Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 169 - 71 Epub 2003 Dec 18. Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of recombinant human phosphodiesterase 3B; Patel SB et al.; The catalytic domain of human phosphodiesterase 3B has been cloned, expressed in Escherichia coli and purified in the presence of the PDE3 inhibitors IBMX (3-isobutylmethylxanthine) or MERCK1 by affinity chromatography . Initial screening of crystallization conditions for these complexes in the hanging-drop vapor-diffusion mode resulted in three different crystal forms, all characterized by quite large unit-cell parameters, elevated solvent content and poor diffraction quality . Subsequent optimization of these conditions led to crystals that diffract to 2.4 A and belong to space group C2, with unit-cell parameters a = 146.7, b = 121.5, c = 126.3 A, beta = 100.6 degrees . Rotation-function analysis indicates that the asymmetric unit contains four copies of the monomeric enzyme, corresponding to a solvent content of 64% . To solve the structure of the PDE3B catalytic domain, molecular replacement as well as multiple isomorphous replacement methods are currently being utilized. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 140 - 3 Epub 2003 Dec 18. Crystallization of the crenarchaeal SRP core; Rosendal KR et al.; Protein translocation across or targeting to membranes mediated by the signal recognition particle (SRP) is a universal mechanism conserved in all domains of life . SRP54 from the crenarchaeon Sulfolobus solfataricus has been recombinantly expressed and crystallized with and without SRP RNA helix 8 . The RNA has been transcribed in vitro using ribozyme technology . Both crystal forms are perfect merohedral twins . While SRP54 alone is hemihedrally twinned, the crystals of the SRP54-helix 8 complex indicate tetartohedral twinning, which has not previously been observed in protein crystals . The tetartohedral twinning is enabled by a special diamond-like packing in a trigonal crystal. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 135 - 6 Epub 2003 Dec 18. Expression, purification, crystallization and preliminary crystallographic analysis of the diarrhoea-causing and virulence-determining region of rotaviral nonstructural protein NSP4; Deepa R et al.; The region spanning the tetrameric coiled-coil domain and the interspecies-variable virulence-determining region of the cytoplasmic tail of rotaviral nonstructural protein NSP4 has been crystallized . The crystals belong to space group I222, with unit-cell parameters a = 30.70, b = 38.07, c = 181.62 A, and contain two molecules in the asymmetric unit . Diffraction data have been collected utilizing a MAR imaging plate to a resolution of 2.2 A . The tetramer is generated by the crystallographic dyad along the c axis. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 132 - 4 Epub 2003 Dec 18. Crystallization and preliminary X-ray analysis of tryptophan synthase alpha-subunits from Escherichia coli; Jeong MS et al.; Tryptophan synthase alpha-subunit (alphaTS) catalyzes the cleavage of indole-3-glycerolphosphate to glyceraldehyde-3-phosphate and indole, which is channelled to the active site of the associated beta-subunit (betaTS), where it reacts with serine to yield the amino acid tryptophan in tryptophan biosynthesis . The alphaTS from Escherichia coli is a 268 amino-acid protein with no disulfide bonds or prosthetic groups . Although the crystallization of the subunits from E . coli has been attempted over many years, there have been no reports of an X-ray structure . To explore the molecular origin of the conformational stabilization mechanism of alphaTS, the alpha-subunit protein was overexpressed in E . coli and crystallized using the hanging-drop vapour-diffusion method at 298 K . A native data set to 2.8 A resolution was obtained from a flash-cooled crystal upon exposure to Cu Kalpha X-rays . The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 162.27, b = 44.48, c = 71.52 A, beta = 106.56 degrees . The asymmetric unit contains two molecules of alphaTS, giving a crystal volume per protein mass (V(M)) of 2.16 A(3) Da(-1) and a solvent content of 43.18%. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 122 - 4 Epub 2003 Dec 18. Purification, crystallization and preliminary X-ray diffraction data of L7Ae sRNP core protein from Pyrococcus abyssii; Charron C et al.; The L7Ae sRNP core protein from Pyrococcus abyssii was crystallized using the sitting-drop vapour-diffusion method . Crystals were obtained in the presence of MgCl(2), PEG 2000 MME and acetate buffer at pH 4.0 . A native data set has been collected at 2.9 A resolution using a rotating-anode generator at room temperature . Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 70.7, b = 112.9, c = 34.8 A . There are two monomers of MW 14 200 Da per asymmetric unit and the packing density V(M) is 2.45 A(3) Da(-1) . A molecular-replacement analysis gave solutions for the rotation and translation functions. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 112 - 5 Epub 2003 Dec 18. Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family; Olsen AN et al.; The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops . Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop . One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis . Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K . The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis . Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form . Data sets were collected from four potential heavy-atom derivatives of the form III crystals . The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 54 - 60 Epub 2003 Dec 18. Refinement of the structure of human Rab5a GTPase domain at 1.05 A resolution; Terzyan S et al.; Rab5 is a GTPase that regulates early endosome fusion . Its GTPase domain crystal structure is reported here at 1.05 A resolution in complex with a GTP-analog molecule . It provides the highest resolution three-dimensional model so far obtained for proteins from the Ras-like GTPase family . This study allows extension of structural examination of the GTPase machinery as well as of high-resolution protein structures in general . For example, a buried water-molecule network was observed underneath the switch regions, which is consistent with the functional roles of these regions in the molecular-switching process . Furthermore, residues of multiple conformation and clustered distribution of anisotropic thermal motions of the protein molecule may have general implications for the function of Ras-like GTPases. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 13 - 21 Epub 2003 Dec 18. Substrate recognition and selectivity of plant glycerol-3-phosphate acyltransferases (GPATs) from Cucurbita moscata and Spinacea oleracea; Tamada T et al.; Stromal glycerol-3-phosphate acyltransferases (GPAT) are responsible for the selective incorporation of saturated and unsaturated fatty-acyl chains into chloroplast membranes, which is an important determinant of a plant's ability to tolerate chilling temperatures . The molecular mechanisms of plant chilling tolerance were elucidated by creating chimeric GPATs between squash (Cucurbita moscata, chilling-sensitive) and spinach (Spinacea oleracea, chilling-tolerant) and the results were interpreted using structural information on squash GPAT determined by X-ray crystallography at 1.55 A resolution . Enzymatic analysis of the chimeric GPATs showed that the chimeric GPATs containing the spinach region from residues 128 to 187 prefer the 18:1 unsaturated fatty acid rather than 16:0 saturated fatty acid . Structure analysis suggests that the size and character of the cavity that is formed from this region determines the specific recognition of acyl chains. Plant Physiol, 2004 Jan, 134(1), 92 - 100 Epub 2003 Dec 18. Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance; Rippert P et al.; Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets . Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content . Here, we have explored a new strategy to reach this goal . In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids . In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E . Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate . This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence . A massive accumulation of tocotrienols was observed in leaves . These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines . An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed . This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants. Science, 2003 Dec 19, 302(5653), 2115 - 7 Maize genome sequencing by methylation filtration; Palmer LE et al.; Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize . We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences . Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences . About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified . Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome. J Biol Chem, 2004 Mar 12, 279(11), 10374 - 81 Epub 2003 Dec 18. Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase; Pirneskoski A et al.; Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI) . The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues . It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates . Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains . We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries . We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it . Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect. Bioorg Med Chem Lett, 2004 Jan 5, 14(1), 137 - 41 An electrochemical device for the assay of the interaction between a dioxin receptor and its various ligands; Murata M et al.; Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of a variety of chemicals . Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, information on the actual spectrum of chemical structures that can bind to and activate the AhR is insufficient . In order to determine the binding affinities of chemicals to the human AhR (hAhR), we constructed an electrochemical system which carries the hAhR ligand-binding domain on the electrode surface . The recombinant hAhR ligand-binding domain that was expressed in Escherichia coli using a T7 expression system was immobilized on a gold electrode . The specificity of this biosensor based on a ligand-receptor interaction was comparable to other in vitro screening methods . The receptor-modified electrode can rapidly detect the binding of ligands to hAhR . The electrochemical measurement can be carried out within just 5 min . This electrochemical screening system is rapid, low in cost, and adaptable to high-throughput applications without sacrificing either sensitivity or selectivity. Bioorg Med Chem Lett, 2004 Jan 5, 14(1), 77 - 9 Peptidyl hydroxamic acids as methionine aminopeptidase inhibitors; Hu X et al.; A new class of methionine aminopeptidase (MetAP) inhibitors, which contain an internal hydroxamate (N-acyl-N-alkylhydroxylamine) core as the metal-chelating group, has been designed, synthesized, and tested . The compounds exhibited reversible, competitive inhibition against Escherichia coli MetAP as well as human MetAP-1 and MetAP-2 . The most potent inhibitor had a K(i) value of 2.5 microM and >20-fold selectivity toward E . coli MAP. Biochem Biophys Res Commun, 2004 Jan 9, 313(2), 343 - 50 Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras; Gu B et al.; The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication . NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b . Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein . Differences in thermal stability may partially account for the inability to efficiently oligomerize . Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration . To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function. Biochem Biophys Res Commun, 2004 Jan 9, 313(2), 308 - 13 Selective production of rat mutant selenoprotein W with and without bound glutathione; Bauman AT et al.; Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da) . Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography . Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein. Biochem Biophys Res Commun, 2004 Jan 9, 313(2), 294 - 9 Escherichia coli rpoS gene has an internal secondary translation initiation region; Subbarayan PR et al.; Sigma S (sigma(s)) encoded by rpoS in Escherichia coli is a stationary phase specific sigma subunit of the RNA polymerase holoenzyme . Widespread among the E . coli K12 strains is an amber mutation that prematurely terminates sigma(s) . These rpoSAm mutants would be expected to show no sigma(s) activity . However, suppressor free rpoSAm mutants retain an intermediate catalase activity, a sigma S controlled function . By analyzing the sequence of the rpoS gene we hypothesize that a 277 amino acids long delta1-53 sigma(s) of about 30 kDa can be translated from an internal secondary translation initiation region (STIR, AGGGAGN11GUG) that is located downstream of the amber codon . By cloning this rpoSAm gene, following the expression, function, and N-terminal sequence of this mutant protein, we report the presence of a functional internal STIR in E . coli rpoS, from where a truncated but nevertheless functional form of sigma(s) can be synthesized. Biosens Bioelectron, 2004 Jan 15, 19(6), 537 - 46 Electric chips for rapid detection and quantification of nucleic acids; Gabig-Ciminska M et al.; A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids . A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps . The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface . Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe . p-Aminophenol phosphate (pAPP) was used as a substrate . The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode . The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte . The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract . The assay time depends on the sensitivity required . Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively. Hybrid Hybridomics, 2003 Dec, 22(6), 397 - 400 Generation of a rat monoclonal antibody specific for importin alpha3/Qip1; Sakaguchi N et al.; Importin alpha, which mediates the nuclear import of nuclear localization signal (NLS)-containing proteins, is a member of nuclear transport factors . Importin alpha binds directly NLS and functions as an adapter for accessing the importin beta-dependent import pathway . To date, several isoforms of importin alpha have been identified and classified into three subfamilies in higher eukaryotes . In this study, we report on the production of a rat monoclonal antibody (MAb) against importin alpha3/Qip1, a member of the importin alpha family, using a rat medial iliac lymph node method . The MAb 3D10 produced, reacted with both recombinant and endogenous importin alpha 3/Qip1 . Immunoblotting analysis revealed that MAb 3D10 exclusively recognizes importin alpha3/Qip1 among members of the importin alpha family, in various mammalian cells. Protein Pept Lett, 2003 Dec, 10(6), 599 - 606 The site-directed mutagenesis of gastrodia anti-fungal protein mannose-binding sites and its expression in Escherichia coli; Wang P et al.; Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens . Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli . The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one . The double and triplicate amino acids mutated GAFPs have very low inhibitory activity . For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process. Protein Pept Lett, 2003 Dec, 10(6), 581 - 90 Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B; Su-Xia L et al.; A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies . The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved . Further research showed that the obtained recombinant carboxypeptidase B could substitute carboxypeptidase B isolated from pancreas. Plant Mol Biol, 2003 Aug, 52(6), 1203 - 13 Dual positional specificity and expression of non-traditional lipoxygenase induced by wounding and methyl jasmonate in maize seedlings; Kim ES et al.; Lipoxygenases (LOXs) catalyze the formation of fatty acid hydroperoxides involved in responses to stresses . This study examines the expression of a non-traditional dual positional specific maize LOX in response to wounding or methyl jasmonate (MeJA) . Full-length maize LOX cDNA was expressed in Escherichia coli, and recombinant LOX was purified and characterized enzymatically . RP-HPLC and GC-MS analysis showed that the purified LOX converts alpha-linolenic acid into 13-hydroperoxylinolenic acid and 9-hydroperoxylinolenic acid in a 6:4 ratio . LOX mRNA accumulated rapidly and transiently in response to wounding reaching a peak of expression about 3 h after wounding . This increase followed an initial increase in endogenous jasmonic acid (JA) 1 h after wounding (JA burst) . However, the expression of LOX induced by MeJA lasted longer than the expression induced by wounding, and the MeJA-induced expression seemed to be biphasic pattern composed of early and late phases . The expression of LOX in the presence of inhibitors of JA biosynthesis was not completely inhibited, but delayed in wound response and the expression period was shortened in MeJA response . These results suggest that wound-responsive JA burst may trigger the early phase of LOX expression which facilitates biosynthesis of endogenous JA through its 13-LOX activity, and subsequently leads to the activation of the late phase LOX expression in MeJA-treated maize seedlings . Implications of dual positional specificity of maize LOX in the observed expression kinetics are discussed. Cancer Gene Ther, 2004 Jan, 11(1), 16 - 27 Combined suicide gene therapy for pancreatic peritoneal carcinomatosis using BGTC liposomes; Hajri A et al.; Peritoneal dissemination is a common end-stage complication of pancreatic cancer for which novel therapeutic modalities are actively investigated, as there is no current effective therapy . Thus, we evaluated, in a mouse model of pancreatic peritoneal carcinomatosis, the therapeutic potential of a novel nonviral gene therapy approach consisting of bis-guanidinium-tren-cholesterol (BGTC)-mediated lipofection of a combined suicide gene system . Human BxPC-3 pancreatic cells secreting the carcinoembryonic antigen (CEA) tumor marker were injected into the peritoneal cavity of nude mice . After 8 days, intraperitoneal (i.p.) lipofection was performed using BGTC/DOPE cationic liposomes complexed with plasmids encoding the two prodrug-activating enzymes Herpes Simplex Virus thymidine kinase and Escherichia coli cytosine deaminase, the latter being expressed from a bicistronic cassette also encoding E . coli uracil phosphoribosyltransferase . Administration of the lipoplexes was followed by treatment with the corresponding prodrugs ganciclovir and 5-fluorocytosine . The results presented herein demonstrate that BGTC/DOPE liposomes can efficiently mediate gene transfection into peritoneal tumor nodules . Indeed, HSV-TK mRNA was detected in tumor nodule tissues by semiquantitative reverse transcription-polymerase chain reaction analysis . In addition, green fluorescent protein (GFP) fluorescence and X-gal staining were observed in the peritoneal tumor foci following lipofection of the corresponding EGFP and LacZ reporter genes . These expression analyses also showed that transgene expression lasted for about 2 weeks and was preferential for the tumor nodules, this tumor preference being in good agreement with the absence of obvious treatment-related toxicity . Most importantly, mice receiving the full treatment scheme (BGTC liposomes, suicide genes and prodrugs) had significantly lower serum CEA levels than those of the various control groups, a finding indicating that peritoneal carcinomatosis progression was strongly reduced in these mice . In conclusion, our results demonstrate the therapeutic efficiency of BGTC-mediated i.p . lipofection of a combined suicide gene system in a mouse peritoneal carcinomatosis model and suggest that BGTC-based prodrug-activating gene therapy approaches may constitute a potential treatment modality for patients with peritoneal carcinomatosis and minimal residual disease. RNA, 2004 Jan, 10(1), 102 - 13 EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding; Sharma D et al.; Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site . Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit . Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated . These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G. RNA, 2004 Jan, 10(1), 7 - 11 Aptamer redesigned tRNA is nonfunctional and degraded in cells; Lee D et al.; An RNA aptamer derived from tRNA(Gln) isolated in vitro and a rationally redesigned tRNA(Gln) were used to address the relationship between structure and function of tRNA(Gln) aminoacylation in Escherichia coli . Two mutant tRNA(Gln) sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNA(Gln) in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm . Both mutants were tested in a tRNA(Gln) knockout strain of E . coli, but neither supported knockout cell growth . It was later found that both mutant tRNAs were present in very low amounts in the cell . These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D303 - 6 RegulonDB (version 4.0): transcriptional regulation, operon organization and growth conditions in Escherichia coli K-12; Salgado H et al.; RegulonDB is the primary database of the major international maintained curation of original literature with experimental knowledge about the elements and interactions of the network of transcriptional regulation in Escherichia coli K-12 . This includes mechanistic information about operon organization and their decomposition into transcription units (TUs), promoters and their sigma type, binding sites of specific transcriptional regulators (TRs), their organization into 'regulatory phrases', active and inactive conformations of TRs, as well as terminators and ribosome binding sites . The database is complemented with clearly marked computational predictions of TUs, promoters and binding sites of TRs . The current version has been expanded to include information beyond specific mechanisms aimed at gathering different growth conditions and the associated induced and/or repressed genes . RegulonDB is now linked with Swiss-Prot, with microarray databases, and with a suite of programs to analyze and visualize microarray experiments . We provide a summary of the biological knowledge contained in RegulonDB and describe the major changes in the design of the database . RegulonDB can be accessed on the web at the URL: http://www.cifn.unam.mx/Computational_Biology/regulondb/. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D300 - 2 GenProtEC: an updated and improved analysis of functions of Escherichia coli K-12 proteins; Serres MH et al.; Using more than one approach to characterizing functions of unknown proteins, we now present in GenProtEC some level of function information for 87% of Escherichia coli K-12 proteins . A new approach that has yielded new information entails assigning content of structural domains and their functions to E.coli proteins . In addition, some earlier methods have been further refined to provide more meaningful data . The process of identifying and separating multimodular or fused proteins into component modules has been improved . As a result, groups of sequence-similar (paralogous) proteins have been refined . Experimental information from recent literature on previously unknown genes has been incorporated . We now use a rich system of characterizing cell roles which accents the fact that many proteins play more than one cellular role and therefore carry more than one designation from our detailed catalog of roles, MultiFun. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D293 - 5 The CyberCell Database (CCDB): a comprehensive, self-updating, relational database to coordinate and facilitate in silico modeling of Escherichia coli; Sundararaj S et al.; The CyberCell Database (CCDB: pharmacy.ualberta.ca/CCDB) is a comprehensive, web-accessible database designed to support and coordinate international efforts in modeling an Escherichia coli cell on a computer . The CCDB brings together both observed and derived quantitative data from numerous independent sources covering many aspects of the genomic, proteomic and metabolomic character of E.coli (strain K12) . The database is self-updating but also supports 'community' annotation, and provides an extensive array of viewing, querying and search options including a powerful, easy-to-use relational data extraction system. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D168 - 70 RPG: the Ribosomal Protein Gene database; Nakao A et al.; RPG is a new database that provides detailed information about ribosomal protein (RP) genes . It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli . Users can search the database by gene name and organism . Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs . In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments . RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes. J Biol Chem, 2004 Mar 12, 279(11), 10584 - 92 Epub 2003 Dec 17. Structure of the catalytic fragment of translation initiation factor 2B and identification of a critically important catalytic residue; Boesen T et al.; Eukaryotic initiation factor (eIF) 2B catalyzes the nucleotide activation of eIF2 to its active GTP-bound state . The exchange activity has been mapped to the C terminus of the eIF2Bepsilon subunit . We have determined the crystal structure of residues 544-704 from yeast eIF2Bepsilon at 2.3-A resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes also found in eIF4G and eIF5 . The eight helices are organized in a manner similar to HEAT repeats . The molecule is highly asymmetric with respect to surface charge and conservation . One area in the N terminus is proposed to be directly involved in catalysis . In agreement with this hypothesis, mutation of glutamate 569 is shown to be lethal . An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2 . Two mutations causing the fatal human genetic disease leukoencephalopathy with vanishing white matter are buried and appear to disrupt the structural integrity of the catalytic domain rather than interfering directly with catalysis or binding of eIF2. J Biol Chem, 2004 Mar 12, 279(11), 9840 - 6 Epub 2003 Dec 16. Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins; Schneider D et al.; The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes . A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data . We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers . Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association. Mol Genet Metab, 2003 Nov, 80(3), 315 - 20 Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria; Desviat LR et al.; Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism . This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans . MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized . Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions . We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect . All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones . The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase . The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype. Protein Expr Purif, 2004 Jan, 33(1), 145 - 52 Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3; Weng L et al.; A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli . The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da . Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones . The HPhA was purified to homogeneity by heat precipitation and affinity chromatography . Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA . The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C . Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C . The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography. Protein Expr Purif, 2004 Jan, 33(1), 80 - 91 High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli; Jaiswal S et al.; Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine . The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity . We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli . A 6x His tag was inserted at the N terminus to facilitate its one-step purification . The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol . We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of approximately 30 mg/L . The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 neutralizing epitope known to reside in domain III . The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope . In addition, murine and human polyclonal sera also recognized the recombinant protein . The in vitro refolded recombinant protein preparation was biologically functional . It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells . This expression/purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts. Protein Expr Purif, 2004 Jan, 33(1), 72 - 9 Expression, purification, and structural study of the EC4 domain of E-cadherin; Zheng K et al.; The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides . The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E . coli . This protein contains a Streptag sequence at the N-terminus, and thus was purified using a Strep-Tactin affinity column . However, at high concentrations the 15N-labeled EC4 protein showed an unstable conformation . Conditions for stabilizing the conformation of this protein were evaluated using CD spectroscopy . The CD results showed that this protein has high conformational stability in Tris buffer at pH 6.0 in the presence of 10 mM calcium chloride. Protein Expr Purif, 2004 Jan, 33(1), 66 - 71 High-level expression of recombinant rabbit cytochrome P450 2E1 in Escherichia coli C41 and its purification; Cheng D et al.; Cytochrome P450 2E1 (CYP2E1) is of great interest because of its important role in the oxidation of