cytoplasmic water decrease and cytoplasmic concentrations of biopolymers and K+, increase with increasing osmolality, even for cells grown in the presence of osmoprotectants like glycine betaine . We observe general correlations between the amount of cytoplasmic water, growth rate and cytoplasmic K+ concentration in osmotically stressed cells grown both with and without osmoprotectants . To explain these correlations, we propose that crowding increases with increasing growth osmolality, which in turn buffers the binding of proteins to nucleic acids against changes in cytoplasmic K+ concentration and (by affecting biopolymer diffusion rates and/or assembly equilibria) is a determinant of growth rate of osmotically stressed cells . Changes in biopolymer concentration and crowding may also explain the increase of the activity coefficient of cytoplasmic water with increasing osmolality of growth in E . coli. J Mol Recognit, 2004 Sep-Oct, 17(5), 448 - 55 Applications of time-resolved resonance energy transfer measurements in studies of the molecular crowding effect; Ittah V et al.; The native structures of many globular proteins are only weakly stabilized and form in solution ensembles of multiple conformers . The energy differences between the conformers are assumed to be small . This is the case of flexible multidomain proteins where domain motions were observed . High concentrations of inert macrosolute, which create a crowded or confined environment, can cause shifts of the distribution of the conformers of such proteins towards the more compact structures . This effect may also promote compact structures in partially folded proteins . Time-resolved dynamic non-radiative excitation energy transfer (tr-RET) is suitable for detection of either subtle or major changes in distributions of intramolecular distances in protein molecules in solutions . Two experiments were performed which demonstrated the applicability of tr-RET for detection of the effect of macrosolutes on the conformational ensembles of flexible states of protein molecules . The distribution of distances between residues 203 and 169 in the CORE domain of E . coli adenylate kinase (AK) in the denatured state was determined in the presence of high concentrations of dextran 40 . A significant shift of the mean of the distribution was observed without reduction of its width . This was interpreted as a shift to compact structure without change of the degree of disorder of the chain . In a second experiment the distribution of the distance between residues 55 and 169 in AK, which spans the cleft between the CORE and the AMPbind domains, was monitored . No clear effect of high concentrations of dextran 40 was found . These experiments show the strength of the application of tr-RET in investigation of changes in the sub-states of flexible conformations of globular protein . Networks of pairs of labeled sites can be prepared and tr-RET experiments can be performed in order to search for the segments of the protein molecules, which respond to the presence of inert macromolecules in their environment. Gastroenterology, 2004 Sep, 127(3), 859 - 69 Differing roles of protein kinase C-zeta in disruption of tight junction barrier by enteropathogenic and enterohemorrhagic Escherichia coli; Tomson FL et al.; BACKGROUND & AIMS: Enteropathogenic Escherichia coli and enterohemorrhagic E . coli harbor highly homologous pathogenicity islands yet show key differences in their mechanisms of action . Both disrupt host intestinal epithelial tight junctions, but the effects of enteropathogenic E . coli are more profound than those of enterohemorrhagic E . coli . The basis for this is not understood . The atypical protein kinase C isoform, protein kinase C-zeta, associates with and regulates the tight junction complex . The aim of this study was to compare the role of protein kinase C-zeta in the disruption of tight junctions after infection with enteropathogenic E . coli and enterohemorrhagic E . coli . METHODS: Model intestinal epithelial monolayers infected by enteropathogenic E . coli or enterohemorrhagic E . coli were used for these studies . RESULTS: Neither bisindolylmaleimide nor Go6976, which block several protein kinase C isoforms but not protein kinase C-zeta, protected against the decrease in transepithelial electrical resistance after enteropathogenic E . coli infection . Rottlerin at concentrations that block novel and atypical isoforms, including protein kinase C-zeta, significantly attenuated the decrease in transepithelial electrical resistance . The specific inhibitory peptide, myristoylated protein kinase C-zeta pseudosubstrate, also significantly decreased the enteropathogenic E . coli -associated decrease in transepithelial electrical resistance and redistribution of tight junction proteins . In contrast to enteropathogenic E . coli, the level of protein kinase C-zeta enzyme activity stimulated by enterohemorrhagic E . coli was transient and minor, and protein kinase C-zeta inhibition had no effect on the decrease in transepithelial electrical resistance or the redistribution of occludin . CONCLUSIONS: The differential regulation of protein kinase C-zeta by enteropathogenic E . coli and enterohemorrhagic E . coli may in part explain the less profound effect of the latter on the barrier function of tight junctions. Nat Biotechnol, 2004 Oct, 22(10), 1291 - 6 Epub 2004 Sep 07. Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis; Zhong H et al.; The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches . However, current techniques are not efficient in determining protein modifications . We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity . We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively . Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein . As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells . This technique can potentially be automated for large-scale protein annotation. Nat Struct Mol Biol, 2004 Oct, 11(10), 1015 - 20 Epub 2004 Sep 12. Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture; Korndorfer IP et al.; The 17-kDa protein (Skp) of Escherichia coli is a homotrimeric periplasmic chaperone for newly synthesized outer-membrane proteins . Here we present its X-ray structure at a resolution of 2.35 A . Three hairpin-shaped alpha-helical extensions reach out by approximately 60 A from a trimerization domain, which is composed of three intersubunit beta-sheets that wind around a central axis . The alpha-helical extensions approach each other at their distal turns, resulting in a fold that resembles a 'three-pronged grasping forceps' . The overall shape of Skp is reminiscent of the cytosolic chaperone prefoldin, although it is based on a radically different topology . The peculiar architecture, with apparent plasticity of the prongs and distinct electrostatic and hydrophobic surface properties, supports the recently proposed biochemical mechanism of this chaperone: formation of a Skp(3)-Omp complex protects the outer membrane protein from aggregation during passage through the bacterial periplasm. Science, 2004 Sep 10, 305(5690), 1587 - 94 Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A; Khademi S et al.; The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times . Two structurally similar halves span the membrane with opposite polarity . Structures with and without ammonia or methyl ammonia show a vestibule that recruits NH4+/NH3, a binding site for NH4+, and a 20 angstrom-long hydrophobic channel that lowers the NH4+ pKa to below 6 and conducts NH3 . Favorable interactions for NH3 are seen within the channel and use conserved histidines . Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3. J Biol Chem, 2004 Nov 19, 279(47), 48742 - 50 Epub 2004 Sep 10. Are respiratory enzymes the primary sources of intracellular hydrogen peroxide? Seaver LC, Imlay JA. Endogenous H2O2 is believed to be a source of chronic damage in aerobic organisms . To quantify H2O2 formation, we have generated strains of Escherichia coli that lack intracellular scavenging enzymes . The H2O2 that is formed within these mutants diffuses out into the medium, where it can be measured . We sought to test the prevailing hypothesis that this H2O2 is primarily generated by the autoxidation of redox enzymes within the respiratory chain . The rate of H2O2 production increased when oxygen levels were raised, confirming that H2O2 is formed by an adventitious chemical process . However, mutants that lacked NADH dehydrogenase II and fumarate reductase, the most oxidizable components of the respiratory chain in vitro, continued to form H2O2 at normal rates . NADH dehydrogenase II did generate substantial H2O2 when it was when overproduced or quinones were absent, forcing electrons to accumulate on the enzyme . Mutants that lacked both NADH dehydrogenases respired very slowly, as expected; however, these mutants showed no diminution of H2O2 excretion, suggesting that H2O2 is primarily formed by a source outside the respiratory chain . That source has not yet been identified . In respiring cells the rate of H2O2 production was approximately 0.5% the rate of total oxygen consumption, with only modest changes when cells used different carbon sources. Biochem J, 2005 Jan 15, 385(Pt 2), 389 - 97 Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates; Molinaro G et al.; APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P(1)' . Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP . The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP) . Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone . This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli . Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples . These new substrates represent a helpful analytical tool for rapid and reliable screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors. Transgenic Res, 2004 Jun, 13(3), 295 - 8 Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes; Joensuu JJ et al.; Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed . Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants . In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein . Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment . For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast . The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein . The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions . Plant-produced FaeG proved to be stable up to 2 h under these conditions . The binding and inhibition properties were tested with isolated piglet villi . These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner . Thus, the first two prerequisites for the development of an oral vaccine have been met. Mol Cells, 2004 Aug 31, 18(1), 71 - 8 Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC15835; Kharel MK et al.; The organization of the 2-deoxystreptamine (DOS) biosynthetic gene cluster of Micromonospora echinospora has been determined . Sequencing of a 14.04 kb-region revealed twelve open reading frames (ORFs): four putative DOS biosynthetic genes (gtmA, B, C, and D), five amino sugars biosynthetic genes (gtmE, G, H, I, and gacB), two aminoglycoside resistance genes (gtmF and J) as well as a hypothetical ORF (gacA) . One of the putative DOS biosynthetic genes, gtmA, was expressed in Escherichia coli, and the purified protein was shown to convert glucose-6-phosphate (G-6-P) to 2-deoxy-scyllo-inosose (DOI), a key step in DOS biosynthesis . In addition gtmJ was expressed in Streptomyces lividans and shown to confer gentamicin resistance . Thus gtmA and gtmJ are implicated in the biosynthesis of gentamicin and in resistance to it, respectively. J Med Microbiol, 2004 Oct, 53(Pt 10), 959 - 64 The distribution of, and antibody response to, the core lipopolysaccharide region of Escherichia coli isolated from the faeces of healthy humans and cattle; Gibbs RJ et al.; There are five different core types of Escherichia coli lipopolysaccharide (LPS), and enterohaemorrhagic E . coli tend to have the R3 core type . It has been hypothesized that increased carriage of bacteria with a specific core type will induce higher levels of antibodies and protect against disease caused by bacteria carrying that specific LPS core . Approximately 320 isolates of E . coli, half from healthy human faeces and half from healthy bovine faeces have been core typed both by core-specific monoclonal antibodies, and by PCR for genes encoding the enzymes responsible for the biosynthesis of the specific core structures . Results showed that E . coli possessing R1 core LPS were most frequently detected in both human and cattle populations (63 and 49%, respectively) . Compared to the human isolates a significantly higher level of bacteria with R3 core LPS was detected among the bovine commensal E . coli (11% compared to 4%; P < 0.05) . Antibody levels to each of the specific core types were measured in serum samples from healthy humans (n = 91) and healthy cattle (n = 39) . In each population the highest level of antibody detected was reactive to the R4 core . In cattle the level of anti-R3 core antibody was significantly higher than the level of anti-R1, -R2 and -K12 antibodies (P < 0.01) . In summary there was a greater proportion of E . coli with R3 core type in cattle, together with a corresponding higher anti-R3 antibody level . This suggests that cattle may have greater immunity to E . coli strains with an LPS of R3 core type. J Biol Chem, 2004 Nov 19, 279(47), 48846 - 54 Epub 2004 Sep 09. Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures; Muller L et al.; The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer . p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner . The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo . Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail . Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity . Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation . Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation. J Biol Chem, 2004 Dec 3, 279(49), 50717 - 25 Epub 2004 Dec 3. Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure; Piotrowski M et al.; The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions . To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed . Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a . Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves . Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive . AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli . The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs) . They accept a broad range of dsGSs as substrates . However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c . Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin . Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo. J Biol Chem, 2004 Nov 19, 279(47), 49151 - 9 Epub 2004 Sep 08. Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus; Okamoto H et al.; Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA . Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene . The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis . Although the overall amino acid sequence of the aq065 protein differs considerably from that of E . coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity . The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy . To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E . coli tRNAThr . It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer . We found that the enzyme specificity was critically dependent on the size of the variable loop . Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem . These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity . It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity. Front Biosci, 2004 Sep 01, 9, 2548 - 55 Dual functional regulators coordinate DNA replication and gene expression in proliferating cells; Tye BK et al.; Gene products for cell growth must meet the pace of DNA replication and vice versa during the cell division cycle, therefore coordination of DNA replication and gene expression is vital to proliferating cells . During development in multicellular organisms when rapid cell divisions must be accompanied by the expression of particular gene sets in differentiating tissues, this coordination is even more crucial . Undoubtedly, multiple strategies are used to ensure the coordination of gene expression and DNA replication . In this review, we focus on the strategy that uses dual functional factors to serve both the functions of replication initiator and transcription regulator . Classical examples are the dual functional replication initiator/transcription regulators, DnaA of E . coli and T antigen of SV40, which bind replication origins and regulate their own synthesis . Emerging examples in eukaryotes are the growth responsive transcription factor E2f, the MADS domain combinatorial transcription factor Mcm1, and a subunit of the MCM2-7 helicase, Mcm7. FEBS Lett, 2004 Sep 10, 574(1-3), 203 - 7 Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates; Kawano K et al.; Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates . These hemoglobins appear to be the result of gene duplication and fusion . Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids . These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form . Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined . The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate . On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively) . The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin . These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen. FEBS Lett, 2004 Sep 10, 574(1-3), 161 - 6 Mutational analysis of conserved AAA+ residues in the archaeal Lon protease from Thermoplasma acidophilum; Besche H et al.; The Lon protease from the archaeon Thermoplasma acidophilum (TaLon) is composed of an N-terminal ATPase associated with various cellular activities (AAA+) domain and a C-terminal Lon protease domain . Although related in sequence to the soluble Lon proteases, TaLon was shown to be membrane-bound in its native host and also when expressed in Escherichia coli . Recombinant TaLon was purified as a functional high-molecular weight complex displaying ATPase and proteolytic activity . Mutagenesis of conserved AAA+ residues revealed that the Walker A and B motifs, and the sensor 1 and sensor 2' residues were essential for the ATPase activity, while the sensor 2 and the arginine finger were involved in activation of the protease domain. FEBS Lett, 2004 Sep 10, 574(1-3), 151 - 5 RecQ helicase enhances homologous recombination in plants; Li HQ et al.; RecQ helicase is a key component in the RecF pathway of Escherichia coli for initiation of homologous recombination . Here, we demonstrate that transient expression of RecQ gene in rice embryogenic cell increases the homologous recombination efficiency as much as 4-fold . Further experiments reveal that this effect is influenced by the RecQ dosage . Stable expression of RecQ in rice dramatically increases the homologous recombination events 20- to 40-fold in leaf tissue from different transgenic lines . This is the first evidence indicating that overexpression of RecQ gene can stimulate homologous recombination in plants. FEBS Lett, 2004 Sep 10, 574(1-3), 131 - 7 Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs; Lin CW et al.; Severe acute respiratory syndrome (SARS) has been globally reported . A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease . SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication . In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro . Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity . In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency . Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates . The results will be useful for the rational development of the anti-SARS drugs. FEBS Lett, 2004 Sep 10, 574(1-3), 62 - 8 Functional identification of AtFao3, a membrane bound long chain alcohol oxidase in Arabidopsis thaliana; Cheng Q et al.; The Arabidopsis thalina genome database was searched for homologues of the Candida cloacae fao1 gene which encodes a membrane bound, flavin-containing, hydrogen peroxide generating, long chain alcohol oxidase . This gene has not been isolated from plants or animals . Four putative candidates were found in the database but their function has not been proven . The cDNAs for two of them were cloned by RT-PCR from Arabidopis suspension culture and one of them {AtFAO3} was overexpressed in Escherichia coli and shown to functionally express long chain alcohol oxidase activity . The protein has been solubilised and retains biological activity thereby preparing the way for crystallographic studies . This is the first functional proof identifying a long chain alcohol oxidase in higher plants. Biochim Biophys Acta, 2004 Sep 17, 1679(3), 214 - 21 Characterization of GhRac1 GTPase expressed in developing cotton (Gossypium hirsutum L.) fibers; Kim HJ et al.; Cytoskeleton assembly plays an important role in determining cotton fiber cell length and morphology and