Biosci Biotechnol Biochem, 2004 Mar, 68(3), 615 - 21 Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis dehydratase inhibitor (MBI-D) resistant strains of the rice blast fungus; Yamada N et al.; Carpropamid ((1RS,3SR)-2,2-dichloro-N-{(R)-1-(4-chlorophenyl)ethyl}-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae . In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid . The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide . We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers . The variant enzyme retained a significant level of enzymatic activity . Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type . Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity. Biosci Biotechnol Biochem, 2004 Mar, 68(3), 477 - 87 SigC, the group 2 sigma factor of RNA polymerase, contributes to the late-stage gene expression and nitrogen promoter recognition in the cyanobacterium Synechocystis sp . strain PCC 6803; Asayama M et al.; We examined the role of SigC (Sll0184), a sigma factor of RNA polymerase (RNAP), in a unicellular cyanobacterium, Synechocystis sp . strain PCC 6803 . On the inactivation of sigC, which is an Escherichia coli rpoD homolog, cells were viable but had a low survival rate in the stationary phase of growth under normal physiological conditions, indicating that SigC is a group 2 type sigma factor . In analyses of transcript and protein levels using the sigC knockout strain, it was found that expression of glnB, a nitrogen key regulatory gene, is controlled by SigC in the stationary phase . Primer extension revealed that the glnB nitrogen promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of nitrogen starvation . In vitro studies with purified enzymes indicated effective transcription, on supercoiled DNA templates, from P2 by SigC-RNAP with NtcA which is an activator for nitrogen gene transcription . DNase I footprinting also indicated binding and related sites of NtcA and/or RNAP with SigC on the nitrogen promoter . The unique promoter architecture and the mechanism of transcription by RNAP with SigC are also discussed. Mol Endocrinol, 2004 Jun, 18(6), 1363 - 75 Epub 2004 Mar 31. The transcriptional repressor Nkx6.1 also functions as a deoxyribonucleic acid context-dependent transcriptional activator during pancreatic beta-cell differentiation: evidence for feedback activation of the nkx6.1 gene by Nkx6.1; Iype T et al.; In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes . Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself . In this report, we identify a novel beta-cell-specific enhancer element in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches . Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element . We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1 . We show by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in betaTC3 cells . We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events. Cell Microbiol, 2004 May, 6(5), 447 - 58 Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit; McSweeney LA et al.; Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens . Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis . Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin . Mutant CdtBs lacking DNase activity are devoid of biological activity . Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity . Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS) . Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II . Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells . Our results indicated that EGFP-EcCdtB-II localized to the nucleus . The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively . Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants . When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II . These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II. Biochem J, 2004 Jul 15, 381(Pt 2), 489 - 94 Human hereditary glutathione synthetase deficiency: kinetic properties of mutant enzymes; Njalsson R et al.; Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction . To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations {L188P (Leu188-->Pro), D219A, D219G, Y270C, Y270H, R283C and P314L} were expressed using a His-tagged, Escherichia coli-based expression system . Effects of the mutations on kinetic properties, including negative co-operative binding of gamma-glutamyl substrate, were evaluated . The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation . The remaining mutations decreased V(max) to 2-27% of wild-type activity . Negative co-operativity for gamma-gluABA (L-gamma-glutamyl-L-alpha-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive . The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme. Anal Sci, 2004 Feb, 20(2), 329 - 34 Use of Escherichia coli immobilized on amberlite XAD-4 as a solid-phase extractor for metal preconcentration and determination by atomic absorption spectrometry; Turker AR et al.; A sensitive solid-phase extraction technique (SPE) for the enrichment of Fe(III), Co(II), Mn(II) and Cr(III) prior to atomic absorption spectrometric analysis is described . Escherichia coli immobilized on Amberlite XAD-4 was used as a solid-phase extractor . The effects of the pH, amount of solid-phase, eluent type and volume of the sample solution on the recovery of the metal ions were investigated . The effect of diverse ions was also investigated . The recoveries of Fe(III), Co(II), Mn(II) and Cr(III) under the optimum conditions were found to be 99 +/- 2, 99 +/- 3, 98 +/- 2, 98 +/- 3%, respectively, at the 95% confidence level . The detection limits of the metal ions were found as to be 2.4, 3.8, 1.3 and 1.7 ng ml(-1) for Fe(II), Co(II), Mn(II) and Cr(III) respectively, by applying a preconcentration factor of 25 . The proposed enrichment method was applied to the determination of analytes in Ataturk Dam water samples, and alloy samples (RSD < 5%) . The accuracy of the method was verified on certified alloy samples (NBS SRM 85b and NBS SRM 59a) . The analytes were determined with a relative error lower than 5% in water and alloy samples. Mol Cells, 2004 Feb 29, 17(1), 62 - 6 Formation of an alpha-helix in human tumor necrosis factor-alpha by guanidine hydrochloride-induced unfolding; Jeong W et al.; Human tumor necrosis factor-alpha (TNF-alpha) is a trimeric protein consisting primarily of beta-sheet . GdnHCl-induced unfolding of TNF-alpha was investigated at room temperature by circular dichroism (CD) and size exclusion chromatography . The secondary and tertiary structure of TNF-alpha persisted up to 0.9N GdnHCl regardless of incubation time, but, in the range of 1.2 N to 2.1 N GdnHCl, there was loss of tertiary structure accompanied by the formation of an alpha-helix, as revealed by far- and near-UV CD spectra . The structural changes occurred gradually in 1.2 and 2.1 N GdnHCl, but were rapid in 1.5 and 1.8 N GdnHCl . The GdnHCl-induced state of TNF-alpha is an unfolded, alpha-helical aggregate of about 130 monomers, as shown by size exclusion chromatography . We suggest the most likely pathway for the transition from beta-sheet to alpha-helix. J Biol Chem, 2004 Jun 4, 279(23), 24552 - 60 Epub 2004 Mar 31. Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct; Gomez-Pinto I et al.; The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics . This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one . The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants . The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions . These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation . Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide . Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein. Biopolymers, 2004, 76(2), 119 - 28 Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure; Naider F et al.; The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells . The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors . The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure . Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments . Extension of these studies requires preparation of longer fragments of Ste2p . This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p . Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation . Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain . Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches . Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences . Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions . Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis . Appl Biochem Biotechnol, 2004 Spring, 113-116, 469 - 83 Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values; Penna TC et al.; The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied . The GFPuv (3.5-9.0 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees C) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0) . The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv . For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl . At 85 degrees C, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) . The stability of GFPuv in Tris-HCl (pH >8.0) was constant at 90 and 95 degrees C, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively . The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100 degrees C. Appl Biochem Biotechnol, 2004 Spring, 113-116, 373 - 9 Biosynthesis of R-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli; Park SJ et al.; An efficient system for the production of (R)-hydroxyalkanoic acids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli . Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs . In recombinant E . coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA . When the recombinant E . coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers . R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment . A stable recombinant E . coli strain in which the R . eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB . When the R . eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced. Appl Biochem Biotechnol, 2004 Spring, 113-116, 287 - 97 Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca; Escovar-Kousen JM et al.; Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material . Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities . Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4) . Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties . Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation . In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling . A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model . Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD) . The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected . Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-alpha, and the QuickChange mutagenesis method . E . coli BL21-DE3 was used for protein production and expression . The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC) . Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose. Appl Biochem Biotechnol, 2004 Spring, 113-116, 233 - 50 Properties of a recombinant beta-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis; Li XL et al.; A beta-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced . The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial beta-glucosidases but lacked significant similarity to those from aerobic fungi . Neither cellulose- nor protein-binding domains were found in BglA . When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells . Km and Vmax values of the secreted BglA were 0.762 mM and 8.20 micromol/(min x mg), respectively, with p-nitrophenyl-beta-D-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 micromol/(min.mg), respectively, for the hydrolysis of cellobiose . Glucose competitively inhibited the hydrolysis of pNPG with a Ki of 3.6 mM . Beta-glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production. J Biol Chem, 2004 Jun 11, 279(24), 25364 - 73 Epub 2004 Mar 30. Functional characterization of a second porin isoform in Drosophila melanogaster . DmPorin2 forms voltage-independent cation-selective pores; Aiello R et al.; Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane . Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes . No functional information was available for the different gene products . In this work we have studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2) . Its coding sequence was expressed in Escherichia coli . The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes . Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective . Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2 . We have mutated two of them (Glu-66 and Glu-163) to lysines to investigate their role in the functional features of the pore . The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity . Both single mutations partially restore the voltage dependence of the pore . We found that an additional effect with the double mutant E66K/E163K was the restoration of voltage dependence . Protein structure predictions highlight a 16 beta-strand pattern, typical for porins . In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides . DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested . Our main conclusions are as follows . 1) The CG17137 gene may express a porin with a functional role in D . melanogaster . 2) We have identified two amino acids of major relevance for the voltage dependence of the porin pore. Clin Chem, 2004 Jun, 50(6), 988 - 95 Epub 2004 Mar 30. Antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein; Chen Z et al.; BACKGROUND: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use . For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein . METHODS: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli . After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments . RESULTS: The full-length protein and two other fragments reacted with all 52 sera tested . Four important regions with possible epitopes were identified and named as EP1 (amino acids 51-71), EP2 (134-208), EP3 (249-273), and EP4 (349-422), respectively . EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera . EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested . CONCLUSION: The N protein is a highly immunogenic protein of the SARS-CoV . Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH(2) terminus . The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV. Am J Public Health, 2004 Apr, 94(4), 565 - 7 Health effects associated with recreational coastal water use: urban versus rural California; Dwight RH et al.; We compared rates of reported health symptoms among surfers in urban North Orange County (NOC) and rural Santa Cruz County (SCC), California, during 2 winters (1998 and 1999) to determine whether symptoms were associated with exposure to urban runoff . NOC participants reported almost twice as many symptoms as SCC participants during the 1998 winter . In both study years, risk increased across symptom categories by an average of 10% for each 2.5 hours of weekly water exposure . Our findings suggest that discharging untreated urban runoff onto public beaches can pose health risks. Mol Cell, 2004 Mar 26, 13(6), 829 - 41 Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition; Swairjo MA et al.; Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs . A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant . Further understanding has been limited due to lack of a crystal structure of the enzyme . We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS . It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold . Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data . It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s. Parasite Immunol, 2003 Nov-Dec, 25(11-12), 531 - 43 Antigenic cross-reactivity between different alleles of the Plasmodium falciparum merozoite surface protein 2; Felger I et al.; The polymorphic domain of the gene encoding Plasmodium falciparum merozoite surface protein 2 (MSP2) was PCR amplified from blood of malaria patients, genotyped, and 19 distinct fragments were cloned and expressed in E . coli . The reactivity of naturally occurring antibodies against this panel of recombinant MSP2 antigens was tested using 67 homologous or heterologous sera from a serum bank of travel clinic patients . Sera from semi-immune individuals strongly recognized almost all recombinant antigens . Sera from primary infected patients either did not react at all (9 sera), or reacted weakly against varying numbers of antigens (39 sera) . The antigens that showed reactions were mostly of the allelic family corresponding to the infecting clone, but in very few cases also of the alternative allelic family . Single clone infections and repeated samples from the same individual were analysed in greater detail . Thus, we were able to quantify cross-reactivity induced by a single P . falciparum infection . Within the two allelic families of MSP2, cross-reactivity was observed between some but not all alleles of the same family, whereas antibodies cross-reactive between variants belonging to different allelic families were detected in only a few cases. Plant J, 2004 Apr, 38(1), 80 - 92 Molecular regulation of sinapate ester metabolism in Brassica napus: expression of genes, properties of the encoded proteins and correlation of enzyme activities with metabolite accumulation; Milkowski C et al.; Members of the Brassicaceae family accumulate specific sinapate esters, i.e . sinapoylcholine (sinapine), which is considered as a major antinutritive compound in seeds of important crop plants like Brassica napus, and sinapoylmalate, which is implicated in UV-B tolerance in leaves . We have studied the molecular regulation of the sinapate ester metabolism in B . napus, and we describe expression of genes, some properties of the encoded proteins and profiles of the metabolites and enzyme activities . The cloned cDNAs encoding the key enzymes of sinapine biosynthesis, UDP-glucose (UDP-Glc):B . napus sinapate glucosyltransferase (BnSGT1) and sinapoylglucose:B . napus choline sinapoyltransferase (BnSCT), were functionally expressed . BnSGT1 belongs to a subgroup of plant GTs catalysing the formation of 1-O-hydroxycinnamoyl-beta-d-glucoses . BnSCT is another member of serine carboxypeptidase-like (SCPL) family of acyltransferases . The B . napus genome contains at least two SGT and SCT genes, each derived from its progenitors B . oleracea and B . rapa . BnSGT1 and BnSCT activities are subjected to pronounced transcriptional regulation . BnSGT1 transcript level increases throughout early stages of seed development until the early cotyledonary stage, and stays constant in later stages . The highest level of BnSGT1 transcripts is reached in 2-day-old seedlings followed by a dramatic decrease . In contrast, expression of BnSCT is restricted to developing seeds . Regulation of gene expression at the transcript level seems to be responsible for changes of BnSGT1 and BnSCT activities during seed and seedling development of B . napus . Together with sinapine esterase (SCE) and sinapoylglucose:malate sinapoyltransferase (SMT), activities of BnSGT1 and BnSCT show a close correlation with the accumulation kinetics of the corresponding metabolites. J Am Chem Soc, 2004 Apr 7, 126(13), 4157 - 66 The nature of the exchange coupling between high-spin Fe(III) heme o3 and CuBII in Escherichia coli quinol oxidase, cytochrome bo3: MCD and EPR studies; Cheesman MR et al.; Fully oxidized cytochrome bo3 from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies . In each form, the spin-coupled high-spin Fe(III) heme o3 and CuB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra . Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of J 1 cm-1 . A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo3 fluoride and azide derivatives . New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b . Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with J approximately 1 cm-1 . These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S' = 2 ground state resulting from strong exchange coupling (J > 10(2) cm-1) within the active site. J Agric Food Chem, 2004 Apr 7, 52(7), 1950 - 7 Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the flavedo of fruit but responsive to cold and water stress in leaves; Sanchez-Ballesta MT et al.; A cDNA encoding a dehydrin was isolated from the flavedo of the chilling-sensitive Fortune mandarin fruit (Citrus clementina Hort . Ex Tanaka x Citrus reticulata Blanco) and designed as Crcor15 . The predicted CrCOR15 protein is a K2S member of a closely related dehydrin family from Citrus, since it contains two tandem repeats of the unusual Citrus K-segment and one S-segment (serine cluster) at an unusual C-terminal position . Crcor15 mRNA is consistently and highly expressed in the flavedo during fruit development and maturation . The relative abundance of Crcor15 mRNA in the flavedo was estimated to be higher than 1% of total RNA . The high mRNA level remained unchanged during fruit storage at chilling (2 degrees C) and nonchilling (12 degrees C) temperatures, and it was depressed by a conditioning treatment (3 days at 37 degrees C) that induced chilling tolerance . Therefore, the expression of Crcor15 appears not to be related to the acquisition of chilling tolerance in mandarin fruits . However, Crcor15, which was barely detected in unstressed mandarin leaves, was rapidly induced in response to both low temperature and water stress . COR15 protein was expressed in Escherichia coli, and the purified protein conferred in vitro protection against freezing and dehydration inactivation . The potential role of Citrus COR15 is discussed. Curr HIV Res, 2004 Jan, 2(1), 1 - 10 Mucosal HIV vaccines: where are we now? Stevceva L, Strober W. Around the world, approximately 5 million people became infected with HIV in 2001, an estimated 70% via sexual transmission . Numerous studies have demonstrated that it is difficult to achieve total protection from vaginally or rectally acquired HIV/SIV when using parenteral immunization . Mucosal immunization was seen as the best approach to achieve sustainable immune responses at mucosal sites of viral entry . This was further emphasized when several studies implicated rectal and vaginal mucosa as latent reservoirs for the HIV virus and virus-specific CD8+ T cell immune responses in gastrointestinal mucosa were shown to be less efficient than in systemic tissues . Mucosal vaccines utilizing various routes of immunization including intranasal, intrarectal, intravaginal and oral immunization have been tested for their potency to induce virus-specific immune responses systemically but especially at mucosal sites of viral entry . The unsatisfactory results in initiating simultaneously sufficient immune responses at mucosal and systemic sites are being overcomed by use of appropriate and novel adjuvants such as Cholera toxin, Escherichia coli heat-labile toxin, immunostimulatory CpG motifs, coinjection of cytokines and others . Various routes of immunization are now being compared and combinations of mucosal immunization and parenteral boost and vice versa have also been tested . Generations of new vaccines, such as DNA-based vaccines, multipeptide, lipopeptide and alphavirus replicon particles-based vaccines have been created and studied for their efficiency. Biotechnol Bioeng, 2004 Apr 20, 86(2), 196 - 200 Screening of large protein libraries by the cell immobilized on adsorbed bead approach; Freeman A et al.; Screening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development . Currently, most frequently employed methods are labor-intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library . In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates . This new screening method is based on the "cell immobilized on adsorbed bead" approach: the cell population to be screened is mixed with an excess of medium pre-equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption . The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support . After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix . These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies . This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous "naturally immobilized" enzyme population with minimal substrate diffusion limitations . Biotechnol Bioeng, 2004 Apr 20, 86(2), 188 - 95 Enhancing multiple disulfide bonded protein folding in a cell-free system; Yin G et al.; A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system . Due to the unstable and reducing environment in the initial E . coli-based cell-free system, disulfide bonds could not be formed efficiently . By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized . Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E . coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA . Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction . Biotechnol Bioeng, 2004 Apr 20, 86(2), 149 - 62 Fundamental Escherichia coli biochemical pathways for biomass and energy production: creation of overall flux states; Carlson R et al.; We have previously shown that the metabolism for most efficient cell growth can be realized by a combination of two types of elementary modes . One mode produces biomass while the second mode generates only energy . The identity of the four most efficient biomass and energy pathway pairs changes, depending on the degree of oxygen limitation . The identification of such pathway pairs for different growth conditions offers a pathway-based explanation of maintenance energy generation . For a given growth rate, experimental aerobic glucose consumption rates can be used to estimate the contribution of each pathway type to the overall metabolic flux pattern . All metabolic fluxes are then completely determined by the stoichiometries of involved pathways defining all nutrient consumption and metabolite secretion rates . We present here equations that permit computation of network fluxes on the basis of unique pathways for the case of optimal, glucose-limited Escherichia coli growth under varying levels of oxygen stress . Predicted glucose and oxygen uptake rates and some metabolite secretion rates are in remarkable agreement with experimental observations supporting the validity of the presented approach . The entire most efficient, steady-state, metabolic rate structure is explicitly defined by the developed equations without need for additional computer simulations . The approach should be generally useful for analyzing and interpreting genomic data by predicting concise, pathway-based metabolic rate structures . J Appl Toxicol, 2004 Mar-Apr, 24(2), 99 - 106 Glioma cell antioxidant capacity relative to reactive oxygen species produced by dopamine; Mazzio EA et al.; The pathology of Parkinson's disease involves oxidative damage to dopaminergic neurons of the substantia nigra . Oxidation of the dopamine (DA) neurotransmitter itself may contribute to the generation of a reactive oxygen species (ROS) and subsequent neurodegeneration . Glia cells can either exacerbate injury or exert protective properties on local neurons in the brain . We investigate glial antioxidant enzyme systems relative to ROS generated during cytokine activation, monoamine oxidase (MAO) activity and autoxidation of DA in glioma cells . Rat C6 glioma cells stimulated with lipopolysaccharide Escherichia coli 0111:B4 and interferon gamma (LPS/IFN-g) produced high levels of nitric oxide (241 nmol mg(-1) protein 24 h(-1)) but not superoxide (O(-) (2)) or hydrogen peroxide (H(2)O(2)) . Basal C6 cells exhibited a rapid and robust capacity to remove exogenous H(2)O(2) within minutes . Preincubation with sodium azide but not buthionine-{S, R}-sulfoximine attenuated this response, indicating catalase as the primary enzyme responsible for this effect . The glioma catalase reaction rate was slightly attenuated by exposure to LPS/IFN-g for 24 h . However, the reduction in catalase activity was not due to nitric oxide, because both the supernatant and sodium nitroprusside had no effect on isolated catalase enzyme activity . Hydrogen peroxide was produced only through substrate-driven MAO activity in prepared lysate . However, the quantity of H(2)O(2) produced per unit time (0.46 nmol mg(-1) protein min(-1)) was negligible compared with the enormous capacity for its removal by catalase (213.9 nmol mg(-1) protein min(-1)) (> or =462 x greater) . Similarly, H(2)O(2) generated by DA autoxidation per unit time (0.28 nmol mg(-1) protein equiv . min(-1)), was rapidly dissolved by glioma cells at high capacity (> or =750 x greater) . In conclusion, C6 cells produce nitric oxide under cytokine/endotoxin-stimulated conditions . Moreover, C6 cells exhibit a dynamic H(2)O(2) scavenging capacity, with ample facility to dispose of the peroxide generated by both MAO activity and spontaneous DA autoxidation . Lab Chip, 2004 Apr, 4(2), 141 - 7 Epub 2004 Feb 04. Microfluidic chip for high efficiency DNA extraction; Chung YC et al.; A high efficiency DNA extraction microchip was designed to extract DNA from lysed cells using immobilized beads and the solution flowing back and forth . This chip was able to increase the extraction efficiency by 2-fold when there was no serum . When serum existed in the solution, the extraction efficiency of immobilized beads was 88-fold higher than that of free beads . The extraction efficiency of the microchip was tested under different conditions and numbers of E . coli cells . When the number of E . coli cells was between 10(6) and 10(8) in 25 microl of whole blood, the extraction efficiency using immobilized beads was only slightly higher than that using free beads (10(0) to 10(1) fold) . When the number of E . coli cells was in the range 10(4) to 10(6) in 25 microl of whole blood, the extraction efficiency of immobilized beads was greater than that of the free beads (10(1) to 10(2) fold) . When the number of E . coli cells was lower, in the range 10(3) to 10(4) in 25 microl of whole blood, the extraction efficiency of immobilized beads was much higher than that of the free beads (10(2) to 10(3) fold) . This study indicated that DNA could be efficiently extracted even when the number of bacterial cells was smaller (10(5) to 10(3)) . This microfluidic extraction chip could find potential applications in rare sample genomic study. Lab Chip, 2004 Apr, 4(2), 104 - 8 Epub 2004 Mar 10. Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement; Lin YC et al.; Simulation and experimental demonstration of the in vitro gene delivery enhancement using electrostatic forces and electroporation (EP) microchips were conducted . Electroporation is a technique with which DNA molecules can be delivered into cells using electric field pulses . This study demonstrates that plasmid DNA can be attracted to the cell surfaces at the specific regions using an electrostatic force . Therefore, the DNA concentration on the cell surface is dramatically increased, which highly enhances the gene transfection efficiency compared to that without an attracting-electric field . The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to, to provide the region-targeting function . In this micro-device, the top electrode and the interdigitated electrodes provided the DNA attracting-electric field, and the interdigitated electrodes provided adequate electric fields for the electroporation process on the chip surface . Using the EP microchip, cells could be manipulated in situ without detachment if adherent cells were used for electroporation . Five different cells of two different types, primary cell and cell line, were successfully transfected under multi-pulse or single pulse electric field stimulation without applying an attracting-electric field . This study simulated and analyzed the electric field distributions at the DNA attracting and electroporation processes, and successfully demonstrated that the electrostatic force attracted DNA plasmids to specific regions and highly enhanced the gene delivery . In summary, this EP microchip should provide many potential applications for gene therapy. Lab Chip, 2004 Apr, 4(2), 83 - 6 Epub 2004 Feb 27. Motor-protein "roundabouts": microtubules moving on kinesin-coated tracks through engineered networks; Clemmens J et al.; Nanotechnology promises to enhance the functionality and sensitivity of miniaturized analytical systems . For example, nanoscale transport systems, which are driven by molecular motors, permit the controlled movement of select cargo along predetermined paths . Such shuttle systems may enhance the detection efficiency of an analytical system or facilitate the controlled assembly of sophisticated nanostructures if transport can be coordinated through complex track networks . This study determines the feasibility of complex track networks using kinesin motor proteins to actively transport microtubule shuttles along micropatterned surfaces . In particular, we describe the performance of three basic structural motifs: (1) crossing junctions, (2) directional sorters, and (3) concentrators . We also designed track networks that successfully sort and collect microtubule shuttles, pointing the way towards lab-on-a-chip devices powered by active transport instead of pressure-driven or electroosmotic flow. Can J Microbiol, 2004 Feb, 50(2), 133 - 6 Over-expression of rexA nullifies T4rII exclusion in Escherichia coli K(lambda) lysogens; Slavcev RA et al.; Dosage and relative cellular levels of RexA and RexB proteins encoded by the rexA-rexB genes of a lambda prophage are important for the Rex+ phenotype, which was nullified when greater RexA or RexB was provided than was necessary for the complementation of a rexA- or a rexB- prophage. J Clin Oncol, 2004 May 1, 22(9), 1546 - 52 Epub 2004 Mar 29. Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer; Palmer DH et al.; PURPOSE: Virus-directed enzyme prodrug therapy depends on selective delivery of virus encoding a prodrug-activating enzyme to tumor, followed by systemic treatment with prodrug to achieve high levels of the activated cytotoxic at the intended site of action . The use of the bacterial enzyme nitroreductase to activate CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a short lived, highly toxic DNA cross-linking agent has been demonstrated in tumor xenografts . In this study, we report the first clinical trial investigating the feasibility, safety, and transgene expression of a replication-defective adenovirus encoding nitroreductase (CTL102) in patients with liver tumors . PATIENTS AND METHODS: Patients with resectable primary or secondary (colorectal) liver cancer received a single dose of CTL102 delivered by direct intratumoral inoculation 3 to 8 days before surgical resection . RESULTS: Eighteen patients were treated with escalating doses of CTL102 (range, 10(8)-5 x 10(11) virus particles) . The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response . Dose-related increases in tumoral nitroreductase expression measured by immunohistochemical analysis have been observed . CONCLUSION: Direct intratumoral inoculation of CTL102 to patients with primary and secondary liver cancer is feasible and well tolerated . The high level of nitroreductase expression observed at 1 to 5 x 10(11) virus particles mandates further studies in patients with inoperable tumors who will receive CTL102 and CB1954. Mol Aspects Med, 2004 Feb-Apr, 25(1-2), 141 - 54 Nitric oxide and iron: effect of iron overload on nitric oxide production in endotoxemia; Galleano M et al.; The amount of iron within the cell is carefully regulated in order to provide an adequate level of micronutrient while preventing its accumulation and toxicity . Iron excess is believed to generate oxidative stress, understood as an increase in the steady-state concentration of oxygen radical intermediates . Nitric oxide (NO) is an inorganic free-radical gaseous molecule which has been shown over the last decade to play an unprecedented variety of roles in biological systems . The effect of nitrogen reactive species may explain the iron sequestration pattern that characterizes macrophages under inflammatory conditions . From a patho-physiological viewpoint, further studies are required to assess the usefulness of this mechanism to minimize formation and release of free radicals in diseased tissues . However, contrary to the deleterious effects of the reactive nitrogen oxide species formed from either NO/O(2) and NO/O(2)(-), it has been pointed out that NO shows antioxidant properties . A number of studies have described the complex relationships between iron and NO, but controversy remains as to the influence and significance of iron on inflammatory NO production . To explore the initial steps of the effects triggered by LPS administration in the presence of excess iron, male Wistar rats were treated with: lipopolysaccharide from Escherichia coli (serotype 0127:B8) (LPS); iron-dextran; or iron-dextran plus LPS and liver samples were taken after 6 h . EPR spectra of NO-Hb in the venous blood were determined at 77 K . Iron-dextran administered to rats intraperitoneally resulted predominantly in iron uptake by the liver Kupffer cells and led to an increased NO level in blood in the presence of LPS . Further studies will be required to assess the complex role of the Kupffer cells on iNOS induction and NO production. Hear Res, 2004 Apr, 190(1-2), 25 - 30 Morphology of auditory hair cells in guinea pig cochlea after transgene expression; Han D et al.; It is very important to determine if recombinant adenoviral vector (Ad) can damage the auditory hair cells in guinea pig cochlea after transgene expression . In this study, the scanning electron microscope was used to determine if there was loss of the auditory hair cells after Ad.LacZ (Ad5 containing Escherichia coli galactosidase) was inoculated into the cochlea through the round window membrane . Seven days later all inner and outer hair cells were found to express the LacZ gene . Except for the sparse loss of outer hair cells in the basal turn and the second turn, there was insignificant loss in the other turns . All inner hair cells were present . The damage to auditory hair cells resulting from intracochlear inoculation of Ad is limited, and this vector can be used as one of the ideal delivery tools in gene therapy of the cochlea. Mol Genet Metab, 2004 Apr, 81 Suppl 1, S4 - 11 Mammalian N-acetylglutamate synthase; Morizono H et al.; N-Acetylglutamate synthase (NAGS, E.C . 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) . The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries . These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay . The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus . The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified . Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway . Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity . We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible . A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu). Comp Biochem Physiol C Toxicol Pharmacol, 2004 Feb, 137(2), 115 - 22 Expression and characterization of ARSP1 from Eisenia fetida; Zhang L et al.; To study the characterization of a protease ARSP1 (apoptosis-related serine protease) of Eisenia fetida, a recombinant ARSP1 was constructed . ARSP1 was produced in E . coli BL21-CodonPlus (DE3)-RIL after IPTG induction and exited in inclusion body . After refolding in vitro, the protein was purified by DEAE-Sepharose F.F . and Sephacryl S-100 chromatography in sequence . ARSP1 showed high sequence identity to other chymotrypsin-like serine proteases and the catalytic triad was His41-Asp90-Ser188 . ARSP1 could degrade casein following Michaelis-Menten kinetics with a Vmax of 43.9 U/mg protein and a Km for casein of 0.83 g/l . Studies with inhibitors indicated that ARSP1 was a chymotrypsin-like serine protease . Experiments in vitro demonstrated that ARSP1 could not only hydrolyze fibrinogen and fibrin directly, but also activate plasminogen to plasmin . ARSP1 inhibited thrombin activity and ADP-induced platelet aggregation in a dose-response correlation . These results showed that ARSP1 has thrombolytic activity and also an anti-thrombus function. J Mol Biol, 2004 Apr 16, 338(1), 127 - 37 Crystal structure of IscA, an iron-sulfur cluster assembly protein from Escherichia coli; Cupp-Vickery JR et al.; IscA, an 11 kDa member of the hesB family of proteins, binds iron and {2Fe-2S} clusters, and participates in the biosynthesis of iron-sulfur proteins . We report the crystal structure of the apo-protein form of IscA from Escherichia coli to a resolution of 2.3A . The crystals belong to the space group P3(2)21 and have unit cell dimensions a=b=66.104 A, c=150.167 A (alpha=beta=90 degrees, gamma=120 degrees ) . The structure was solved using single-wavelength anomalous dispersion (SAD) phasing of a selenomethionyl derivative, and the IscA model was refined to R=21.4% (Rfree=25.4%) . IscA exists as an (alpha1alpha2)2 homotetramer with the (alpha1alpha2) dimer comprising the asymmetric unit . Cys35, implicated in Fe-S cluster assembly, is located in a central cavity formed at the tetramer interface with the gamma-sulfur atoms of residues from the alpha1 and alpha2' monomers (and alpha1'alpha2) positioned close to one another (approximately equal 7 A) . C-terminal residues 99-107 are disordered, and the exact positions of Cys99 and Cys101 could not be determined . However, computer modeling of C-terminal residues in the tetramer suggests that Cys99 and Cys101 in the alpha1 monomer and those of the alpha1' monomer (or alpha2 and alpha2') are positioned sufficiently close to coordinate {2Fe-2S} clusters between the two dimers, whereas this is not possible within the (alpha1alpha2) or (alpha1'alpha2') dimer . This symmetrical arrangement allows for binding of two {2Fe-2S} clusters on opposite sides of the tetramer . Modeling further reveals that Cys101 is positioned sufficiently close to Cys35 to allow Cys35 to participate in cluster assembly, formation, or transfer. J Mol Biol, 2004 Apr 16, 338(1), 77 - 91 Solution structure of a DNA duplex containing an alpha-anomeric adenosine: insights into substrate recognition by endonuclease IV; Aramini JM et al.; The cytotoxic alpha anomer of adenosine, generated in situ by radicals, must be recognized and repaired to maintain genomic stability . Endonuclease IV (Endo IV), a member of the base excision repair (BER) enzyme family, in addition to acting on abasic sites, has the auxiliary function of removing this mutagenic nucleotide in Escherichia coli . We have employed enzymatic, thermodynamic, and structural studies on DNA duplexes containing a central alpha-anomeric adenosine residue to characterize the role of DNA structure on recognition and catalysis by Endo IV . The enzyme recognizes and cleaves our alphaA-containing DNA duplexes at the site of the modification . The NMR solution structure of the DNA decamer duplex establishes that the single alpha-anomeric adenosine residue is intrahelical and stacks in a reverse Watson-Crick fashion consistent with the slight decrease in thermostability . However, the presence of this lesion confers significant changes to the global duplex conformation, resulting from a kink of the helical axis into the major groove and an opening of the minor groove emanating from the alpha-anomeric site . Interestingly, the conformation of the flanking base-paired segments is not greatly altered from a B-type conformation . The global structural changes caused by this lesion place the DNA along the conformational path leading to the DNA structure observed in the complex . Thus, it appears that the alpha-anomeric lesion facilitates recognition by Endo IV. J Mol Biol, 2004 Apr 16, 338(1), 33 - 41 Ribosome rescue by tmRNA requires truncated mRNAs; Ivanova N et al.; tmRNA targets ribosomes, stalled either on truncated mRNAs or on mRNAs with slowly read sense or stop codons, tags the newly synthesized peptide chains for degradation and allows for their release by a class-1 release factor . We have studied in vitro how the rate of trans-transfer of a peptide from the P-site tRNA to tmRNA and the efficiency by which tmRNA competes with peptide release factors depend on the length of the mRNA downstream from the P-site . We show that the rate and efficiency of tmRNA action decrease rapidly with increasing down stream length and approach zero when it exceeds 15 bases . We demonstrate that tmRNA action is strongly stimulated by RelE cleavage of mRNA in the A site . We conclude that tmRNA action in vivo must always be preceded by mRNA truncation, and suggest that cleavage of ribosome bound mRNAs is a common element in different bacterial stress responses. J Pept Res, 2004 Mar, 63(3), 200 - 6 Structural features of Escherichia coli heat-stable enterotoxin that activates membrane-associated guanylyl cyclase; Sato T et al.; Heat-stable enterotoxin (ST), a small peptide of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli, is the cause of acute diarrhea in infants and travelers in developing countries . ST triggers a biological response by binding to a membrane-associated guanylyl cyclase C (GC-C) which is located on intestinal epithelial cell membranes . This binding causes an increase in the concentration of cGMP as a second messenger in cells and activates protein kinase A and cystic fibrosis transmembrane conductance regulator . Here we describe the crystal structure of an ST at 0.89 A resolution . The molecule has a ring-shaped molecular architecture consisting of six peptide molecules with external and internal diameters of approximately 35 and 7 A, respectively and a thickness of approximately 11 A . The conserved residues at the central portion of ST are distributed on the outer surface of the ring-shaped peptide hexamer, suggesting that the hexamer may be implicated in the association with GC-C through these invariant residues. Mol Microbiol, 2004 Apr, 52(1), 243 - 56 Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli; Yang J et al.; The tyrP gene of Escherichia coli encodes a tyrosine specific transporter . Its synthesis is repressed by tyrosine but is activated by phenylalanine and to a lesser extent by tryptophan . Both of these effects are mediated by the TyrR protein when it binds to one or both of its cognate binding sites (TyrR boxes) which encompass nucleotides -30 to -75 . Activation in the presence of phenylalanine or tryptophan involves a dimer binding to the upstream box and interacting with the alpha subunit (alphaCTD) of RNA polymerase (RNAP) . Repression in the presence of tyrosine involves a hexamer binding to both TyrR boxes . The molecular basis for this repression has been studied in vitro . Whereas initial gel shift experiments fail to show the exclusion of RNAP from the promoter region when TyrR hexamer is bound, a DNase I analysis of slices from the gel shows that in the presence of TyrR, RNAP now binds to a previously unrecognized upstream promoter . Although this upstream promoter is bound strongly by RNAP and forms an open complex on linear DNA templates, it fails to form an open complex on supercoiled templates in vitro and is unable to initiate transcription in vivo . A subsequent gel shift assay using a tyrP fragment which eliminates the upstream RNAP binding site confirms conclusively that, in the presence of tyrosine and ATP, the TyrR protein prevents RNAP from binding to the tyrP promoter . In vitro studies have also been carried out in the presence of TyrR protein and phenylalanine . Binding of TyrR protein to the upstream TyrR box in the presence of phenylalanine is shown to increase the affinity of RNAP for the promoter and stimulate open complex formation at the -10 region of the tyrP promoter . This observation coupled with the results from mutational analysis supports the proposal that TyrR-phenylalanine activates tyrP transcription by stimulating the onset of open complex formation. Mol Microbiol, 2004 Apr, 52(1), 119 - 32 RecG helicase promotes DNA double-strand break repair; Meddows TR et al.; Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved . This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair . In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood . To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome . We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase . Both pathways rely on PriA, presumably to facilitate DNA replication . We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA. Mol Microbiol, 2004 Apr, 52(1), 53 - 65 Control of Cre recombination by regulatory elements from Xer recombination systems; Gourlay SC et al.; Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products . In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane . Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination . We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites . Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected . PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity . Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA . We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry . This may occur without the need for protein-protein interactions between accessory proteins and the recombinases. Biochemistry, 2004 Apr 6, 43(13), 3844 - 52 A linear correlation between the energetics of allosteric communication and protein flexibility in the Escherichia coli cyclic AMP receptor protein revealed by mutation-induced changes in compressibility and amide hydrogen-deuterium exchange; Gekko K et al.; Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive . In addition, the binding to DNA is concomitantly affected . To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 degrees C . These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)) . These changes in global structural properties correlated with the rate of amide proton exchange . A linear correlation was established between beta(s)(o) and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive . This linear correlation indicates that the nature and magnitude of cooperativity are a continuum . A similar linear correlation was established between compressibility and DNA binding affinity . In addition, linear correlations were also found among the dynamics of CRP and functional energetics . Double mutation (K52N/H159L) at positions 52 and 159, whose alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o) . These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP. Biochemistry, 2004 Apr 6, 43(13), 3835 - 43 Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions; Mancusso R et al.; Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability . We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH) . We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin . DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ) . The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69 . Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C . However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes . These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69 . Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups. Biochemistry, 2004 Apr 6, 43(13), 3802 - 13 Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change; Marks GT et al.; Two detailed mechanisms {Marks et al . (2001) Biochemistry 40, 6805} have been proposed to explain the activity of methylglyoxal synthase (MGS), a homohexameric allosterically regulated enzyme that catalyzes the elimination of phosphate from DHAP to form enol pyruvaldehyde . This enol then tautomerizes to methylglyoxal in solution . In one of these mechanisms His 98 plays an obligate role in the transfer of a proton from the O(3) oxygen of DHAP to the O2 oxygen . To test this hypothesized mechanism, the variants H98N and H98Q were expressed and purified . Relative to the wild-type enzyme, the H98N variant shows a 50-fold decrease in k(cat) with all other kinetic parameters (i.e., K(m), K(PGA), etc.) being nearly the same . By contrast, the apparent catalytic rate for the H98Q variant is 250-fold lower than that of the wild-type enzyme . Inorganic phosphate acts as a competitive inhibitor (with a K(i) of 15 microM) rather than as an allosteric-type inhibitor as it does in the wild-type enzyme, and the competitive inhibitor phosphoglyolate (PGA) acts as an activator of this variant . These facts are explained by a shift in the conformational equilibrium toward an "inactive" state . When activation by PGA is accounted for, the catalytic rate for the "active" state of H98Q is estimated to be only 6-fold less than that of the wild-type enzyme, and thus His 98 is not essential for catalysis . Primary deuterium isotope effect data were measured for the wild-type enzyme and the two variants . At pH 7.0, the (D)V isotope effect (1.5) and the absence of a (D)(V/K) isotope effect for the wild-type enzyme suggest that the rate for the isotopically sensitive step is partially rate limiting but greater than the rate of substrate dissociation . Both the (D)V (2.0) and (D)(V/K) (3.4) isotope effects were more pronounced in the H98N variant, while the H98Q variant displayed an unusual inverse (D)V (0.8) isotope effect and a normal (D)(V/K) (1.5) isotope effect . The X-ray crystal structures of PGA bound to the H98Q and H98N variants were both determined to a resolution of 2.2 A . These mutations had little effect on the overall structure . However, the pattern of hydrogen bonding in the active site suggests an explanation as to how in the wild-type and H98N mutated enzymes the "inactive to active" equilibrium lies toward the active state, while with the H98Q mutated enzyme the equilibrium lies toward the inactive state . Thus, the role of His 98 appears to be more as a regulator of the enzyme's conformation rather than as a critical contributor to the catalytic mechanism. Poult Sci, 2004 Mar, 83(3), 485 - 94 N(omega)-nitro-L-arginine methyl ester (L-NAME) amplifies the pulmonary hypertensive response to endotoxin in broilers; Wideman RF et al.; The pulmonary hypertensive response to bacterial lipopolysaccharide (LPS, endotoxin) varies widely among individual broilers, leading to the suggestion that innate variability may exist in the proportions or profiles of chemical mediators released during the ensuing inflammatory cascade . LPS induces the expression of nitric oxide synthase (iNOS), which produces the vasodilator nitric oxide (NO) to modulate the responses to concurrently produced vasoconstrictors . In experiment 1, broilers were given the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), followed by a supra-maximal dose of LPS while the pulmonary arterial pressure was recorded . In experiment 2 the cardiac output also was recorded before and following the i.v . injection of L-NAME . In both experiments, injection with L-NAME modestly increased the pulmonary arterial pressure when compared with control values, confirming previous reports that tonic/basal NO synthesis is required to promote flow-dependent pulmonary vasodilation in chickens . This response to L-NAME occurred in spite of a tendency for cardiac output and stroke volume to decline and, therefore, can be attributed to pulmonary vasoconstriction (an increase in the pulmonary vascular resistance) rather than an increase in pulmonary blood flow . When L-NAME was used to block NO synthesis induced by LPS, an early peak of pulmonary hypertension was revealed that rarely develops in broilers in the absence of L-NAME, and that has been correlated with the release of platelet activating factor and thromboxane A2 in mammals . The control group responded to LPS with a delayed-onset pulmonary hypertension that was typical in timing, amplitude, and duration of the responses previously observed in broilers and that has been attributed to endothelin-mediated thromboxane A2 synthesis in mammals . This delayed-onset pulmonary hypertensive response to LPS was longer in duration and higher in amplitude in the L-NAME group when compared with the control group . These observations are consistent with the hypothesis that NO modulates the responses to vasoconstrictors released concurrently during the LPS-mediated inflammatory cascade . Inhibition of NOS by L-NAME apparently reduced the modulatory influence of NO and exposed a more dramatic pulmonary hypertensive response to LPS. Can J Microbiol, 2001 Jan, 47(1), 72 - 6 Construction of a multiframe vector to express coding sequences in Escherichia coli; Dominguez-Martinez V et al.; Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors . To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner . Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream . To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons . Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons . In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products . We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame. Biotechnol Lett, 2004 Feb, 26(3), 229 - 33 Enhanced production of nikkomycin X by over-expression of SanO, a non-ribosomal peptide synthetase in Streptomyces ansochromogenes; Wang G et al.; A recombinant strain was constructed by introducing pWO6 which contains the sanO gene encoding a non-ribosomal peptide synthetase (NRPS) into Streptomyces ansochromogenes 7100 (wild-type strain) . The bioassay of nikkomycin indicated that the anti-fungal activity was enhanced in the recombinant strain in comparison with the wild-type strain . HPLC analysis revealed that 1200 mg nikkomycin X l(-1) was produced in the recombinant strain after growth for 6 d, which was about twice that of the wild-type strain . RT-PCR also indicated that higher transcription of the sanO gene was observed in the recombinant strain in contrast with wild-type strain. Proteomics, 2004 Apr, 4(4), 1007 - 13 Screening protein refolding using surface plasmon resonance; Jones DB et al.; Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol . The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths . One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody . This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity . The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions . SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design . The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host. Proteomics, 2004 Apr, 4(4), 961 - 9 A computational method for assessing peptide- identification reliability in tandem mass spectrometry analysis with SEQUEST; Razumovskaya J et al.; High-throughput protein identification in mass spectrometry is predominantly achieved by first identifying tryptic peptides by a database search and then by combining the peptide hits for protein identification . One of the popular tools used for the database search is SEQUEST . Peptide identification is carried out by selecting SEQUEST hits above a specified threshold, the value of which is typically chosen empirically in an attempt to separate true identifications from false ones . These SEQUEST scores are not normalized with respect to the composition, length and other parameters of the peptides . Furthermore, there is no rigorous reliability estimate assigned to the protein identifications derived from these scores . Hence, the interpretation of SEQUEST hits generally requires human involvement, making it difficult to scale up the identification process for genome-scale applications . To overcome these limitations, we have developed a method, which combines a neural network and a statistical model, for normalizing SEQUEST scores, and also for providing a reliability estimate for each SEQUEST hit . This method improves the sensitivity and specificity of peptide identification compared to the standard filtering procedure used in the SEQUEST package, and provides a basis for estimating the reliability of protein identifications. Proteins, 2004 May 1, 55(2), 236 - 44 Two histidines of the coat protein of turnip yellow mosaic virus at the capsid interior are crucial for viability; Bink HH et al.; RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups . Two different types of interaction have been proposed . In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA . The type II interaction involves charged histidines interacting with phosphates of the RNA backbone . Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction . The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked . Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation . Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly . Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability . The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein . Proteins, 2004 May 1, 55(2), 229 - 35 Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE-MS; Zhang X et al.; White spot syndrome virus (WSSV) is a major shrimp pathogen that also infects many other species of crustaceans . Its 305-kb double-stranded DNA genome has the capacity to encode 181 presumptive proteins . In an attempt to identify the viral proteins from the 181 theoretical proteins, proteins of the purified WSSV were separated by two-dimensional electrophoresis (2-DE) . More than 60 protein spots were revealed, as detected by silver staining, from which 12 viral proteins were identified by mass spectrometry . In total, 25 WSSV proteins, including those reported in one of our earlier studies (Huang et al., Mol Cell Proteomics 2002;1:223-231), were revealed by this proteomic approach, and their corresponding genes were further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) . Two of them were characterized to be WSSV envelope proteins using immuno-electron microscopy . Our study showed that the proteomic approach is a powerful method for discovering the viral structural proteins and their corresponding genes . Proteins, 2004 May 1, 55(2), 223 - 8 The mechanism of proton exclusion in aquaporin channels; Ilan B et al.; The mechanism of proton exclusion in aquaporin channels is elucidated through free energy calculations of the pathway of proton transport . The second generation multistate empirical valence bond (MS-EVB2) model was applied to simulate the interaction of an excess proton with the channel environment . Jarzynski's equality was employed for rapid convergence of the free energy profile . A barrier sufficiently high to block proton transport is located near the channel center at the NPA motif-a site involved in bi-orientational ordering of the embedded water-wire in absence of the excess proton . A second and lower barrier is observed at the selectivity filter near the periplasmic outlet where the channel is narrowest . This secondary barrier may be essential in filtering other large solutes and cations . Biopolymers, 2004 Apr 15, 73(6), 705 - 15 Study of protein aggregation using two-dimensional correlation infrared spectroscopy and spectral simulations; Lefevre T et al.; Two-dimensional (2D) correlation spectroscopy establishes correlations between intensity variations in a series of spectra obtained by the application of an external perturbation . However, spectral effects (wavenumber shift or bandwidth change) are known to generate apparent asynchronisms in 2D maps . Surprisingly, spectral effects are often neglected in the literature when interpreting experimental maps, which can lead to erroneous conclusions . In an attempt to evaluate the contribution of these effects and that of true asynchronisms on 2D maps, the heat-induced aggregation of glutamyl-tRNA synthetase (GluRS) was studied as a typical example of the application of Fourier transform infrared (FTIR) spectroscopy in the amide I region . The data were compared with those obtained from a mutant protein that differs by one amino acid . To determine whether the aggregation mechanisms are identical for both proteins, the experimental 2D maps were compared to simulations based on curve fitting of the initial and final spectra of the series, which allows change in position and bandwidth of the components to be taken into account . Intermediate spectra were generated using a convenient function that mimics the spectral evolution . The speed and the delay of each component were controlled . Apart from the appearance of turns that occur for the mutant and not for GluRS, the aggregation mechanisms of both proteins seems to be essentially identical . In particular, the loss of alpha-helices seems to be concomitant with the formation of intermolecular beta-sheets, whereas the loss of intramolecular beta-sheets is delayed . Since the experimental maps are satisfactorily simulated when almost all the components are in phase, it appears that many of the asynchronous features are mainly due to spectral effects . Thus, one has to be aware that true asynchronisms are not necessarily at the origin of peaks observed in asynchronous maps . Bone Marrow Transplant, 2004 May, 33(9), 963 - 7 Escherichia coli-nitroreductase suicide gene control of human telomerase reverse transcriptase-transduced minor histocompatibility antigen-specific cytotoxic T cells; Verdijk RM et al.; Adoptive immunotherapy with ex vivo generated cytotoxic T lymphocytes (CTLs) is applied for the treatment of leukemia relapses or viral infections after allogeneic stem cell transplantation . A common problem of adoptive immunotherapy strategies is the ex vivo expansion of the generated T cells to sufficient numbers . CTLs can be efficiently expanded by ectopic expression of the human telomerase gene (hTert) . However, hTert transduction may also increase the chance for malignant transformation . Therefore, we explored the feasibility of suicide gene control of ex vivo generated CTLs expanded through the ectopic expression of hTert . To this end, we compared the efficacy of the new Escherichia coli-nitroreductase (E . coli-Ntr) suicide gene with the well-known herpes simplex virus-thymidine kinase (HSV-Tk) . Introduction of hTert provided the transduced CTLs with a distinct growth advantage over the nontransduced CTLs . The hTert-E . coli-Ntr double-transduced CTLs retained their antigen-specific functions . Treatment of hTert-E . coli-Ntr double-transduced CTLs with metronidazole significantly inhibited the proliferation to a similar extent to the treatment of hTert-HSV-Tk double-transduced CTLs with ganciclovir . This is the first application of the E . coli-nitroreductase gene for the elimination of human T cells with metronidazole. J Biochem (Tokyo), 2004 Feb, 135(2), 185 - 91 Interaction of the Escherichia coli lipoprotein NlpI with periplasmic Prc (Tsp) protease; Tadokoro A et al.; Escherichia coli spr (suppressor of prc) mutants and nlpI mutants show thermosensitive growth . The thermosensitivity of the spr mutants was suppressed by the nlpI mutations . Expression of the fusion genes encoding hexa-histidine-tagged NlpI (NlpI-His) and purification of the tagged NlpI showed that NlpI-His bound with Prc protease and IbpB chaperone . NlpI-His with the amino acid substitution of G103D did not bind with either of these proteins, while NlpI-His variants (NlpI-284-His, NlpI-Q283-His, and NlpI-G282-His) lacking 10 to 12 residues from the carboxy terminus bound with both proteins . The tagged NlpI lacking 11 amino acid residues from the carboxy terminus was processed by Prc, but that lacking 12 residues was not . The thermosensitivity of the nlpI mutant was corrected by the production of the former NlpI variant, but not by production of the latter . Expression of the truncated NlpI that lacked 10 or 11 residues from the carboxy terminus corrected the thermosensitivity of the prc nlpI double mutant, while expression of the full-length NlpI did not . Thus, it was suggested that NlpI was activated by Prc protease processing. Antimicrob Agents Chemother, 2004 Apr, 48(4), 1281 - 8 Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity; Aubry A et al.; Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis . Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M . tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action . To address these issues, we separately overexpressed the M . tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes . The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity . The drug concentration that inhibited DNA supercoiling by 50% (IC(50)) was measured for 22 different quinolones, and values ranged from 2 to 3 microg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 microg/ml (pipemidic acid and nalidixic acid) . By comparison, MICs measured against M . tuberculosis ranged from 0.12 microg/ml (for gatifloxacin) to 128 microg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC(50)s (R(2) = 0.9) . Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion . Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R(2) = 0.7) . These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M . tuberculosis . The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M . tuberculosis gyrase. Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 253 - 8 Molecular cloning, overexpression, and characterization of the ligand-binding D2 domain of fibroblast growth factor receptor; Hung KW et al.; Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes . The biological activities of FGFs are mediated by cell surface receptors (FGFRs) . In the present study for the first time we report the cloning, expression, and characterization of the ligand (FGF)-binding D2 domain of human FGFR2 . D2 domain is expressed in Escherichia coli in high yields (10 mg/L) as inclusion bodies . The protein is recovered by dissolving the inclusion bodies in 8 M urea and subsequently refolding on nickel affinity column . The protein is purified (to approximately 97% purity) to homogeneity using heparin-Sepharose affinity column . Far-UV circular dichroism data and chemical shift index plot based on 1H-alpha, 13C-alpha, 13C-beta, and 13carbonyl group chemical shifts suggest that D2 domain is an all beta-sheet protein consisting of 9 beta-strands . Isothermal titration calorimetry and equilibrium urea unfolding experiments show that recombinant D2 domain is in a biologically active conformation and binds strongly to its ligand (FGF) and to the heparin analog, sucrose octasulfate (SOS) . Using a variety of triple resonance NMR experiments, complete assignment of 1H, 15N, and 13C resonances in D2 domain has been accomplished . The findings of the present study not only pave way for an in-depth investigation of the molecular mechanism(s) underlying the activation of FGF signaling but also provide avenues for the rational design of potent inhibitors against FGF-mediated pathogenesis. Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 218 - 22 Peptide binding to Geminin and inhibitory for DNA replication; Yoshida K et al.; Geminin binds to Cdt1 to ensure that DNA replication occurs only once during the cell cycle . To identify the peptide that binds to Geminin and thereby modifies the latter's ability to alter the DNA replication activity in human cancer cells, we screened a phage display library of random peptides in successive cycles of phage library panning and found one peptide sequence that bound to the 31-111 amino acid residues of Geminin . Delivery of this peptide sequence into the nucleus of HCT116 human colon cancer cells resulted in the suppression of BrdU incorporation . These results provide new insights into the function of Geminin and further validate Geminin as a potential therapeutic target in tumors. Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 198 - 204 Hemangiopoietin inhibits apoptosis of MO7e leukemia cells through phosphatidylinositol 3-kinase-AKT pathway; Zhan M et al.; Hemangiopoietin (HAPO) is a growth factor that significantly stimulates proliferation and survival of the primitive cells of hematopoietic and endothelial lineages . To determine the mechanism of action of HAPO, the anti-apoptotic activity and signal transduction pathway of HAPO were investigated using a factor-dependent leukemia cell line, the MO7e cells . Recombinant human HAPO (rhHAPO) was produced in Escherichia coli and purified by a series of column chromatography with a purity of more than 95% . rhHAPO significantly supported the survival of MO7e cells after deprivation of granulocyte-macrophage colony stimulating factor and activated phosphatidylinositol 3-kinase (PI3K) . When the MO7e cells were treated with two specific inhibitors to PI3K (LY294002 or wortmannin), a significant loss of cell viability with evidence of apoptosis was observed . Moreover, the protein kinase B (Akt), one of the downstream effectors of PI3K-dependent survival signaling, was activated in HAPO-stimulated MO7e cells . Phosphorylation of Akt at serine 473 and its downstream molecular Bad at serine 136 was induced by HAPO, but was blocked by two PI3K inhibitors, LY294002 and wortmannin . In addition, HAPO inhibited caspase-3 activities and poly(ADP-ribose) polymerase degradation . Such an effect of HAPO was also significantly blocked by either LY294002 or wortmannin . These results indicate that HAPO protects MO7e cells from apoptotic death through a PI3K-Akt pathway. Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 157 - 61 The interaction between apolipoprotein serum amyloid A and high-density lipoprotein; Wang L et al.; Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins (HDLs) via its N-terminus . The murine isoform SAA2.2 forms a hexamer in solution and the N-terminus is shielded from the solvent . Therefore, it is unclear how the SAA2.2 hexamer might bind HDL . In this study, the binding of SAA2.2 to murine HDL was investigated by glutaraldehyde cross-linking and polyacrylamide gel electrophoresis . The hexamer did not bind HDL significantly at 20 degrees C . However, at temperatures between 25-30 degrees C, SAA2.2 became destabilized and its monomeric form bound to HDL . SAA2.2 binding did not significantly replace Apo A-I in HDL particles . At 37-45 degrees C SAA2.2 binds less to HDL, suggesting that its binding is weak and sensitive to physiological and pathological temperatures, and thereby, potentially modulated, in vivo, by other factors. J Surg Res, 2004 Apr, 117(2), 272 - 82 Altered glycosylation of surfactant and brush border membrane of the small intestine in response to surgical manipulation; Prabhu R et al.; BACKGROUND: Surgical stress can lead to bacterial translocation from the intestine into systemic circulation . Adherence of bacteria onto the glycoconjugates of the brush border membrane (BBM) and surfactant coat (SLP) of the mucosal cells is the first step in the translocation of luminal bacteria . Our earlier study showed that surgical manipulation of the intestine results in oxidative stress leading to structural and functional alterations in the mucosa . This study looks at the effect of surgical manipulation on the glycoconjugate alterations of SLP and BBM . MATERIALS AND METHODS: SLP and BBM were isolated from control and after surgical manipulation and the sugar composition was analyzed . Bacterial adherence using E . coli isolated from cecum was compared after coating microtiter plates with SLP or BBM isolated from control and after surgical manipulation . RESULTS: An increase in various sugars was seen after surgical manipulation both in SLP and BBM and this increase was maximum at 12 h after surgery . These alterations increased bacterial adherence onto SLP and BBM . Inhibiting superoxide generation by allopurinol treatment prior to surgical manipulation prevented glycosylation alteration and bacterial adherence . CONCLUSION: Surgical manipulation results in altered glycoconjugates of SLP and BBM which leads to increased bacterial adherence . These alterations are probably brought about by oxygen-free radicals . This is clinically significant because postsurgical complications such as sepsis may be brought about by altered glycosylation. J Surg Res, 2004 Apr, 117(2), 223 - 31 Levamisole modulates prostaglandin E2 production and cyclooxygenase II gene expression in human colonic cancer cells; Liu CY et al.; BACKGROUND: Colorectal adenocarcinoma is one of the leading causes of cancer mortality in the world . Recent studies have demonstrated that prostaglandin E(2) (PGE(2)) and cyclooxygenase (COX) are involved in the early development of colorectal cancer . Levamisole together with 5-fluorouracil has been shown to improve the survival of patients with Duke's C colon cancer . This study examined the effect of levamisole on PGE(2) production and COX-2 gene activation in immortalized human colon cells . MATERIALS AND METHODS: Colon 205 cells, a human colonic cancer cell line, were exposed to Escherichia coli LPS (1 microg/mL) in the presence of levamisole (1 microm to 1 mm) . The PGE(2) production was determined by enzyme-linked immunosorbent assay . In addition, cyclooxygenase II (COX-2) mRNA expression and COX-2 protein were measured by the real-time reverse transcription polymerase chain reaction and Western blot assay, respectively . Transcription factor nuclear factor (NF)-kappaB activity of the nuclear and cytoplasmic proteins was determined by Western blot assay with a P65 antibody . RESULTS: Colon 205 cells produced significantly more PGE(2) when stimulated by LPS . Levamisole inhibited PGE(2) production by LPS-stimulated colon 205 cells in a dose-dependent fashion . In addition, COX-2 mRNA expression and COX-2 protein induced by LPS were also reduced by levamisole . Finally, NF-kappaB activation of LPS-stimulated colon cells was inhibited by levamisole . CONCLUSION: Levamisole inhibits PGE(2) production by LPS-stimulated colon 205 cells . The inhibition of levamisole occurs at the transcription level of COX-2 gene and is regulated through NF-kappaB activation. Res Vet Sci, 2004 Jun, 76(3), 219 - 25 Antibody responses against avian reovirus nonstructural protein sigmaNS in experimentally virus-infected chickens monitored by a monoclonal antibody capture enzyme-linked immunosorbent assay; Chen PN et al.; Crude antigen preparations from avian reovirus (ARV)-infected chicken embryo fibroblasts (sigmaNS) or from bacterially expressed protein sigmaNS (esigmaNS) were captured by monoclonal antibody 1E1(MAb 1E1) against ARV nonstructural protein sigmaNS immobilized on the ELISA plates and were used as the MAb capture ELISA for antibody detection . Sixty one-week-old specific pathogenic free (SPF) chickens were divided into six groups and were vaccinated with live or inactivated ARV vaccine preparations in different combinations or inoculated with a virulent ARV strain . Sera collected from the birds were tested for their antibody responses to ARV nonstructural protein sigmaNS . Using the MAb capture ELISAs, the level of nonspecific binding reactions was tested on the serum samples obtained weekly from mock-infected SPF chickens from 1 to 25 weeks and compared to the results tested by the conventional ELISA . The results indicated that both MAb capture ELISAs had lower nonspecific bindings than those in the conventional ELISA, even in older birds . Antibody responses against ARV sigmaNS of the birds which received the inactivated vaccine twice (group I), inactivated vaccine followed by a live vaccine (group II), or a live vaccine followed by boosting with an inactivated vaccine (group III) were detected by MAb captured ELISA with sigmaNS crude antigens . The absorbance values increased rapidly at 1-2 weeks after boosting, approximated a peak at 5-6 weeks of age, and maintained this throughout the length of the experiment . The absorbance values of the MAb capture ELISA showed a good correlation to the SN titers ( r value > 0.85) . On the other hand, serum samples from the birds which received the live vaccine twice (group IV) or were inoculated with a virulent ARV (group V) did not show antibody responses to sigmaNS, similar to those from the mock-infected birds (group VI), as the absorbance values maintained at a low level (below 0.5) throughout the length of the experiment . Similar results were obtained in the sera detected by MAb capture ELISA with crude esigmaNS antigens, except that the absorbance values in the sera from the birds in group III were gradually increased and later approximated a peak at 11 weeks of age and maintained this throughout the length of the experiments . The results suggest that MAb capture ELISAs can be readily used to detect antibody responses of the birds against ARV nonstructural protein sigmaNS which may reflect an immune status of a chicken flock, receiving ARV vaccine as long as including an inactivated vaccine. Biosens Bioelectron, 2004 May 15, 19(10), 1169 - 74 Rectified photocurrent in a protein based molecular photo-diode consisting of a cytochrome b562-green fluorescent protein chimera self-assembled monolayer; Lee B et al.; We fabricated a self-assembled monolayer (SAM) of a chimeric protein created as a novel model protein for an artificial light-harvesting complex (LHC) composed of two proteins, cytochrome b(562) (cytb(562)) mutated for SAM fabrication (cytb(562), N22C, G82C) and enhanced green fluorescent protein (EGFP) . The SAM formation of chimeric protein on a single-crystalline Au(111) substrate was confirmed by atomic force microscopy (AFM) measurement . The rectified photocurrent of the chimeric protein SAM on a gold substrate was detected by light-illumination scanning tunneling microscopy (LI-STM) co-operated with a lock-in technique . The photocurrent generation of the chimeric protein SAM was wavelength-specific to the light-illumination (488 nm), which indicated that the EGFP part of the chimera plays a role as a sensitizer in the photo-induced electron transfer process. Artif Organs, 2004 Mar, 28(3), 271 - 7 Lentivirus-based gene delivery in mouse embryonic stem cells; Kosaka Y et al.; BACKGROUND: Embryonic stem (ES) cells are widely used in therapeutic research as an unlimited source of cell therapy . Therefore, it is of great value to find a way to efficiently manipulate ES cells . HIV-1-derived lentiviral vectors are now considered to be an efficient vehicle for delivering genes into a variety of cells . In this study, we examined the efficacy of lentivirus-based gene delivery into mouse ES (mES) cells . MATERIALS AND METHODS: Recombinant HIV-I-based lentiviral vectors Lt-GFP, expressing green fluorescent protein (GFP), and Lt-LacZ, expressing E . coli LacZ gene in conjunction with neomycin resistance gene, were generated using a FuGENE 6 transduction method and used for transducing ES cells derived from 129Sv mice . Lentiviral transduction efficacy was evaluated by GFP expression assay using flow cytometry and by X-gal staining . The in vivo potential of developing teratoma of such transduced mES cells was examined in severe combined immunodeficiency (SCID) mice . RESULTS: FuGENE 6 showed no considerable transduction-associated cytotoxicity . The expression rate of GFP and LacZ of mES cells increased on a multiplicity of infection (MOI)-dependent manner with the amount of Lt-GFP and Lt-LacZ used . Approximately 42% of mES cells were positive for GFP after infection of Lt-GFP at an MOI of 30 . Notably, after G418 selection, nearly 100% of Lt-LacZ-transduced mES cells were positive for LacZ and formed teratomas in SCID mice . CONCLUSIONS: This work demonstrates that HIV-I-based lentiviral vectors are capable of transducing mES cells . Lentiviral vectors may facilitate an advance in the field of gene transfer and expression in various types of ES cells, including human ES cells. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 255 - 8 Hyperthermophilic dehydrogenase enzymes; Littlechild JA et al.; Archaeal dehydrogenases are often found to be of a specific class of dehydrogenase which has low sequence identity to the equivalent bacterial and eukaryotic counterparts . This paper focuses on two different types of hyperthermophilic dehydrogenase enzyme that have been cloned and over-expressed in Escherichia coli . The crystallographic structures of the apo form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from Sulfolobus solfataricus and the related holo form of GAPDH from Methanothermus fervidus have been solved to high resolution . The zinc-containing structure of ADH (alcohol dehydrogenase) from Aeropyrum pernix has also been solved as a quaternary complex with the cofactor NADH and the inhibitor octanoic acid . The results show that despite the low sequence identity to the related enzymes found in other organisms the fold of the protein chain is similar . The archaeal GAPDH enzymes show a relocation of the active site which is a feature of evolutionary interest . The high thermostability of these three archaeal dehydrogenases can be attributed to a combination of factors including an increase in the number of salt bridges and hydrophobic interactions, a higher percentage of secondary structure and the presence of disulphide bonds. Acta Paediatr, 2004 Feb, 93(2), 169 - 73 Serum procalcitonin and C-reactive protein in children with diarrhoea of various aetiologies; Korczowski B et al.; AIM: Procalcitonin (PCT) and C-reactive protein (CRP) are two acute-phase reactants with different clinical features . The study aimed to compare the diagnostic value of admission serum PCT and CRP concentrations as indicators of aetiology and intensity of inflammation in children hospitalized with diarrhoea . METHODS: Serum PCT and CRP concentrations were determined on admission in 129 children hospitalized with diarrhoea . They were divided into four groups: group A: 37 children with diarrhoea as one of symptoms of ongoing systemic bacterial infection (sepsis/ meningitis): group B: 36 children with bacterial enterocolitis; group C: 43 children with rotaviral enterocolitis; and group D: 13 children with active inflammatory bowel disease (IBD) . For comparison serum PCT and CRP concentrations were determined in 30 healthy controls . RESULTS: PCT concentration was > 0.5 ng ml(-1) in all 37 (100%) children with diarrhoea and systemic bacterial infection (mean 18.5 +/- 3.2 ng ml(-1)) and CRP was above 2 mg dl(-1) in 33 (89%) of these children (11.7 +/- 1.5 mg dl(-1)) . PCT concentration was > or = 0.5 ng ml(-1) in 22 of 36 (61%) children with bacterial enterocolitis (2.2 +/- 0.6 ng ml(-1)), in 3 of 43 (7%) children with rotaviral infection (0.2 +/- 0 ng ml(-1)) and in 3 of 13 (23%) patients with IBD (0.3 +/- 0.1 ng ml(-1)) . CRP value was > or =2 mg dl(-1) in 22 (61%) children from group B (5.4 +/- 1.0 mg dl(-1)), in 8 (19%) children from group C (l.3 +/- 0.3 mg dl(-1)) and in 6 (46%) patients from group D (3.3 +/- 0.9 mg dl(-1)) . In the control group the PCT (0.1 +/- 0.1 ng ml(-1)) and CRP (0.03 +/- 0.1 mg dl(-1)) levels were low or undetectable . CONCLUSION: In this study PCT was a more reliable marker than CRP of systemic bacterial infection in children with diarrhoea . PCT was more specific but less sensitive in the differentiation of bacterial and non-bacterial aetiology of inflammation. Arch Virol, 2004 Apr, 149(4), 699 - 712 Epub 2003 Dec 08. Structural characterization of Tobacco etch virus coat protein mutants; Voloudakis AE et al.; The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied . CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs) . These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP . CP from which 175 N-terminal amino acids were removed, failed to polymerize . A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E . coli.To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated . The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule . No differences were observed in spectra obtained from fluorescence spectroscopy . The point mutants bind RNA in vitro to the same degree as the wild type protein . However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E . coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not . These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions. Histochem Cell Biol, 2004 Apr, 121(4), 291 - 7 Epub 2004 Mar 24. Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide; Miyauchi M et al.; This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus . In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half . After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed . Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7 . Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE . These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction. J Mol Microbiol Biotechnol, 2003, 6(2), 67 - 75 Cloning, expression, activity and folding studies of serine hydroxymethyltransferase: a target enzyme for cancer chemotherapy; Agrawal S et al.; All the members of pyridoxal-5'-phosphate-dependent enzymes are involved in the metabolism of amino acids . The sequence homology studies further divide this family into three distinct groups . A fine scrutiny of the reactions catalyzed by these enzymes shows their regio specificity; they have been considered as the largest group of enzymes having tendency to affect the valency of the same carbon atom that carries the amino group forming an amine linkage with the coenzyme . Thus, this group was named 'alpha-class of enzymes' . Serine hydroxymethyltransferase (SHMT) is a member of this alpha-class; it reversibly catalyses the conversion of serine into glycine while the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate . The resultant compound is the sole precursor of purine biosynthesis . Henceforth, this enzyme greatly affects nucleic acid biosynthesis in all the organisms . It is obvious that SHMT plays an indispensable role in nucleic acid biosynthesis; therefore, designing and developing a repressor/inhibitor of the SHMT gene/protein may resolve the problem of drug resistance to cancer chemotherapy . SHMT has been widely studied in many living systems (e.g . Escherichia coli, humans, sheep, rabbits, Trypanosoma, Arabidopsis, peas, tobacco) in terms of its structure, cloning, expression, purification and folding patterns . Such studies have enabled one to assess the pattern of overall kinetic and activity behaviour of the enzyme, which may further help in developing a suitable cancer therapeutic molecule . Protein Sci, 2004 Apr, 13(4), 1031 - 42 Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain; Hansen M et al.; The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP . The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis . The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone . These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents . A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two alpha-helices . This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for