numerous drugs and carcinogens . The yields of CYP2E1 obtained by the traditional recombinant expression systems have been relatively poor . We report here the development of a system for high-level expression of rabbit CYP2E1 in Escherichia coli strain C41 (DE3) . Expression of the membrane-bound CYP2E1 by the pLW01-P450 expression plasmid, which utilizes a T7 promoter, is markedly improved by employing E . coli strain C41 (DE3) . The pLW01/2E1 expression plasmid was successfully constructed and high-level expression of CYP2E1 was achieved, which ranged between 900 and 1400 nmol (liter culture)(-1) . This yield was 9-14-fold higher than other reports of CYP2E1 expression in other E . coli strains . This system provides a highly efficient tool for expressing CYP2E1 . An improved purification procedure for the expressed CYP2E1 involving chromatography on diethylaminoethyl cellulose (DE52), Reactive Red-agarose (type 1000-CL), and hydroxyapatite is also reported . This procedure allowed recovery of 45% of the expressed protein and CYP2E1 with a specific content of 14 nmol/mg protein, which showed a single band on a polyacrylamide gel stained with Coomassie brilliant blue. Protein Expr Purif, 2004 Jan, 33(1), 48 - 56 The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase; Reilly TJ et al.; The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase . The hppA gene of H . pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli . The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity . The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE . Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE . The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75 . While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity . Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+) . Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates . The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively . The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM . Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase. Protein Expr Purif, 2004 Jan, 33(1), 39 - 47 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins; Lamla T et al.; We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins . The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag . The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM . We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate . No significant influence of the Nano-tag(15) and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag(9) revealed a slight decline in the amount and activity of the synthesized proteins . The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state . Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate. Protein Expr Purif, 2004 Jan, 33(1), 34 - 8 Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.); Branco AT et al.; The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress . Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein . After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein . The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution . After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration) . The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay. Protein Expr Purif, 2004 Jan, 33(1), 25 - 33 Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons; Schiedel AC et al.; The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II . So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons . Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length . Here, we report on the cloning of thiolase II from sunflower cotyledons . The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons . RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE . Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region . The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level . The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography . The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein . Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization. Protein Expr Purif, 2004 Jan, 33(1), 19 - 24 In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein; Araujo MS et al.; Azospirillum brasilense is a diazotroph which associates with important agricultural crops . The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ . Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation . In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A . brasilense strain FP2 . The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein . After purification, we obtained 30-40 mg of purified GlnZ per liter of culture . This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity . Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction . This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation. Protein Expr Purif, 2004 Jan, 33(1), 1 - 10 In vitro protein refolding by chromatographic procedures; Li M et al.; In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli . This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results. Protein Expr Purif, 2003 Nov, 32(1), 119 - 25 Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha; Kurata M et al.; Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor . The protein sequence is homologous to the proregion of mouse cathepsin L . Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha) . CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS . The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography . Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium . The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy . Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM) . Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM) . Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases. Protein Expr Purif, 2003 Nov, 32(1), 110 - 8 Optimized gene synthesis and high expression of human interleukin-18; Li A et al.; Human interleukin-18 (hIL-18), originally known as an IFN-gamma-inducing factor, is a recently cloned cytokine that is secreted by Kupffer cells of the liver and by stimulated macrophages . We have previously established a method of expression and purification of IL-18 . The yield however remains low and the insufficient expression of a heterologous protein could be due to skewed codon usage between the expression host and the cDNA donor . The sequence of mature hIL-18 has 37 a.a . rare codons for Escherichia coli in a total of 157 a.a . To overcome this problem, gene synthesis was performed with optimized codons for the expression host E . coli . The final yield of the hIL-18 protein with optimized codons was about five times higher than the yield with the native sequence . Using a minimal medium, this system produces large quantities of labeled proteins that can be used in NMR analysis . Our simple and efficient production system can be applied to the production of other cytokines for new structural and therapeutic use. Protein Expr Purif, 2003 Nov, 32(1), 104 - 9 On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag; Yin SM et al.; Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases . Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies . Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column . Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations . Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process . The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy . Its correct folding was confirmed from circular dichroism (CD) experiments . Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc). Protein Expr Purif, 2003 Nov, 32(1), 89 - 94 High-level expression of the Arabidopsis thaliana ethylene receptor protein ETR1 in Escherichia coli and purification of the recombinant protein; Voet-van-Vormizeele J et al.; Ethylene responses in plants are mediated by a small family of membrane integral receptors including the ETR1 gene product which are similar to the two-component bacterial histidine kinase regulators . Detailed biochemical and structural analysis of the ethylene-receptor family was hampered by the scarce amount of pure protein . Here, we report the construction, expression, and single-step purification of the ETR1 receptor protein from Arabidopsis thaliana in a bacterial expression system . The DNA fragment encoding the mature ETR1 receptor protein was subcloned into the pET15b expression vector and highly expressed in derivatives C41(DE3) and C43(DE3) of the Escherichia coli strain BL21(DE3) . The recombinant protein was solubilised from the bacterial cells using mild non-denaturing detergents and purified to homogeneity by Ni-NTA affinity chromatography, yielding approximately 2-3 mg pure protein per litre of cells . The molecular mass of the purified protein was estimated to be 78 kDa by SDS-PAGE . The expression and purification of recombinant ETR1 reported here provide a basis for detailed functional and structural studies of the receptor protein, which might help to understand signal perception and signal transduction of the phytohormone ethylene on the molecular level. Protein Expr Purif, 2003 Nov, 32(1), 75 - 82 Expression, purification, and characterization of His20 mutants of rat mevalonate kinase; Chu X et al.; Mevalonate kinase plays a key role in regulating the biosynthesis of cholesterol in animal cells . Human mevalonate kinase His20Pro has been reported as one of the three common mutations in the mevalonate kinase gene in mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome patients . His20 is also a highly conserved residue among all aligned mevalonate kinase sequences . To study the role of His20 of mevalonate kinase, a variety of mutant expression plasmids of rat mevalonate kinase including pRMK(H20L), pRMK(H20Y), and pRMK(H20K) were constructed using site-directed mutagenesis, and mutant proteins were overexpressed and purified . CD spectroscopy of wild-type protein and mutants indicated that mutations H20L and H20Y did not induce significant secondary structural changes . The results from kinetic studies showed that this highly conserved histidine is an important residue for the function of the enzyme. Protein Expr Purif, 2003 Nov, 32(1), 68 - 74 Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor; Xie Q et al.; Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis . To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone . The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain . Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL . Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding . This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique . Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments . Thus, unglycosylated CTLD is sufficient for binding modified LDL. Protein Expr Purif, 2003 Nov, 32(1), 61 - 7 Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa; Paula DP et al.; The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression . The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically important plant diseases, including the citrus variegated chlorosis (CVC) . The putative H-NS ORF was cloned into a pET32-Xa/LIC vector in order to overexpress it coupled with fusion tags in Escherichia coli BL21(DE3) . The expressed recombinant protein was purified by immobilized metal affinity chromatography (Ni-NTA resin) and its identity verified by mass spectrometry (MALDI-TOF) . Final purification was performed by cation-exchange chromatography (SP Sepharose Fast Flow) and the purified protein was found as a single band on SDS-PAGE . The folding and its DNA binding activity were verified by circular dichroism and fluorescence spectroscopy, respectively. Protein Expr Purif, 2003 Nov, 32(1), 52 - 60 Characterization of a variant of PAC-1 in large granular lymphocyte leukemia; Kothapalli R et al.; Phosphatase in activated T cells (PAC-1) is a mitogen-induced early responsive gene . It encodes a 32 kDa tyrosine-threonine dual specificity phosphatase . Constitutive expression of PAC-1 leads to an inhibition of MAP kinase activity in vivo . Such constitutive expression was reported in HTLV-1 infected cell lines . In the present study, we observed the constitutive over-expression of two transcripts related to PAC-1 in large granular lymphocyte (LGL) leukemia . By screening a LGL leukemia cDNA library using the 3' end of a PAC-1 probe, we obtained a clone (clone 8) which retains one and one half introns, excludes two exons, and matches one hundred percent with a DNA sequence on chromosome 2 . The deduced amino acid sequence of the predicted protein contains 170 amino acids and is 144 amino acids shorter than PAC-1 . When we expressed this protein in Escherichia coli as a GST-fusion protein, a 45 kDa (19 kDa PAC-1 variant+26 kDa GST protein) protein was obtained . The expressed protein was purified to near homogeneity by using a glutathione affinity column . The purified protein did not have any intrinsic phosphatase activity when assayed in vitro . But when this purified protein was added to a phosphatase assay system in combination with a recombinant dual specificity phosphatase, CL100, enhanced phosphatase activity was observed . The significance of the constitutive over-expression and its physiological role of this protein remain to be established in leukemic LGL. Protein Expr Purif, 2003 Nov, 32(1), 18 - 27 Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli; Halbhuber Z et al.; In this work, we featured an expression system that enables the production of sufficient quantities ( approximately mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies . PsbH gene from cyanobacterium Synechocystis sp . PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione-S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells . A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism . As a result, the majority of fusion protein was obtained in a soluble state and could be purified from crude bacterial lysate by affinity chromatography on immobilized glutathione under non-denaturing conditions . The PsbH protein was cleaved from the carrier protein with Factor Xa protease and purified on DEAE-cellulose column with yields of up to 2.1 microg protein/ml of bacterial culture . The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 825 - 30 A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs; Nakayama M et al.; Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli . Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E . coli . This system experimentally identified any random cDNA fragments producing soluble protein domains . Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag . These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins . The latter is useful as a fluorescence indicator of protein folding . The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag . This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 801 - 5 Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth; He GA et al.; Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis . Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al . Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay . In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis . The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment . Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day . These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 761 - 6 cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus; Wu F et al.; SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus . It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins . cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family . The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli . The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro . Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed . SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state . The ANS anion is a much-utilized "hydrophobic probe" for proteins . This binding activity indicated another biological function of SPE-16. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 733 - 40 Stability and apoptotic activity of recombinant human cytochrome c; Olteanu A et al.; An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress . Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c . We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis . Yields of greater than 8 mg of pure protein per liter culture were attained . Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c . Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay . We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 615 - 22 Molecular modeling of RecX reveals its mode of interaction with RecA; Mishra S et al.; The protein RecA is involved in homologous recombination, DNA repair and also catalyzes DNA strand exchange . RecX gene is downstream of recA and the gene product RecX is supposed to be important for RecA regulation . Recombinant RecX is purified to homogeneity, and circular dichroism (CD) and FTIR spectroscopy show the protein to exist mostly in helical conformation . The fluorescence emission maxima of the native and the denatured protein and the steady-state fluorescence quenching studies with acrylamide indicate the presence of tryptophan residues partially exposed to the bulk solvent . Denaturation studies with urea and guanidine hydrochloride by use of spectroscopic methods, fluorescence, and CD also confirm the instability of the protein and unfolding occurs following a two-state model . Mass spectrometry and gel permeation chromatography suggest the monomeric form of the protein . Molecular modeling of RecX represents the molecule as extended and helical bundle in conformity with the spectroscopic results . To understand the mechanism of RecX in the regulation of RecA the structural model of RecA-RecX has been discussed . In this proposed model, entry of RecX into hexameric RecA filament prevents binding of ssDNA and also inhibits ATPase activity. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 562 - 70 Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea; Jiang X et al.; Src I is the first reported acidic actinoporin from sea anemone Sagartia rosea with a pI value of 4.8 and comprises 13.9% alpha-helix, 65.1% beta-sheet, and 18.2% random coil . For structure-function studies, Src I was expressed in Escherichia coli as a cleavable fusion protein . Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic alpha-helix for its functional activity . The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells . Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID(50) 2.8 microg/ml and BGC-823 was the least sensitive cells with ID(50) 7.4 microg/ml . After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent . When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein . According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly. Chem Commun (Camb), 2003 Dec 7, (23), 2870 - 1 Cell-permeable small molecule probes for site-specific labeling of proteins; Yeo DS et al.; We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells . Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo. Mikrobiologiia, 2003 Sep-Oct, 72(5), 658 - 65 {The transformation of the unicellular alga Chlamydomonas reinhardtii by electroporation}; Ladygin VG; The cell wall-lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase gene hpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter . Under optimal conditions (10(6) mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10(3) HygR transformants per 10(6) recipient cells . The exogenous DNA integrated into the nuclear genome of Ch . reinhardtii was persistently inherited through more than 350 cell generations . The advantages of this system for the transformation of Ch . reinhardtii with heterologous genes are discussed. Mikrobiologiia, 2003 Sep-Oct, 72(5), 609 - 15 {The status and the role of glutathione under disturbed ionic balance and pH homeostasis in Escherichia coli}; Smirnova GV et al.; The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSHin + GSSGin) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5 . The incubation of E . coli cells in the presence of 50 mM acetate or 10 micrograms/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively . The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSHin:GSSGin) ratio . The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSHout:GSSGout) ratio by 2.1 times . The gramicidin S treatment and acetate stress inhibited the growth of mutant E . coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells . The pH downshift and the exposure of E . coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA. J Bacteriol, 2004 Jan, 186(1), 240 - 3 Identification of residues of the kid toxin involved in autoregulation of the parD system; Lemonnier M et al.; The toxin-antitoxin system parD (kis kid) of plasmid R1 is coregulated by the coordinated action of its two gene products . Here we describe the isolation and the in vivo characterization of three single-amino-acid changes in the Kid toxin, G4E, C74Y, and E91K, that affect the coregulatory activity but preserve the toxicity of the protein. J Bacteriol, 2004 Jan, 186(1), 207 - 11 Effect of the CopB auxiliary replication control system on stability of maintenance of Par(+) plasmid R1; Olsson JA et al.; Plasmid R1 is a low-copy-number plasmid that is present at a level of about four or five copies per average cell . The copy number is controlled posttranscriptionally at the level of synthesis of the rate-limiting initiator protein RepA . In addition to this, R1 has an auxiliary system that derepresses a second promoter at low copy numbers, leading to increased repA mRNA synthesis . This promoter is normally switched off by a constitutively synthesized plasmid-encoded repressor protein, CopB; in cells with low copy numbers, the concentration of CopB is low and the promoter is derepressed . Here we show that the rate of loss of a Par(+) derivative of the basic replicon of R1 increased about sevenfold when the cells contained a high concentration of the CopB protein formed from a compatible plasmid. J Bacteriol, 2004 Jan, 186(1), 8 - 14 Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown; Ruan B et al.; Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS) . However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS . It was reported that M . jannaschii prolyl-tRNA synthetase (C . Stathopoulos, T . Li, R . Longman, U . C . Vothknecht, H . D . Becker, M . Ibba, and D . Soll, Science 287:479-482, 2000; R . S . Lipman, K . R . Sowers, and Y . M . Hou, Biochemistry 39:7792-7798, 2000) or the M . jannaschii MJ1477 protein (C . Fabrega, M . A . Farrow, B . Mukhopadhyay, V . de Crecy-Lagard, A . R . Ortiz, and P . Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation . These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene . Here we show that E . coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP . While transformants of E . coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E . coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium . An E . coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D . radiodurans DR0705 gene . Construction of a D . radiodurans DR0705 deletion strain showed this gene to be dispensable . However, attempts to delete D . radiodurans cysS failed, suggesting that this is an essential Deinococcus gene . These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M . jannaschii . Thus, the mechanism of Cys-tRNA(Cys) formation in M . jannaschii still remains to be discovered. Cancer Res, 2003 Dec 1, 63(23), 8366 - 76 The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene; Porosnicu M et al.; Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo . To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT) . rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluorouracil (5-FU), which exhibited considerable bystander effect . Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone . Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-gamma-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing . In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease. Di Yi Jun Yi Da Xue Xue Bao, 2003 Dec, 23(12), 1314 - 6 {Vector construction for the intracellular domain of human receptor for advanced glycation end- products and expression of the fusion protein}; Zhao SC et al.; OBJECTIVE: To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor for advanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its interacting proteins . METHODS: The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted into pGEX-KG vector, a general GST fusion protein expression vector . After PCR identification, endonuclease digestion and DNA sequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced by isopropyl-beta-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin . RESULTS: The recombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing . After protein expression was achieved in E . coli, a molecular mass of 35 kD GST fusion protein was purified, whose molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . CONCLUSION: The expression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusion protein is obtained, which can be of value for further studies. Di Yi Jun Yi Da Xue Xue Bao, 2003 Dec, 23(12), 1273 - 6 {Refolding and purification of Plasmodium falciparum glutamate dehydrogenase fusion protein}; Li Y et al.; OBJECTIVE: To establish the method for renaturation and purification of the fusion protein of Plasmodium falfiparum (FCC1/HN ) glutamate dehydrogenase (GDH) with glutathione S-transferase (GST) . METHODS: The recombinant plasmid GDH/pGEX-4T-1, encoding the full-length GDH gene, was transformed into E.coli BL21 (DE3) to achieve IPTG-induced high expression of GDH/GST in the form of inclusion bodies identified by SDS-PAGE . After denaturation with 8 mol/L urea, the inclusion bodies were subjected to 3 different renaturation methods, namely Sephacryl S-200 chromatography, dialysis and dilution, for refolding of the fusion protein . The refolded GDH/GST was then purified by different chromatographic approaches . RESULTS: SDS-PAGE analysis showed that the expression GDH/GST fusion protein mounted up to approximately 25% of the total bacterial protein . The dilution was better than the other two methods for the refolding of the fusion protein, with the optimized renaturation condition necessitating the presence of 20 mmol/L Tris-HCl and 1 mmol/L EDTA at pH8.5 with GSSG/GSH ratio of 1 10, which resulted in a recovery rate exceeding 90% . Two-step ion exchange chromotography was optimal for purification of the fusion protein . CONCLUSION: The high-purity and biologically active GDH/GST can be acquired by dilution renaturation followed by two-step ion exchange chromatography. Di Yi Jun Yi Da Xue Xue Bao, 2003 Dec, 23(12), 1245 - 8 {A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation}; Ma JF et al.; OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro . METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds . This K5 mutant (Mut1) was expressed in E . coli and affinity purified . The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay . RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5 . In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition . CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor. Arch Biochem Biophys, 2004 Jan 1, 421(1), 143 - 8 Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein; Liu Y et al.; The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell . Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins . In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil . The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix) . Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins . CD spectral results show that HR1 forms an alpha-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature . Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2 . The 2-Helix protein also shows characteristic properties of the 6-helix bundle . Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors. Arch Biochem Biophys, 2004 Jan 1, 421(1), 135 - 42 Kinetic analysis of human homogentisate 1,2-dioxygenase; Amaya AA et al.; Homogentisate 1,2-dioxygenase (HGD) is a mononuclear Fe(II)-dependent oxygenase that catalyzes the third step in the pathway for the catabolism of tyrosine, the conversion of homogentisate (HG) to maleylacetoacetate (MAA) . We have heterologously expressed and purified native human HGD in the apo form . Steady-state analysis varying the concentration of both HG and molecular oxygen shows that the purified enzyme has a turnover number of 16 s(-1) . Our data suggest that HG binds to the apo-enzyme and that the apo-HGD.HG complex does not bind Fe(II) and dissociates slowly at approximately 0.028 s(-1) . The rate constant for the dissociation of Fe(II) from the holo-enzyme as measured under anaerobic conditions is 0.00004 s(-1) and indicates that this process is not relevant in steady-state turnover . The addition of HG and molecular oxygen to the holo-enzyme is formally random as the holo-enzyme reduces molecular oxygen at a rate of 1.35x10(3) M(-1) s(-1) at 4 degrees C . The term ordered with respect to the addition of substrates is most descriptive as the rate of reduction of molecular oxygen must increase in the presence of HG to sustain the observed turnover number. Arch Biochem Biophys, 2004 Jan 1, 421(1), 77 - 84 Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements; Cisneros DA et al.; Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics . These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site . In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value . For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves . These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response. Astrobiology, 2003 Fall, 3(3), 583 - 96 Supraglacial sulfur springs and associated biological activity in the Canadian high arctic-signs of life beneath the ice; Grasby SE et al.; Unique springs, discharging from the surface of an arctic glacier, release H(2)S and deposit native sulfur, gypsum, and calcite . The presence of sulfur in three oxidation states indicates a complex series of redox reactions . Physical and chemical conditions of the spring water and surrounding environment, as well as mineralogical and isotopic signatures, suggest biologically mediated reactions . Cell counts and DNA analyses confirm bacteria are present in the spring system, and a limited number of sequenced isolates suggests that complex communities of bacteria live within the glacial system. J Physiol, 2004 Jan 1, 554(Pt 1), 78 - 88 Transference of recombinant VE-cadherin cytoplasmic domain alters endothelial junctional integrity and porcine microvascular permeability; Guo M et al.; VE-cadherin constitutes endothelial adherens junctions through a homophilic binding of its extracellular domain and by the anchoring of its intracellular domain to actin cytoskeleton via catenins . The aim of this study was to determine the functional importance of VE-cadherin-cytoskeleton association in the maintenance of endothelial junctional integrity . A recombinant VE-cadherin cytoplasmic domain (rVE-cad CPD) was expressed in E . coli and purified through Ni-NTA spin columns . Immunoprecipitation assays showed that rVE-cad CPD was able to bind beta-catenin in vitro and to compete with endogenous VE-cadherin for binding of beta-catenin in human umbilical vein endothelial cells . A significant increase in the transendothelial flux of albumin was observed in the endothelial cell monolayers transfected with rVE-cad CPD . Importantly, transfection of rVE-cad CPD into intact isolated coronary venules markedly elevated the albumin permeability of the venular endothelium . In addition, immunofluorescence microscopic analysis revealed a conformational change of VE-cadherin from a uniform, continuous distribution along the cell membrane under control conditions to a diffuse, stitch-like pattern after rVE-cad CPD transfection . The effects were likely due to an attenuated anchorage of endogenous VE-cadherin to the cytoskeleton, as evidenced by a decreased partitioning of VE-cadherin in the detergent-insoluble cytoskeletal pool . The results suggest that the intracellular association of VE-cadherin with beta-catenin-linked cytoskeleton is essential to the maintenance of endothelial junctional integrity and microvascular permeability. Oral Microbiol Immunol, 2004 Feb, 19(1), 6 - 15 Characterization of two outer membrane protein antigens of Porphyromonas gingivalis that are protective in a murine lesion model; Ross BC et al.; Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis . The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis . Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked . Analysis of 14 laboratory and clinical isolates of P . gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro . Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation . While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P . gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease. Clin Exp Immunol, 2004 Jan, 135(1), 114 - 8 Nicotinamide does not influence cytokines or exhaled NO in human experimental endotoxaemia; Soop A et al.; This study examined the hypothesis that nicotinamide could attenuate endotoxin-induced inflammatory responses in humans as indicated by levels of cytokines and nitric oxide . Ten healthy male volunteers participated in a randomised, double-blind, cross-over design with regard to the effects of nicotinamide . The volunteers received orally 4 g nicotinamide or placebo at 14 h and at 2 h preceding the experiment (total dose of 8 g) . Endotoxin (E . coli, 2 ng/kg), was administered intravenously . Blood samples and haemodynamic data were collected prior to and up to 6 h after the endotoxin infusion . Orally exhaled NO was measured hourly . Following endotoxin, body temperature increased from baseline 36.3 +/- 0.09 degrees C to a maximum of 38.0 +/- 0.1 degrees C for all (mean +/- SEM, P < 0.001) and heart rate increased from 59 +/- 1.9 to 87.0 +/- 2.6 beats/min after 3 h (mean +/- SEM, P < 0.001) . Endotoxin challenge also markedly elevated the TNF-alpha, IL-6, IL-8 and IL-10 concentrations (P < 0.001 versus baseline for all) during the study period . Orally exhaled NO also increased (P < 0.01) compared to baseline . Nicotinamide treatment did not influence the patterns of cytokine and NO response to endotoxin . In conclusion, there was no effect on the inflammatory parameters by oral nicotinamide at a dose of 8 g, limiting the potential use of this agent for anti-inflammatory purpose in man. J Am Chem Soc, 2003 Dec 24, 125(51), 15822 - 30 Variable coordination geometries at the diiron(II) active site of ribonucleotide reductase R2; Voegtli WC et al.; The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue . After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical . Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism . Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6 . To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH . The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals . Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects . The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms. Ital J Biochem, 2003 Jun, 52(2), 98 - 103 Imaging transcription complexes with the Atomic Force Microscope; Rivetti C et al.; Recent developments in sample deposition and image analysis have shown that the Atomic Force Microscope is a valuable tool for the structural investigation of transcription complexes . When deposited under conditions that allow molecular equilibration onto the substrate, transcription complexes behave as worm-like chains and the mean square end-to-end distance can readily be used to determine the protein induced DNA bend angle . Measurements of the DNA contour length by means of accurate image processing procedures have revealed a DNA compaction in transcription complexes which is compatible with wrapping of the DNA against the surface of the RNA Polymerase . The methods presented have to be considered of general practical use for imaging protein-DNA complexes. Klin Padiatr, 2003 Nov-Dec, 215(6), 321 - 6 Effects of dose-reduced Medac L-asparaginase on coagulation in trial ALL-BFM 2000; Attarbaschi A et al.; BACKGROUND: Glucocorticoids and L-asparaginase (L-ASP) are essential elements of contemporary chemotherapy of childhood acute lymphoblastic leukemia (ALL) . Both cytotoxic drugs are well-known to induce significant alterations in hemostasis, especially affecting the inhibitors of coagulation including antithrombin III (AT III), protein C and protein S . PATIENTS AND METHODS: The objectives of the present prospective study were to analyze the course and degree of the changes of several coagulation proteins during induction therapy of 16 patients treated according to the Berlin-Frankfurt-Munster (BFM) ALL protocol 2000 . The induction protocol included a 7-day mono-therapy with glucocorticoids followed by 4 weeks with additional vincristine, daunorubicin and E . coli L-ASP (Medac) which was administered at a dosage of 5000 IU/m (2) 8-times at 3-day intervals . RESULTS AND CONCLUSIONS: This analysis is the first to show that 5000 IU/m (2) of the Medac L-ASP leads to a less pronounced decrease of the plasma AT III and fibrinogen concentrations during induction therapy (after the 5 (th) L-ASP dose), as compared to previous BFM protocols which used the Medac L-ASP in a dosage of 10 000 IU/m (2) . Our results confirmed that following a mono-therapy with glucocorticoids the AT III, protein C and protein S levels increased while the fibrinogen level decreased . As the D-Dimers remained within the normal range during the 3 weeks of L-ASP combination chemotherapy and none of the patients suffered a thromboembolic event, we also concluded that despite of the significant decrease of anticoagulant proteins, there might be a balance between coagulation and fibrinolysis; thus the D-Dimers may eventually serve as a helpful indicator for therapeutic interventions. J Korean Med Sci, 2003 Dec, 18(6), 869 - 75 Effects of dopamine infusion on cerebral blood flow, brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet; Park WS et al.; In the present study, we tested whether maintenance of adequate cerebral perfusion pressure (CPP) by pharmacologically preventing systemic hypotension with dopamine infusion would prevent cerebral ischemia and attenuate energy depletion and neuronal injury even though intracranial pressure remains elevated in a newborn piglet meningitis model . Cerebral blood flow, measured at the end of the experiment using fluorescent microspheres, was significantly increased by dopamine infusion . The decreased cerebral cortical cell membrane Na+, K+ -ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain damage, were significantly attenuated by dopamine infusion . Dopamine also significantly attenuated the meningitis-induced reduction in both brain ATP and phosphocreatine levels and the increase in brain lactate level . In summary, maintenance of adequate CPP with dopamine prevented cerebral ischemia, reduced cerebral energy depletion, and attenuated brain injury in neonatal bacterial meningitis. J Biol Chem, 2004 Feb 20, 279(8), 6315 - 26 Epub 2003 Dec 04. Functional analysis of multiple single-stranded DNA-binding proteins from Methanosarcina acetivorans and their effects on DNA synthesis by DNA polymerase BI; Robbins JB et al.; Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination . We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans . RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain . Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers . Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M . acetivorans RPAs can act as a distinct single-stranded DNA-binding protein . Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M . acetivorans RPAs bind to as few as 10 single-stranded DNA bases . However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 +/- 0.28 nM, 173.6 +/- 105.17 nM, and 5.92 +/- 0.23 nM, for RPA1, RPA2, and RPA3, respectively . The architectures of the M . acetivorans RPAs are different from those of hitherto reported homologs . Thus, these proteins may represent novel forms of replication protein A . Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M . acetivorans DNA polymerase BI . Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon. J Biol Chem, 2004 Mar 5, 279(10), 8769 - 78 Epub 2003 Dec 15. Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth; Cha MK et al.; To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases . p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide . Furthermore, p20 exerted the most potent lipid peroxidase activity . Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress . Expression level of p20 during anaerobic growth was sustained at the approximately 50% level compared with that of the aerobic growth . Viability of aerobic p20Delta without glucose was reduced to the approximately 65% level of isogenic strains, whereas viability of aerobic p20Delta with 0.5% glucose supplement was sustained . The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth . At the stationary phase, the viability of p20Delta was down to approximately 10% level of parent strains . An analysis of the protein carbonyl contents of p20Delta as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20Delta was caused by cumulative oxidative stress . P20Delta showed hypersensitivity toward membrane-soluble organic hydroperoxides . An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20Delta demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane . Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats. FEBS Lett, 2003 Dec 18, 555(3), 521 - 7 Ternary complex formation between DNA-adenovirus core protein VII and TAF-Ibeta/SET, an acidic molecular chaperone; Haruki H et al.; The adenovirus (Ad) genome complexed with viral core proteins designated Ad core is the template for transcription of early genes and the first round of replication in Ad-infected cells . A cellular protein designated template-activating factor-I (TAF-I) is found to be involved in remodeling of the Ad core in vitro . Here we found that TAF-I interacts with the Ad DNA through core protein VII in infected cells in early phases of infection . In vitro binding assays using recombinant proteins showed that TAF-I forms ternary complexes with DNA-protein VII complexes. FEBS Lett, 2003 Dec 18, 555(3), 443 - 8 Evidence for a subgroup of thioredoxin h that requires GSH/Grx for its reduction; Gelhaye E et al.; Poplar thioredoxin h4 (popTrxh4) and a related CXXS type (popCXXS3) are both members of a plant thioredoxin h subgroup . PopTrxh4 exhibits the usual catalytic site WCGPC, whereas popCXXS3 harbors the non-typical active site WCMPS . Recombinant popTrxh4 and popCXXS3 are not reduced either by Arabidopsis thaliana NADPH-dependent thioredoxin reductases (NTR) A and B or by Escherichia coli NTR . We report here evidence that a poplar glutaredoxin as well as three E . coli Grxs are able to reduce popTrxh4 . PopTrxh4 is able to reduce several thioredoxin targets as peroxiredoxins or methionine sulfoxide reductases . On the other hand, popCXXS3 exhibits an activity in the presence of glutathione and hydroxyethyldisulfide . Except for examples of glutathiolation, these are the first two examples of a direct interconnection between the thioredoxin and glutathione/glutaredoxin systems. Virology, 2003 Dec 5, 317(1), 73 - 83 Characterization of a chlorella virus PBCV-1 encoded ribonuclease III; Zhang Y et al.; Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30-35% amino acid identity to ribonuclease III (RNase III) from many bacteria . The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system . The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in a Mg(2+)-dependent manner, into a defined set of products . The substrate cleavage specificity overlaps, but is nonidentical to that of E . coli RNase III . The a464r gene is expressed very early during PBCV-1 infection, within 5-10 min p.i . The RNase III protein appears at 15 min p.i . and disappears by 120 min p.i . The a464r gene is highly conserved among the chlorella viruses . Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniae RNase III. Plant J, 2004 Jan, 37(1), 61 - 72 Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants; Ottenhof HH et al.; Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions . Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively . Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR . The cDNAs were able to complement their respective E . coli auxotrophs, demonstrating that they encoded functional enzymes . Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy . The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol . This implies that there must be transporters for pathway intermediates . KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures . We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E . coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR) . No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E . coli ADC was compared to all the annotated proteins in Arabidopsis . ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria . In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue. Plant J, 2004 Jan, 37(1), 1 - 8 Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor; Cho EM et al.; We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor . OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins . The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves . In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves . The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice. Biochemistry, 2003 Dec 23, 42(50), 15003 - 8 X-ray absorption spectroscopic analysis of reductive {2Fe-2S} cluster degradation in hyperthermophilic archaeal succinate:caldariellaquinone oxidoreductase subunits; Li Z et al.; The biological {2Fe-2S} clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya . The two recombinant hyperthermophilic archaeal {2Fe-2S} cluster-binding proteins, SdhC and the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite . We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible {2Fe-2S} cluster degradation process . Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable {2Fe-2S}(2+) cluster environments, a complete reductive breakdown of the {2Fe-2S} clusters resulted in the appearance of a new Fourier transform (FT) peak at approximately 3.3 A with a concomitant loss of the Fe-Fe interaction at ca . 2.7 A for both proteins . On the basis of the unambiguous assignment of the 3.3 A FT peak, our results suggest that a biological {2Fe-2S} cluster breakdown under reducing conditions generally releases Fe(2+) from the polypeptide chain into the aqueous solution, and the Fe(2+) might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system. Biochemistry, 2003 Dec 23, 42(50), 14877 - 84 Effect of pH on the oxidation-reduction properties of thioredoxins; Setterdahl AT et al.; Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins . These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values . At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide . Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated . The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E . coli thioredoxin and C . reinhardtii thioredoxin h. J Infect Dis, 2003 Dec 15, 188(12), 1951 - 60 Epub 2003 Dec 09. A novel recombinant antigen for immunodiagnosis of human cystic echinococcosis; Li J et al.; A pool of serum samples from mice infected with oncospheres (eggs) of Echinococcus granulosus was used to screen a cDNA library constructed with RNA extracted from protoscolex larvae from sheep hydatid cysts . One immunoreactive clone, designated EpC1, was shown to encode a protein of 76 residues . The complementary DNA (cDNA) fragment was subcloned into an expression vector, pET-41b(+), and the resulting recombinant EpC1 glutathione S-transferase (GST) fusion protein (rEpC1-GST) was expressed in Escherichia coli and was affinity purified against the GST tag . Immunoglobulin G was the dominant antibody isotypes generated against rEpC1-GST . A total of 896 human serum samples were used to evaluate the diagnostic sensitivity and specificity of the fusion protein by immunoglobulin G immunoblotting; 324 serum samples from patients with cystic echinococcosis (CE), 172 from patients with neurocysticercosis, 89 from patients with alveolar echinococcosis, and 241 from patients with other infections or clinical presentations, as well as 70 from confirmed-negative control subjects, yielded an overall sensitivity of 92.2% and an overall specificity of 95.6% . The combined levels of sensitivity and specificity achieved with the rEpC1-GST fusion protein for diagnosis of CE are unprecedented, taking into account the large panel of serum samples that were tested. J Biol Inorg Chem, 2004 Mar, 9(2), 161 - 70 Epub 2003 Dec 13. Effect of phosphate on bacterioferritin-catalysed iron(II) oxidation; Aitken-Rogers H et al.; The iron(III) mineral cores of bacterioferritins (BFRs), as isolated, contain a significant component of phosphate, with an iron-to-phosphate ratio approaching 1:1 in some cases . In order to better understand the in vivo core-formation process, the effect of phosphate on in vitro core formation in Escherichia coli BFR was investigated . Iron cores reconstituted in the presence of phosphate were found to have iron-to-phosphate ratios similar to those of native cores, and possessed electron paramagnetic resonance properties characteristic of the phosphate-rich core . Phosphate did not affect the stoichiometry of the initial iron(II) oxidation reaction that takes place at the intrasubunit dinuclear iron-binding sites (phase 2 of core formation), but did increase the rate of oxidation . Phosphate had a more significant effect on subsequent core formation (the phase 3 reaction), increasing the rate up to five-fold at pH 6.5 and 25 degrees C . The dependence of the phase 3 rate on phosphate was compl