is developmentally regulated . As in other plant cells, it is not clear how cytoskeletal assembly in fibers is regulated . Recently, several Rac/Rop GTPases in Arabidopsis were shown to regulate isotropic and polar cell growth of root hairs and pollen tubes by controlling assembly of the cytoskeleton . GhRac1, isolated from cottonseeds, is a member of the Rac/Rop GTPase family and is abundantly expressed in rapidly growing cotton tissues . GhRac1 shows the greatest sequence similarity to the group IV subfamily of Arabidopsis Rac/Rop genes . Overexpression of GhRac1 in E . coli led to the production of a functional GTPase as shown by in vitro enzyme activity assay . In contrast to other Rac/Rop GTPases found in cotton fiber, GhRac1 is highly expressed during the elongation stage of fiber development with expression decreasing dramatically when the rate of fiber elongation declines . The association of highest GhRac1 expression during stages of maximal cotton fiber elongation suggests that GhRac1 GTPase may be a potential regulator of fiber elongation by controlling cytoskeletal assembly. FEMS Microbiol Lett, 2004 Sep 15, 238(2), 383 - 9 Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli; Deutch CE; 3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid . When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed . There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate . Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine . An E . coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E . coli strain UMM5 . Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine . These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E . coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues. FEMS Microbiol Lett, 2004 Sep 15, 238(2), 297 - 305 Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp . strain A4; Pinyakong O et al.; Sphingomonas sp . strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs) . The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp . strain P2 . The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases . Biotransformation with recombinant E . coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene . Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene . Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain. Protein Expr Purif, 2004 Oct, 37(2), 472 - 8 Expression and purification of His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase wild-type and Arg256 mutant proteins; Zeng J et al.; Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported . We cloned the gene of rat mitochondrial medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 3' of the gene . The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 88% yield to apparent homogeneity . The specific activity of the purified His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase was 4.0 U/mg . Arg256 is a highly conserved amino acid, which may play an important role in enzymatic reaction based on the crystal structure of medium-chain acyl-CoA dehydrogenase . We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis . Mutant proteins were overexpressed in E . coli and purified with a nickel metal affinity column . Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Arg256 is a very important residue of rat mitochondrial medium-chain acyl-CoA dehydrogenase . Our overexpression in E . coli and one-step purification of the highly active rat mitochondrial medium-chain acyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of medium-chain acyl-CoA dehydrogenase. Protein Expr Purif, 2004 Oct, 37(2), 450 - 4 Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags; Banerjee AL et al.; Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer . Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view . We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure . By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76% . The purified enzyme showed a single band on the SDS-PAGE . Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors. Protein Expr Purif, 2004 Oct, 37(2), 434 - 42 Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase; Brizio C et al.; Dimethylglycine dehydrogenase (Me(2)GlyDH) is a mitochondrial enzyme that catalyzes the oxidative demethylation of dimethylglycine to sarcosine . The enzyme requires flavin adenine dinucleotide (FAD), which is covalently bound to the apoprotein via a histidyl(N3)-(8alpha)FAD linkage . In the present study, the mature form of rat Me(2)GlyDH has been over-expressed in Escherichia coli as an N-terminally 6-His-tagged fusion protein . The over-expressed protein distributed almost equally between the soluble and insoluble (inclusion bodies) cell fraction . By applying the soluble cell lysate to a nickel-chelating column, two fractions were eluted, both containing a nearly homogeneous protein with a molecular mass of 93 kDa, on SDS-PAGE . The first protein fraction was identified by Western blotting analysis as the covalently flavinylated Me(2)GlyDH . It showed optical properties and specific activity (240 nmol/min/mg protein) similar to those of the native holoenzyme . The second fraction was identified as an underflavinylated (apo-) form of Me(2)GlyDH, with a 70% lower specific activity . The recombinant holoenzyme exhibited optimal activity at pH 8.5, an activation energy of about 80 kJ/mol, and two KM values for N,N-dimethylglycine (KM1 = 0.05 mM and KM2 = 9.4 mM), as described for the native holoenzyme . Starting from the inclusion bodies, the unfolded flavinylated enzyme was solubilized by SDS treatment and refolded by an 80-fold dilution step, with a reactivation yield of 50-60%. Protein Expr Purif, 2004 Oct, 37(2), 426 - 33 Expression of enterovirus 70 capsid protein VP1 in Escherichia coli; Chen D et al.; The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139 . Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E . coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS) . High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E . coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence . The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively) . However, in E . coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster . The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA) . Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E . coli. Protein Expr Purif, 2004 Oct, 37(2), 419 - 25 Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli; Rea G et al.; The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein . To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies . The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent . Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml . SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers . The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity . Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding . The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%) . The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites. Protein Expr Purif, 2004 Oct, 37(2), 409 - 18 Functional transplantation of the sumoylation machinery into Escherichia coli; Mencia M et al.; Modification by SUMO proteins appears to be very common in eukaryotic cells . Many proteins have been reported to be sumoylated, at least under certain circumstances, in vivo, and new examples get published every month . On the other hand, sumoylation is, in essence, a way to construct branched proteins or protein fusions . Obtention of pure sumoylated proteins from eukaryotic cells is not easy because of the dynamic nature of this modification and the large number of sumoylated proteins in vivo . Production of sumoylated proteins in vitro requires the previous purification of most of the components of the pathway, and has the typical limitations of such systems . In this paper, we describe a method to quantitatively produce sumoylated proteins in vivo in Escherichia coli as a way to obtain large quantities of specifically sumoylated target proteins with a high degree of purity, to generate fusion proteins not limited to N- or C-end additions, and to polymerize proteins by covalent linkage. Protein Expr Purif, 2004 Oct, 37(2), 392 - 8 Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase; Austin CJ et al.; The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism . It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases . Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO) . Significant formation of inclusion bodies was noted at the growth temperature of 37 degrees C, with reduced formation at 30 degrees C . The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees C and purification by nickel-agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 micromol of kynurenine/h/mg of protein (both identical to native human IDO) . The protein was homogeneous in terms of electrophoretic analysis . Yields of soluble protein (3-5 mg/L of bacterial culture) and heme content are greater than previously reported. Protein Expr Purif, 2004 Oct, 37(2), 368 - 76 Expression and characterization of isoform 1 of human mitochondrial elongation factor G; Bhargava K et al.; Elongation factor G (EF-G) catalyzes the translocation step of protein biosynthesis . Genomic analysis suggests that two isoforms of this protein occur in mitochondria . The region of the cDNA coding for the mature sequence of isoform 1 of human mitochondrial EF-G (EF-G1(mt)) has been cloned and expressed in Escherichia coli . The recombinant protein has been purified to near homogeneity by chromatography on Ni-NTA resins and cation exchange high performance liquid chromatography . EF-G1(mt) is active on both bacterial and mitochondrial ribosomes . Human EF-G1(mt) is considerably more resistant to fusidic acid than many bacterial translocases . A molecular model for EF-G1(mt) has been created and analyzed in the context of its relationship to the translocases from other systems. Protein Expr Purif, 2004 Oct, 37(2), 361 - 7 Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin; Guo C et al.; An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG . The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs) . The IBs were denatured and then refolded by step-wise dialysis . About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC . The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage . Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds . This implied that the purified Gel by this method is biologically active and suitable for further studies. Protein Expr Purif, 2004 Oct, 37(2), 352 - 60 A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase; Linster CL et al.; Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E . coli BL21(DE3)pLysS cells and purified to near-homogeneity . The kinetic properties of the two enzymes were investigated . The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+) . Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+) . The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate . This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase. Protein Expr Purif, 2004 Oct, 37(2), 344 - 51 Expression and purification of His-tagged rat mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase wild-type and Ser137 mutant proteins; Liu X et al.; Mitochondrial 3-hydroxyacyl-CoA dehydrogenase is a key enzyme in the beta-oxidation of fatty acids . The deficiency of this enzyme in patients has been previously reported . We cloned the gene of rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase in a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' of the gene . The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity . The specific activity of the purified His-tagged rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase was 452 U/mg . Ser137 is a highly conserved amino acid, which, it has been suggested, is an important residue because of its proximity to the modeled L-3-hydroxyacyl-CoA substrate in the crystal structure of 3-hydroxyacyl-CoA dehydrogenase . We constructed three mutant expression plasmids of the enzyme using site-directed mutagenesis . Mutant proteins were overexpressed in E . coli and purified with a nickel metal affinity column . Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Ser137 is a very important residue of rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase . Our overexpression in E . coli and one-step purification of the highly active rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of 3-hydroxyacyl-CoA dehydrogenase. Protein Expr Purif, 2004 Oct, 37(2), 320 - 6 Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa; Catani CF et al.; In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented . The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49 . The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration . The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing . The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy. Protein Expr Purif, 2004 Oct, 37(2), 311 - 9 Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents; Ahl IM et al.; To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents . We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents . This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+) . The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein . The enzymes were purified to homogeneity and active enzymes were obtained in high yield . The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy. Protein Expr Purif, 2004 Oct, 37(2), 306 - 10 Expression and purification of a novel rice (Oryza sativa L.) mitochondrial ATP synthase small subunit in Escherichia coli; Liu S et al.; To clarify the function of the rice mitochondrial ATP synthase 6 kDa subunit (RMtATP6), a method of producing large quantities of this protein is needed . Here, we describe an Escherichia coli expression system for the rapid and economic expression of RMtATP6 . The RMtATP6 gene (GenBank Accession No . ) was cloned into the pGEX-6p-3 vector to allow expression of RMtATP6 as a glutathione S-transferase (GST) fusion protein . The RMtATP6-GST fusion protein was purified by affinity chromatography using a glutathione-Sepharose 4B column . A Western blot analysis using anti-GST antibody showed that the fusion protein was not degraded . After enzymatic cleavage of the GST tail, the RMtATP6 protein showed a molecular weight of around 6 kDa . The predicted pI of this protein is 10.01 . After improving the conditions of expression and the purification procedures, the final yield of the entire expression and purification process was about 4.6 mg of pure RMtATP6 protein per liter of bacterial culture. Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 234 - 40 Insights into function of PSI domains from structure of the Met receptor PSI domain; Kozlov G et al.; PSI domains are cysteine-rich modules found in extracellular fragments of hundreds of signaling proteins, including plexins, semaphorins, integrins, and attractins . Here, we report the solution structure of the PSI domain from the human Met receptor, a receptor tyrosine kinase critical for proliferation, motility, and differentiation . The structure represents a cysteine knot with short regions of secondary structure including a three-stranded antiparallel beta-sheet and two alpha-helices . All eight cysteines are involved in disulfide bonds with the pattern consistent with that for the PSI domain from Sema4D . Comparison with the Sema4D structure identifies a structurally conserved core comprising the N-terminal half of the PSI domain . Interestingly, this part links adjacent SEMA and immunoglobulin domains in the Sema4D structure, suggesting that the PSI domain serves as a wedge between propeller and immunoglobulin domains and is responsible for the correct positioning of the ligand-binding site of the receptor. Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 183 - 91 Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen; Oyama M et al.; Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses . Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER) . We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts . GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His)6-tagged p53 proteins, indicating direct protein-protein interaction between those proteins . Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1 . These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms. Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 94 - 101 Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli; Jiang Z et al.; Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity . Here, we addressed this question by production of such protein . In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica . Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon . The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs . 16.6 s(-1)), provided that the catalytic Tyr residue was intact . When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity . It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide) . These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E . coli at sufficient amounts for purification . We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity. Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 31 - 7 Chaperone characteristics of PDI-related protein A from Aspergillus niger; Zhou H et al.; The functional properties of a novel protein, protein disulfide isomerase-related protein A (PRPA) from Aspergillus niger T21, have been characterized . (1) PRPA possesses disulfide isomerase activity . (2) In Hepes buffer, at substoichiometric concentrations, PRPA facilitates the formation of inactive lysozyme aggregates associated with PRPA (anti-chaperone activity); while at a high molar excess, PRPA inhibits aggregation by maintaining lysozyme in a soluble, yet inactive, state (chaperone-like activity) . However, PRPA only exhibits chaperone-like activity during lysozyme refolding in phosphate buffer . (3) Experiments have indicated that disulfide cross-linkage is not required for the interaction between PRPA and lysozyme, and hydrophobic interaction may be responsible for PRPA effect on lysozyme . (4) Co-expression of PRPA and prochymosin in Escherichia coli leads to reduction of inclusion bodies, rendering part of prochymosin molecules soluble yet inactive . The structural and functional characteristics of PRPA suggest that PRPA may play an important role in protein folding, aggregation, and retention in the endoplasmic reticulum. Biochem Biophys Res Commun, 2004 Aug 20, 321(2), 355 - 63 Characterization of Smubp-2 as a mouse mammary tumor virus promoter-binding protein; Uchiumi F et al.; A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt11 library by South-Western blot screening using a 50-bp tannic acid responsive element {J . Biol . Chem . 273 (1998) 12499} of the mouse mammary tumor virus (MMTV) promoter region as a probe . The full-length cDNA encodes a protein with a predicted size of 108 kDa . Northern blot analysis revealed that the gene expression of Smubp-2 is comparatively high in testis, moderate in brain, and low in other tissues . The recombinant Smubp-2 protein was expressed as a GST- or Trx-fusion protein in Escherichia coli and purified by affinity column chromatography . Gel mobility shift competition analysis indicated that the recombinant Smubp-2 protein binds to region II (containing the ACTG-motif) in the 50-bp element in the MMTV promoter . A transient transfection assay of the Smubp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Smubp-2 is a negative transcription factor . Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Smubp-2 . Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Smubp-2 . These results suggest that the negative transcriptional effect of Smubp-2 arises from its binding to the 50-bp element located in the MMTV promoter region. J Fish Dis, 2004 Sep, 27(9), 517 - 22 Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans; Yoganandhan K et al.; The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector . The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction . Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs . The antiserum did not recognize any of the other known WSSV structural proteins . Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P . indicus at 12 and 24 h post-infection (p.i.), respectively . The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp. Biochem J, 2005 Jan 1, 385(Pt 1), 255 - 64 Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization; Khorchid A et al.; Escherichia coli EnvZ is a membrane sensor histidine kinase that plays a pivotal role in cell adaptation to changes in extracellular osmolarity . Although the cytoplasmic histidine kinase domain of EnvZ has been extensively studied, both biochemically and structurally, little is known about the structure of its periplasmic domain, which has been implicated in the mechanism underlying its osmosensing function . In the present study, we report the biochemical and biophysical characterization of the periplasmic region of EnvZ (Ala38-Arg162) . This region was found to form a dimer in solution, and to consist of two well-defined domains: an N-terminal a-helical domain and a C-terminal core domain (Glu83-Arg162) containing both a-helical and b-sheet secondary structures . Our pull-down assays and analytical ultracentrifugation analysis revealed that dimerization of the periplasmic region is highly sensitive to the presence of CHAPS, but relatively insensitive to salt concentration, thus suggesting the significance of hydrophobic interactions between the homodimeric subunits . Periplasmic homodimerization is mediated predominantly by the C-terminal core domain, while a regulatory function may be attributed mainly to the N-terminal a-helical domain, whose mutations have been shown previously to produce a high-osmolarity phenotype. J Microbiol, 2004 Jun, 42(2), 103 - 10 Factors influencing preferential utilization of RNA polymerase containing sigma-38 in stationary-phase gene expression in Escherichia coli; Kim EY et al.; In order to understand the molecular basis of selective expression of stationary-phase genes by RNA polymerase containing sigma38 (Esigma38) in Escherichia coli, we examined transcription from the stationary-phase promoters, katEP, bolAP, hdeABP, csgBAP, and mcbP, in vivo and in vitro . Although these promoters are preferentially recognized in vivo by Esigma38, they are transcribed in vitro by both Esigma38 and Esigma70 containing the major exponential sigma, sigma70 . In the presence of high concentrations of glutamate salts, however, only Esigma38 was able to efficiently transcribe from these promoters, which supports the concept that the promoter selectivity of sigma38-containing RNA polymerase is observed only under specific reaction conditions . The examination of 6S RNA, which is encoded by the ssr1 gene in vivo, showed that it reduced Esigma70 activity during the stationary phase, but this reduction of activity did not result in the elevation of Esigma38 activity . Thus, the preferential expression of stationary-phase genes by Esigma38 is unlikely the consequence of selective inhibition of Esigma70 by 6S RNA. J Microbiol, 2004 Mar, 42(1), 42 - 6 Chlorothalonil-biotransformation by glutathione S-transferase of Escherichia coli; Kim YM et al.; It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al, 2003) . GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification . In an attempt to elucidate the relation between chlorothalonil-detoxification and GST, the GST of Escherichia coli was expressed and purified . The drug-hypersensitive E . coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil . The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione . Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST. J Microbiol, 2004 Mar, 42(1), 20 - 4 Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli; Park SH et al.; Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column . The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate . The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C . The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000 . A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis . The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively. Arch Pharm Res, 2004 Jul, 27(7), 776 - 80 Cloning and characterization of directly amplified antiviral gene interferon alpha-2b (HulFNalpha-2b) from human leukocytes chromosomal DNA; Behravan J et al.; Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation . The interferon alpha gene from human blood samples was amplified, cloned and expressed in E . coli (BL21) . Leukocyte chromosomal DNA was used as a source of template DNA . Using specific primers, the gene for HulFNalpha-2b was amplified and inserted into the E . coli vector, pET21b, by ligation of the Hindlll and BamHI linkers of the vector and insert . The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E . coli strain . The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins. Clin Infect Dis, 2004 Sep 1, 39(5), e46 - 8 Epub 2004 Aug 11. Shiga toxin-producing Escherichia coli as a possible etiological agent of chronic diarrhea; Spacek LA et al.; We identified Shiga toxin-producing Escherichia coli (STEC) as the likely etiologic pathogen for chronic diarrhea in 2 patients, 1 of whom was immunocompromised with acquired immunodeficiency syndrome, and 1 of whom was immunocompetent . Both were treated with antibiotics, and neither developed systemic complications of the infection . These cases suggest that STEC infection should be considered in the differential diagnosis of chronic diarrhea. Proc Natl Acad Sci U S A, 2004 Sep 21, 101(38), 13945 - 50 Epub 2004 Sep 08. Structural analysis uncovers a role for redox in regulating FKBP13, an immunophilin of the chloroplast thylakoid lumen; Gopalan G et al.; Change in redox status has long been known to link light to the posttranslational regulation of chloroplast enzymes . So far, studies have been conducted primarily with thioredoxin-linked members of the stroma that function in a broad array of biosynthetic and degradatory processes . Consequently, little is known about the role of redox in regulating the growing number of enzymes found to occur in the lumen, the site of oxygen evolution in thylakoid membranes . To help fill this gap, we have studied AtFKBP13, an FKBP-type immunophilin earlier shown to interact with a redox-active protein of the lumen, and found the enzyme to contain a pair of disulfide bonds in x-ray structural studies . These disulfides, which in protein mutagenesis experiments were shown to be essential for the associated peptidyl-prolyl isomerase activity, are unique to chloroplast FKBPs and are absent in animal and yeast counterparts . Both disulfide bonds were redox-active and were reduced by thioredoxin from either chloroplast or bacterial sources in a reaction that led to loss of enzyme activity . The results suggest a previously unrecognized paradigm for redox regulation in chloroplasts in which activation by light is achieved in concert with oxygen evolution by the oxidation of sulfhydryl groups (conversion of SH to S-S) . Such a mechanism, occurring in the thylakoid lumen, is in direct contrast to regulation of enzymes in the stroma, where reduction of disulfides targeted by thioredoxin (S-S converted to SH) leads to an increase in activity in the light. Nucleic Acids Res . 2004 Sep 08;32(16):e128. Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts; Zhou D et al.; The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming . The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months . We describe a method in which this timeline is typically reduced to 3 months . Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts . We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene . The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes. J Biol Chem, 2004 Nov 26, 279(48), 50197 - 203 Epub 2004 Nov 26. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase from the thermoacidophilic archaeon Sulfolobus solfataricus . Molecular cloning and characterization of a membrane-intrinsic prenyltransferase involved in the biosynthesis of archaeal ether-linked membrane lipids; Hemmi H et al.; The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e . bacteria and eukaryotes . (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids . Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin . The thermoacidophilic archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome . One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized . Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate . The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions . A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S . solfataricus suggests that the two other S . solfataricus homologues, excluding the (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively . We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases. J Biol Chem, 2004 Nov 19, 279(47), 49131 - 7 Epub 2004 Sep 07. Structural and dynamic independence of isopeptide-linked RanGAP1 and SUMO-1; Macauley MS et al.; Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established . To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1 . Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible . Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1 . Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether . Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage . We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries. J Biol Chem, 2004 Nov 19, 279(47), 48663 - 70 Epub 2004 Sep 07. Involvement of the nonhomologous region of subunit A of the yeast V-ATPase in coupling and in vivo dissociation; Shao E et al.; The catalytic nucleotide binding subunit (subunit A) of the vacuolar proton-translocating ATPase (or V-ATPase) is homologous to the beta-subunit of the F-ATPase but contains a 90-amino acid insert not present in the beta-subunit, termed the nonhomologous region . We previously demonstrated that mutations in this region lead to changes in coupling of proton transport and ATPase activity and to inhibition of in vivo dissociation of the V-ATPase complex, an important regulatory mechanism (Shao, E., Nishi T., Kawasaki-Nishi, S., and Forgac, M . (2003) J . Biol . Chem . 278, 12985-12991) . Measurement of the ATP dependence of coupling for the wild type and mutant proteins demonstrates that the coupling differences are observed at ATP concentrations up to 1 mm . A decrease in coupling efficiency is observed at higher ATP concentrations for the wild type and mutant V-ATPases . Immunoprecipitation of an epitope-tagged nonhomologous region from cell lysates indicates that this region is able to bind to the integral V0 domain in the absence of the remainder of the A subunit, an interaction confirmed by immunoprecipitation of V0 . Interaction between the nonhomologous region and V0 is reduced upon incubation of cells in the absence of glucose, suggesting that the nonhomologous region may act as a trigger to activate in vivo dissociation . Immunoprecipitation suggests that the epitope tag on the nonhomologous region becomes less accessible upon glucose withdrawal, possibly due to binding to another cellular target . In vivo dissociation of the V-ATPase in response to glucose removal is also blocked by chloroquine, a weak base that neutralizes the acidic pH of the vacuole . The results suggest that the dependence of in vivo dissociation of the V-ATPase on catalytic activity may be due to neutralization of the yeast vacuole, which in turn blocks glucose-dependent dissociation. Lett Appl Microbiol, 2004, 39(4), 383 - 7 Evaluation of the effectiveness of a commercially available defined substrate medium and enumeration system for measuring Escherichia coli numbers in faeces and soil samples; Muirhead RW et al.; AIMS: To determine if a commercially available defined substrate medium and enumeration system could be utilized as an effective and accurate means of enumerating Escherichia coli in environmental samples containing faeces and soil . METHODS AND RESULTS: The samples tested were either inoculated with laboratory grown E . coli or natural E . coli populations in cow faeces . The number of E . coli recovered from faeces and soil samples using the defined substrate medium and enumeration system and a miniaturized MPN method (using traditional media) was compared by analysing the difference between the two methods in relation to the mean . For four of five groups of samples analysed there was no significant difference in the number of E . coli recovered by the two methods (P > 0.05) . In one batch the difference was 0.30 log, which while being statistically significant (P < 0.01) was not considered to be biologically significant . CONCLUSION: The commercially available enumeration system was significantly more precise than the miniaturized MPN method (P < 0.001) . SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that the commercially available defined substrate medium and enumeration system is a suitable method for the measurement of E . coli numbers in faeces and soil samples and should provide advantages of increased precision and a reduction in laboratory analysis time. Eur J Biochem, 2004 Sep, 271(18), 3794 - 803 Escherichia coli cyclophilin B binds a highly distorted form of trans-prolyl peptide isomer; Konno M et al.; Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation . To date, only short peptides with a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm . The crystal structures analyzed in this study show two complexes in which peptides having a trans-form proline, i.e . succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence . Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Neta2 of Arg is formed to fix the peptide . On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsilon2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro . Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character . Eur J Biochem, 2004 Sep, 271(18), 3785 - 93 Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation; Hwang JY et al.; Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination . GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional . It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase {Ermilov, A.N., Olshevskaya, E.V . & Dizhoor, A.M . (2001) J . Biol . Chem.276, 48143-48148} . In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation . When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type . CaM-GCAP-1 neither activated nor interacted with guanylate cyclase . Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type . Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+-induced conformational changes . The latter mutation also promoted dimerization of the protein . Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation . Eur J Biochem, 2004 Sep, 271(18), 3765 - 75 Identification of intracellular target proteins of the calcium-signaling protein S100A12; Hatakeyama T et al.; In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner . Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogen