J Biol Chem, 2004 Apr 23, 279(17), 17945 - 50 Epub 2004 Jan 14. Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60-kDa Drosophila melanogaster dUTPase trimer; Dubrovay Z et al.; The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme . This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme . Flexible segments of the homotrimer give rise to sharp resonances in the (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule . Binding of the product dUMP or the analogues dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances . This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule . Three-dimensional (15)N-edited (1)H-(15)N HSQC total correlation spectroscopy (TOCSY) and (1)H-(15)N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks . The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate . NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer . The results suggest allosterism in the eukaryotic dUTPase. Di Yi Jun Yi Da Xue Xue Bao, 2004 Jan, 24(1), 1 - 6 Preparation and characterization of monoclonal antibodies against S1 domain at N-terminal residues 249 to 667 of SARS-associated coronavirus S1 protein; Wen K et al.; OBJECTIVE: To prepare and characterize monoclonal antibodies (mAbs) against S1 protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) . METHODS: 6-His-tagged recombinant fragment at N-terminal residues 249 to 667 of SARS-CoV S1 protein including S-protein receptor-binding domain was expressed in E.coli . The immunogenicity of this S1 domain was identified and used to immunize BALB/c mice for the production of hybridomas . The identification of the mAbs against this S1 domain was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and Western blotting, respectively . RESULTS: Three hybridomas producing mAbs specific to the S1 domain was obtained, with a relative molecular mass of 48,500 . None of the 3 mAbs were reactive with human coronaviruses 229E and OC43 . Two of the mAbs were IgG2a isotype, and the other was IgG1 . CONCLUSIONS: This is the first report of mAbs produced against S-protein receptor-binding domain of SARS-CoV . The 3 S1-specific mAbs may be useful for further study of the function of the S protein and for diagnosis of SARS-CoV infection. Zhonghua Er Ke Za Zhi, 2003 Dec, 41(12), 893 - 6 {Experimental study on cerebral white matter damage in neonatal rat after intrauterine Escherichia coli infection}; Yu HM et al.; OBJECTIVE: To investigate the expression of glial fibrillary acidic protein (GFAP), GFAP mRNA and interleukin-1beta mRNA (IL-1beta mRNA), tumor necrosis factor-alpha mRNA (TNF-alpha mRNA) in neonatal rat brain after intrauterine infection . METHODS: Escherichia coli (E . coli) was inoculated into both uterine horns of pregnant rats when gestation was 70% complete (15 days) . The control group was treated with normal saline . The pups were killed on the postnatal day 1 (P1), P3 and P7, respectively . The cerebral white matter damage of the neonatal rats was determined by HE staining . Immunohistochemistry was used for evaluation of GFAP expression in neonatal rat brains and RT-PCR to analyze GFAP mRNA, IL-1beta mRNA and TNF-alpha mRNA expression at P1, P3 and P7 . RESULTS: The major histopathological changes in neonatal cerebral white matter at P7 after intrauterine infections were: weak staining of cerebral white matter and focal rarefaction . GFAP-positive cells were observed in both the control and the E . coli-treated groups . The numbers of GFAP-positive cells of the E . coli-treated group pups were markedly increased in periventricular white matter and hippocampus at P7 compared with those of the control group (periventricular white matter: 9.73 +/- 3.55 vs 5.67 +/- 1.90, P < 0.05 and hippocampus: 7.81 +/- 3.61 vs 2.16 +/- 1.11, P < 0.05, respectively) . No significantly different levels of GFAP expression in corpus callosum were found between two groups (P > 0.05) . The expression of GFAP mRNA in brain of the E . coli-treated neonatal rat was higher than the control at P1, P3 (P1: 0.25 +/- 0.07 vs 0.15 +/- 0.08, P < 0.05 and P3: 0.50 +/- 0.09 vs 0.39 +/- 0.08, P < 0.05, respectively), but the expression of GFAP mRNA in brain of the neonatal rat at P7 had no significant difference between two groups (P > 0.05) . The expression of IL-1beta mRNA and TNF-alpha mRNA in brain of the E . coli-treated neonatal rat were higher than of the control at P1 (IL-1beta mRNA: 0.83 +/- 0.19 vs 0.50 +/- 0.30, P < 0.05 and TNF-alpha mRNA: 0.74 +/- 0.30 vs 0.30 +/- 0.20, P < 0.05, respectively), but the expression of IL-1beta mRNA and TNF-alpha mRNA in brain of the neonatal rat at P3 and P7 had no significant difference between two groups (P > 0.05) . CONCLUSIONS: The intrauterine infection could cause neonatal white matter damage and IL-1beta, TNF-alpha may be a mechanism mediating between the two events. Immunogenetics, 2004 Feb, 55(11), 763 - 9 Epub 2004 Jan 13. Identification of a novel Japanese flounder (Paralichthys olivaceus) CC chemokine gene and an analysis of its function; Khattiya R et al.; A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced . The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues . The full gene was cloned and sequenced from a BAC library . It has a length of approximately 750 bp from the start codon to the stop codon and is composed of four exons and three introns . Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines . Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen . The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified . The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber . On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity . These results indicate that Paol-SCYA104 probably acts as a CC chemokine. J Clin Invest, 2004 Jan, 113(2), 274 - 84 CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations; Scott JW et al.; CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life . Although thought to be regulatory, their exact functions have been unknown . However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (gamma 2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine beta-synthase) . AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized . We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding . This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives. J Biol Chem, 2004 Mar 26, 279(13), 12484 - 94 Epub 2004 Jan 12. Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca2+-calmodulin-dependent protein kinase II; Gaertner TR et al.; Ca(2+)-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (alpha, beta, gamma, and delta) in mammalian cells . We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products . Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 A revealed that all four isoforms were dodecamers with similar but highly unusual architecture . A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core . At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes . Enzymatic analyses showed that the isoforms were similar in their K(m) for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca(2+)-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation . Interestingly, the rank order of CaM binding affinity (gamma > beta > delta > alpha) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (beta > gamma > delta > alpha) . Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform. J Biol Chem, 2004 Mar 19, 279(12), 11253 - 8 Epub 2004 Jan 13. Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase; Weber J et al.; The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque . In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1) . To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)) . With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm . Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm . The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1) . The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer . This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions. J Biol Chem, 2004 Apr 2, 279(14), 13769 - 77 Epub 2004 Jan 13. Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA; Natale P et al.; SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli . SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis . We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex . Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction . Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex . Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein . This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction. Exp Cell Res, 2004 Jan 1, 292(1), 170 - 8 Biochemical evidence for interaction between smoothelin and filamentous actin; Niessen P et al.; The two major isoforms of smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs) . Based on these findings, we hypothesized that smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin . The interaction between smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin {alpha-SKA}) was investigated using several in vitro assays . Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts . In rat embryonic fibroblasts, transfected smoothelins-A and -B associated with stress fibers . In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled smoothelin, showed binding of smoothelin-A to actin filaments, but not to monomeric G-actin . A truncated smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent . ELISA results showed that the binding of smoothelin to actin has no significant isoform specificity . Our results indicate an interaction between smoothelin and actin filaments . Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin. J Infect, 2004 Feb, 48(2), 161 - 7 Frequency and characteristics of diarrhoeagenic Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro, Brazil; Regua-Mangia AH et al.; The frequency of diarrhoeagenic Escherichia coli (DEC) strains was investigated in 253 children up to 3 years old, with (patient group, PG, 199 children) and without (control group, CG, 54 children) diarrhoea, living in Rio de Janeiro, Brazil . DEC strains were detected in 70 (27.6%) children, including 54 (27.1%) with diarrhoea and 16 (29.6%) without diarrhoea . Enteroaggregative E . coli (EAEC) was the most frequent DEC category, accounting for 14.6% of the isolates in the PG and for 11.1% in the CG . E . coli strains carrying enteropathogenic E . coli (EPEC) virulence markers showed higher incidence in the CG (12.9%) than in the PG (8.0%) . E . coli strains belonging to non-classical EPEC groups that carried eae only or eae and bfpA, designated as attaching-effacing E . coli (AEEC) were the most frequent (79.1%) . Simultaneous presence of multiple EPEC virulence factors (EAF/eae/bfpA) were only detected among strains isolated from the PG . Enterotoxigenic E . coli (ETEC) strains were isolated from 5.5% of the children in the CG and from 3.5% of those in the PG . Most of the ETEC isolates were LT-probe positive (70%) and none carried both LT-I and ST-I probe sequences . One enteroinvasive E . coli (EIEC) strain was recovered from a child with diarrhoea . No stx-probe positive E . coli strains were detected . Overall, DEC strains were not found to be significantly associated with diarrhoea (p>0.05) . However, the higher incidence of EAEC, the most frequent DEC category, among children with diarrhoea, suggests a potential role of EAEC as an important enteric pathogen in the community investigated. Biochem J, 2004 Apr 15, 379(Pt 2), 433 - 40 Characterization of iron binding in IscA, an ancient iron-sulphur cluster assembly protein; Ding H et al.; Iron-sulphur clusters are one of the most common types of redox centre in biology . At least six proteins (IscS, IscU, IscA, HscB, HscA and ferredoxin) have been identified as being essential for the biogenesis of iron-sulphur proteins in bacteria . It has been shown that IscS is a cysteine desulphurase that provides sulphur for iron-sulphur clusters, and that IscU is a scaffold for the IscS-mediated assembly of iron-sulphur clusters . The iron donor for iron-sulphur clusters, however, remains elusive . Here we show that IscA is an iron binding protein with an apparent iron association constant of 3.0x10(19) M(-1), and that iron-loaded IscA can provide iron for the assembly of transient iron-sulphur clusters in IscU in the presence of IscS and L-cysteine in vitro . The results suggest that IscA is capable of recruiting intracellular iron and delivering iron for iron-sulphur clusters in proteins. J Am Chem Soc, 2004 Jan 21, 126(2), 496 - 504 Protein-template-driven formation of polynuclear iron species; Malone SA et al.; Ferritins are iron-storage proteins capable of holding up to 4500 Fe(3+) ions within a single water-soluble protein shell made from 24 polypeptide chains . The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimers of polypeptide chains being their stable forms . The aerobic addition to these of up to 8-10 or 14-20 Fe(2+) ions per dimer, respectively, results in the oxidation of the added Fe(2+) to Fe(3+) . Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe(3+) ions are associated with the polypeptide dimer, and the lack of intense EPR signals from magnetically isolated Fe(3+) ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe(3+) ions . The effect of Fe(3+) chelators on iron-loaded subunit dimers is to remove the Fe(3+) from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein . These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe(3+) occurs independent from the protein surface . From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified. Anal Chem, 2004 Jan 15, 76(2), 365 - 72 A robust technique for assembly of nucleic acid hybridization chips based on electrochemically templated chitosan; Yi H et al.; A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface . Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent . Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and DNA:mRNA hybridization detection schemes were evaluated . Hybridization of target nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal . Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich assay . Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities. Anal Chem, 2004 Jan 15, 76(2), 267 - 75 An alternative to tandem mass spectrometry: isoelectric point and accurate mass for the identification of peptides; Cargile BJ et al.; The traditional approach to the identification of peptides in complex biological samples integrally involves the use of tandem mass spectrometry to generate a unique fragmentation pattern in order to accurately assign its identity to a particular protein . In this article we describe the theoretical basis for a new paradigm for the identification of peptides and proteins . This methodology employs the use of accurate mass and peptide isoelectric point (pI) as identification criteria, and represents a change in focus from current tandem mass spectrometry-dominated approaches . A mathematical derivation of the false positive rate associated with accurate mass and pI measurements is presented to demonstrate the utility of the technique . The equations for calculation of the experimental false positive rate allow for the determination of the validity of the data . The false positive rate issue examined in detail here is not restricted to accurate mass-based approaches, but also has application to the tandem mass spectrometry community as well . The theoretical proteomes of Escherichia coli and Rattus norvegicus are used to evaluate the efficacy of this approach . The power of the technique is demonstrated by analyzing a series of peptides with the same monoisotopic masses but with differing isoelectric points . Finally, the speed of algorithm when combined with the experimental peptide analysis has the potential to rapidly accelerate the protein identification process. Biotechnol Lett, 2003 Nov, 25(22), 1917 - 24 Immunological comparison of native and recombinant egg allergen, ovalbumin, expressed in Escherichia coli; Rupa P et al.; Chicken ovalbumin is one of the major egg white allergens which causes IgE-mediated food hypersensitivity . A gene encoding for chicken ovalbumin (Gad dI) was isolated from chicken oviduct by PCR amplification and was cloned under the control of T5 promoter fused with a six-histidine tag at the N-terminal end . Escherichia coli harbouring this construct expressed high quantities of the recombinant protein in the form of soluble fraction . The protein was purified using affinity chromatography on a Ni(2+)-nitrilotriacetic acid agarose column and was further purified to homogeneity by ion exchange chromatography . Homogeneity was confirmed through SDS-PAGE, Western blot and secondary conformation analysis . The reactivity of the recombinant and native protein was tested against six egg allergic human patient's sera and the IgE and IgG binding activity was tested using both Western blot and ELISA . When compared to native ovalbumin, the recombinant protein had similar binding activity in immunoblotting, but slightly increased activity by ELISA . Circular dichroism revealed that the recombinant protein had a slightly less compact structure than the native form . Both antigens exhibited a similar immunogenicity in mice. Proc Natl Acad Sci U S A, 2004 Jan 20, 101(3), 745 - 50 Epub 2004 Jan 12. Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species; Mukhopadhyay P et al.; We examined the genomewide transcriptional responses of Escherichia coli treated with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium nitrite (NaNO(2)) during aerobic growth . These assays showed that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the global repressor of ferric ion uptake, are major regulators of the response to reactive nitrogen species . In contrast, SoxR and OxyR, regulators of the E . coli defenses against superoxide-generating compounds and hydrogen peroxide, respectively, have minor roles . Moreover, additional regulators of the E . coli response to reactive nitrogen species remain to be identified because several of the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant strains . We propose that the E . coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species. Protein Sci, 2004 Feb, 13(2), 466 - 75 Epub 2004 Jan 10. Nucleotide recognition in the ATP-grasp protein carbamoyl phosphate synthetase; Kothe M et al.; Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle on duplicated nucleotide-binding sites (N and C) . To clarify the contributions of sites N and C to the overall reaction, we carried out site-directed mutagenesis aimed at changing the substrate specificity of either of the two sites from ATP to GTP . Mutant design was based in part on an analysis of the nucleotide-binding sites of succinyl-CoA synthetases, which share membership in the ATP-grasp family with CPS and occur as GTP- and ATP-specific isoforms . We constructed and analyzed Escherichia coli CPS single mutations A144Q, D207A, D207N, S209A, I211S, P690Q, D753A, D753N, and F755A, as well as combinations thereof . All of the mutants retained ATP specificity, arguing for a lack of plasticity of the ATP sites of CPS with respect to nucleotide recognition . GTP-specific ATP-grasp proteins appear to accommodate this substrate by a displacement of the base relative to the ATP-bound state, an interaction that is precluded by the architecture of the potassium-binding loop in CPS . Analysis of the ATP-dependent kinetic parameters revealed that mutation of several residues conserved in ATP-grasp proteins and CPSs had surprisingly small effects, whereas constructs containing either A144Q or P690Q exerted the strongest effects on ATP utilization . We propose that these mutations affect proper movement of the lids covering the active sites of CPS, and interfere with access of substrate. Nucleic Acids Res . 2004 Jan 12;32(1):e11. Method to integrate multiple plasmids into the mycobacterial chromosome; Saviola B et al.; In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB . This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene . The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site . This system should be useful in the construction of novel mycobacterial strains . In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested. J Biol Chem, 2004 Mar 26, 279(13), 13148 - 55 Epub 2004 Jan 12. A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK); Forouhar F et al.; Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH . It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins . We report here the crystal structures at up to 2.0-A resolution of YiaK alone and in complex with NAD-tartrate . YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor . In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme . The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site . Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK . In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site . The free enzyme conformation is incompatible with NAD binding . His(44) is likely the catalytic residue of the enzyme. J Biol Chem, 2004 Apr 2, 279(14), 13452 - 60 Epub 2004 Jan 12. Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers; Grant GA et al.; A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange . These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer . They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface . Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector . That is, only two effector sites need to be occupied for maximum inhibition . Binding of the last two effector molecules does not contribute functionally to inhibition of activity . Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector . Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound . Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity . This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers . In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement . These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme. J Exp Bot, 2004 Feb, 55(396), 377 - 85 Epub 2004 Jan 12. A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro; Kim IJ et al.; Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase . Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified . A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis . Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H . brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP . A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized . Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity) . In vitro functional analysis of the recombinant protein overexpressed in E . coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does . These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees. Genome Res, 2004 Feb, 14(2), 301 - 12 Epub 2004 Jan 12. Flux coupling analysis of genome-scale metabolic network reconstructions; Burgard AP et al.; In this paper, we introduce the Flux Coupling Finder (FCF) framework for elucidating the topological and flux connectivity features of genome-scale metabolic networks . The framework is demonstrated on genome-scale metabolic reconstructions of Helicobacter pylori, Escherichia coli, and Saccharomyces cerevisiae . The analysis allows one to determine whether any two metabolic fluxes, v(1) and v(2), are (1) directionally coupled, if a non-zero flux for v(1) implies a non-zero flux for v(2) but not necessarily the reverse; (2) partially coupled, if a non-zero flux for v(1) implies a non-zero, though variable, flux for v(2) and vice versa; or (3) fully coupled, if a non-zero flux for v(1) implies not only a non-zero but also a fixed flux for v(2) and vice versa . Flux coupling analysis also enables the global identification of blocked reactions, which are all reactions incapable of carrying flux under a certain condition; equivalent knockouts, defined as the set of all possible reactions whose deletion forces the flux through a particular reaction to zero; and sets of affected reactions denoting all reactions whose fluxes are forced to zero if a particular reaction is deleted . The FCF approach thus provides a novel and versatile tool for aiding metabolic reconstructions and guiding genetic manipulations. Eur J Biochem, 2004 Jan, 271(2), 439 - 49 Crystal structure of the chi:psi sub-assembly of the Escherichia coli DNA polymerase clamp-loader complex; Gulbis JM et al.; The chi (chi) and psi (psi) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery . In E . coli, the chi:psi heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein . We determined the crystal structure of the chi:psi heterodimer at 2.1 A resolution . Although neither chi (147 residues) nor psi (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(chi) or dinucleotide-(psi) binding proteins, without marked similarity to the structures of the clamp-loader subunits . Genes encoding chi and psi proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the chi:psi interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance . The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the chi:psi complex . One of the conserved regions was found to be located on chi, distal to the psi interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein . The other region of sequence conservation is localized to an N-terminal segment of psi (26 residues) that is disordered in the crystal structure . We speculate that psi is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the chi:psi unit is linked to the single-stranded DNA-binding protein via the distal surface of chi . The base of the clamp-loader complex has an open C-shaped structure, and the shape of the chi:psi complex is suggestive of a loose docking within the crevice formed by the open faces of the delta and delta' subunits of the clamp-loader. Eur J Biochem, 2004 Jan, 271(2), 418 - 28 New substrate analogues of human serotonin N-acetyltransferase produce in situ specific and potent inhibitors; Ferry G et al.; Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N-acetyltransferase catalyzes the rate-limiting step . A previous study reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme . This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites . This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A . In the present report, we describe the potency of new N-halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N-acetyltransferase . The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell . The fate of tritiated-phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of {(3)H}BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N-acetyl{(3)H}PEA and coenzyme A-S{(3)H}acetyltryptamine, respectively) . The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme . In a similar manner we studied the production of another inhibitor generated from N-{2-(7-hydroxynaphth-1-yl)ethyl}bromoacetamide . New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model . Some of these compounds proved to be extremely potent, with IC(50)s of approximately 30 nM . As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N-acetyltransferase, we also show that they are potent ligands at the melatonin receptors . Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin production in vivo. Eur J Biochem, 2004 Jan, 271(2), 349 - 55 GTP cyclohydrolase I utilizes metal-free GTP as its substrate; Suzuki T et al.; GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin . We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli . First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP . Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP . We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein . Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein . These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity . We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well. Eur J Biochem, 2004 Jan, 271(2), 291 - 302 Some properties of human small heat shock protein Hsp20 (HspB6); Bukach OV et al.; Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli . The proteins were obtained in a homogeneous state without utilization of urea or detergents . On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa . Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa . At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa . At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial alpha-crystallin . Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein . At pH 6.0, both alpha-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, alpha-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase . The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperature-dependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits . Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp . A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins. Biochemistry, 2004 Jan 20, 43(2), 569 - 76 Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis; Nes WD et al.; Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity . Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity . Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity . Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding . Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9 . These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction . Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments . Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation. Biochemistry, 2004 Jan 20, 43(2), 518 - 25 Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA; Adler J et al.; According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition . To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized . Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge . Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism . Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets. Biochemistry, 2004 Jan 20, 43(2), 415 - 24 Energetics of domain-domain interactions and entropy driven association of beta-crystallins; Sergeev YV et al.; Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction . They undergo modification as the lens ages, including cleavage of their terminal extensions . The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation . Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr) . The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography . All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures . The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein . Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree . Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2 . The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven . The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens. Biochemistry, 2004 Jan 20, 43(2), 405 - 14 Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations; Mendieta J et al.; The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD) . The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form . The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism . A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level . The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base . To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates . Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate . Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine . For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine . Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form . These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors. Biochemistry, 2004 Jan 20, 43(2), 374 - 83 Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase; Schnell JR et al.; Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis . Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops . In this study, information on dynamics is extended to methyl-containing side chains . To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants . The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+ . Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale . The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex . Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale. Biochemistry, 2004 Jan 20, 43(2), 352 - 61 Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1; Ladner JE et al.; The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix . Its relationship to members of the canonical GSH transferase superfamily has remained an enigma . The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A . The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily . The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms . The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity . The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH . The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1) . Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB) . As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM) . The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB . The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner. J Gene Med, 2004 Jan, 6(1), 43 - 54 Gene-directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease; Martiniello-Wilks R et al.; BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) based on the E . coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa) . Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine . Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis . METHODS: These studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression . Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate ( approximately 600 mg/m(2)/day) once daily for 5 consecutive days . Treated mice received a single intraprostatic injection containing 10(10) particles of OAdV220, an ovine atadenovirus which expresses the E . coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment . The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored . Tumours were also analysed histologically . RESULTS: Preliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice . Animals injected with vector and prodrug showed a significant reduction (36-47%) in GU tract weight (ANOVA p = 0.0002) and a 35-50% reduction in seminal vesicle weight (ANOVA p = 0.0007) . In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007) . PNP-GDEPT mice also showed a survival advantage over control mice . Histological analysis suggested that the cancer progression was slowed in GDEPT-treated animals . CONCLUSION: A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice . J Gene Med, 2004 Jan, 6(1), 16 - 21 Factors influencing immune response after in vivo retrovirus-mediated gene transfer to the liver; Podevin G et al.; BACKGROUND: Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes . This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross-presentation of the transgene product present as a contaminant in the viral stock . In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long-term transduction of hepatocytes using the nls-lacZ marker gene . METHODS: Animals were divided in four groups . In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ . In group 2, using the same strategy, animals received retroviral supernatant that was passed through a beta-galactosidase affinity column to reduce beta-galactosidase contamination . In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection . RESULTS: In group 1, 23.1% of animals had no immune response 2 months after gene delivery vs . 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4 . Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant . This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response . CONCLUSIONS: Our present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus-mediated gene delivery to the liver . Nucleic Acids Res, 2004 Jan 09, 32(1), 263 - 70 Print 2004. Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site; Pearson CG et al.; The formation of clustered DNA damage sites is a unique feature of ionizing radiation . Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites . Using a plasmid-based method, damaged DNA containing uracil positioned at 1-5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY . Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions . The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid . In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site . Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC-->TA transversions . The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis. Proc Natl Acad Sci U S A, 2004 Jan 20, 101(3), 835 - 40 Epub 2004 Jan 08. Evidence for polar positional information independent of cell division and nucleoid occlusion; Janakiraman A et al.; We present evidence that, in Escherichia coli, polar positional information is present at midcell independent of known cell division factors . In filamented cells, IcsA, which is normally polar, localizes at or near potential cell division sites . Because the cell pole is derived from the septum, the sites to which IcsA localizes in filaments correspond to future poles . IcsA localization to these sites is independent of FtsZ, MinCDE, septation, and nucleoid occlusion, indicating that positional information for the future pole is independent of cell division and chromosome positioning . Upon IcsA localization to these sites, septation is inhibited, suggesting that IcsA recognition of this polar positional information may influence cell division. J Biomol Tech, 2003 Dec, 14(4), 298 - 307 Improvement in the detection of low concentration protein digests on a MALDI TOF/TOF workstation by reducing alpha-cyano-4-hydroxycinnamic acid adduct ions; Zhu X et al.; Alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) as a matrix facilitates the ionization of proteins and peptides in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer . The matrix itself also ionizes and so do its sodium and potassium adducts . Matrix clusters and metal ion adducts interfere with peptide ionization and peptide mass spectrum interpretation . These matrix adducts are significantly reduced with addition of ammonium monobasic phosphate or ammonium dibasic citrate to the matrix and sample deposited onto the MALDI target . The reduction of matrix adducts results in the increase of peptide intensity and signal-to-noise ratio as well as in improvement of peptide ionization for samples deposited onto the target at levels of 10 fmol or below . These improvements were particularly significant in the detection of peptides at amol levels when reduced amounts of matrix were also used. J Clin Microbiol, 2004 Jan, 42(1), 398 - 400 Reduced etiological role for enteropathogenic Escherichia coli in cases of diarrhea in Brazilian infants; Rodrigues J et al.; Previously common in Brazil, enteropathogenic Escherichia coli (EPEC) strains of serogroups O55, O111, and O119 are now rare, while enteroadherent strains other than EPEC, belonging to serogroups such as O125, were prevalent among 126 diarrheic infants less than 1 year old who were surveyed . None of these strains had the EPEC bundle-forming pilus (bfpA) gene. J Clin Microbiol, 2004 Jan, 42(1), 36 - 44 Molecular epidemiology of the iron utilization genes of enteroaggregative Escherichia coli; Okeke IN et al.; Enteroaggregative Escherichia coli (EAEC) strains are etiologic agents of acute and persistent diarrhea . In this study, the results of phenotypic assays suggested that EAEC strains possess specialized iron acquisition systems . Genes required for the synthesis (iucA) or transport (fepC) of siderophores, and genes encoding siderophore (fyuA, ireA, and iroN) or heme transport (chu) receptors or hemoglobin proteases (pic and hbp), were sought in EAEC strains which have been characterized with respect to known virulence genes and phylogeny . The chuA, iucA, fyuA, fepC, and pic genes were detected in 33, 76.2, 85.7, 33, and 61.9% of these EAEC strains, respectively, and the other genes were absent . The majority of EAEC strains possessed genes encoding multiple iron transport systems, and there was no phylogenetic correlation in the distribution of the majority of these loci, as is typical for EAEC . The notable exceptions were chuA and fepC (which is associated with the prrA-modA-fepC pathogenicity island); these genes were restricted to the EAEC2 and DAEC2 phylogenetic groups, which could represent pathogenic subsets . When collections of EAEC strains isolated during case-control studies in Nigeria and Brazil were examined, no association of the presence of either chuA or iucA alone with diarrhea was seen, but both genes together were present in significantly more strains from cases than from controls in the Nigerian collection (P < 0.05) . It is possible that the presence of both genes marks at least some virulent strains . The data also demonstrate geographical variation in the association of iron utilization genes with disease in EAEC. J Biol Chem, 2004 Mar 26, 279(13), 12043 - 50 Epub 2004 Jan 10. Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB; Chao Y et al.; ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli . We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes . Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively . Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+ . H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+ . Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients . Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport . Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+ . The exchange stoichiometry of metal ion for proton is likely to be 1:1. J Biol Chem, 2004 Mar 26, 279(13), 13241 - 8 Epub 2004 Jan 07. DNA binding features of human POT1: a nonamer 5'-TAGGGTTAG-3' minimal binding site, sequence specificity, and internal binding to multimeric sites; Loayza D et al.; The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo . We examined the in vitro DNA binding features of POT1 . We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity . The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5'-TAGGGTTAG-3', and the optimal substrate is {TTAGGG}(n (n > or = 2)) . POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A) . Whereas POT1 binding to MBS or {TTAGGG}(2) was enhanced by the proximity of a 3' end, POT1 was able to bind to a {TTAGGG}(5) array when positioned internally . These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3' telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop. J Biol Chem, 2004 Mar 19, 279(12), 11992 - 9 Epub 2004 Jan 07. Camptothecin-sensitive relaxation of supercoiled DNA by the topoisomerase I-like activity associated with poly(ADP-ribose) polymerase-1; Yung TM et al.; Poly(ADP-ribose) polymerase-1 is a highly abundant nuclear enzyme implicated in transcription, DNA replication, and DNA repair through binding of nascent RNA and interactions with various factors . We found that purified fractions of recombinant human poly(ADP-ribose) polymerase-1 expressed in Escherichia coli possess yet another activity, a Mg(2+)-dependent DNA supercoil relaxation activity . Cleavage of recombinant poly(ADP-ribose) polymerase-1 by caspase-3, an apoptotic protease, reduced this activity, as did the removal of either of the two zinc finger motifs located in the N-terminal DNA-binding domain of poly(ADP-ribose) polymerase-1 . In addition, this activity was separated from E . coli topoisomerase I by gel-filtration column chromatography, suggesting that this activity is specifically associated with poly(ADP-ribose) polymerase-1 . Because this relaxation activity did not require ATP and was resistant to VP16, a topoisomerase II inhibitor, this activity is closer to that of topoisomerase I . However, the supercoiled DNA relaxation activity associated with poly(ADP-ribose) polymerase-1 is distinct from that of human or E . coli topoisomerase I, as this activity could not completely remove superhelical tensions from plasmid DNA . Thus, we referred to this activity as topoisomerase I-like activity . This Mg(2+)-dependent DNA supercoil relaxation activity was found to be sensitive to camptothecin, a mammalian topoisomerase I inhibitor. Biomol Eng, 2004 Jan, 21(1), 15 - 20 Cloning and sequence analysis of the Candida utilis HIS3 gene; Basabe L et al.; A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA library by complementation of the E . coli hisB mutant . Its nucleotide sequence was determined and it predicts a single open reading frame of 675 bp (224 aa) . The deduced amino acid sequence is highly homologous to other yeast and fungi HIS3 genes. J Virol Methods, 2004 Mar 1, 116(1), 95 - 102 Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand; Reyes-del Valle J et al.; Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector . The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody . The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells . To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein . After washing with isotonic buffer, elution was accomplished using a high salt buffer . The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay . This procedure allows further characterization of molecules that could be involved in dengue binding and entry. Anal Biochem, 2004 Feb 1, 325(1), 151 - 7 In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting; Bernath K et al.; Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function . The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes . While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity . Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS) . These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact . Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker . The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes. Anal Biochem, 2004 Feb 1, 325(1), 126 - 36 A Biacore biosensor method for detailed kinetic binding analysis of small molecule inhibitors of p38alpha mitogen-activated protein kinase; Casper D et al.; Protein kinases are emerging as one of the most intensely studied classes of enzymes as their central roles in physiologically and clinically important cellular signaling events become more clearly understood . We report here the development of a real-time, label-free method to study protein kinase inhibitor binding kinetics using surface plasmon resonance-based biomolecular interaction analysis (Biacore) . Utilizing p38alpha mitogen-activated protein kinase as a model system, we studied the binding properties of two known small molecule p38alpha inhibitors (SB-203580 and SKF-86002) . Direct coupling of p38alpha to the biosensor surface in the presence of a reversible structure-stabilizing ligand (SB-203580) consistently produced greater than 90% active protein on the biosensor surface . The dissociation and kinetic constants derived using this Biacore method are in excellent agreement with values determined by other methods . Additionally, we extend the method to study the thermodynamics of small molecule binding to p38alpha and derive a detailed thermodynamic reaction pathway for SB-203580 . The Biacore method reported here provides an efficient way to directly and reproducibly examine dissociation constants, kinetics, and thermodynamics for small molecules binding to p38alpha and possibly other protein kinases . Immobilization in the presence of a stabilizing ligand may further represent a broadly applicable paradigm for creation of highly active biosensor surfaces. Anal Biochem, 2004 Feb 1, 325(1), 68 - 76 Use of protein biotinylation in vivo for chromatin immunoprecipitation; Viens A et al.; We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of protein-DNA interactions in vivo by chromatin immunoprecipitation (ChIP) . The system is based on coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from Escherichia coli . The superior strength of the biotin-avidin interaction allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a better signal/noise ratio. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 174 - 80 Stationary phase protein overproduction is a fundamental capability of Escherichia coli; Ou J et al.; Although Escherichia coli is well studied and various recombinant E . coli protein expression systems have been developed, people usually consider the rapid growing (log phase) culture of E . coli as optimum for production of proteins . However, here we demonstrate that at stationary phase three E . coli systems, BL21 (DE3)(pET), DH5alpha (pGEX) induced with lactose, and TG1 (pBV220) induced with heat shock could overexpress diversified genes, including three whose products are deleterious to the host cells, more stably and profitably than following the log phase induction protocol . Physical and patch-clamp assays indicated that characteristics of target proteins prepared from cultures of the two different growth phases coincide . These results not only provide a better strategy for recombinant protein preparation in E . coli, but also reveal that rapid rehabilitation from stresses and stationary phase protein overproduction are fundamental characters of E . coli. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 159 - 65 Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase; Sistla S et al.; EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli . Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized . Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA . Metal ions did not influence DNA binding . Interestingly, M.EcoP1I was able to methylate single-stranded DNA . Kinetic parameters were determined for single- and double-stranded DNA methylation . This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system . This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 1 - 5 Determination of the kinetics of guanine nucleotide exchange on EF-Tu and EF-Ts: continuing uncertainties; Manchester KL; An analysis is made of the rate constants for the reactions involving the interactions of EF-Tu, EF-Ts, GDP, and GTP recently derived by Gromadski et al . {Biochemistry 41 (2002) 162} . Though their measured values appear to allow a reasonable rate of nucleotide exchange sufficient to support rates of protein synthesis in vivo, their data underestimate the thermodynamic barrier involved in nucleotide exchange and therefore cannot be considered definitive . A kinetic scheme consistent with the thermodynamic barrier can be achieved by modification of various rate constants, particularly of those involving the release of EF-Ts from EF-Tu.GTP.EF-Ts, but such constants are markedly different from what are experimentally observed . It thus remains impossible at present satisfactorily to model guanine nucleotide exchange on EF-Tu, catalysed by EF-Ts by a double displacement mechanism, with experimentally derived rate constants . Metabolic control analysis has been applied to determine the degree of flux control of the different steps in the pathway. Biochem J, 2004 May 1, 379(Pt 3), 553 - 62 Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli; Biswas SB et al.; In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form . DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E . coli DNA replication . In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin . We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis . DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides . DnaC bound ssDNA in the complete absence of nucleotides . The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP{S} . In the presence of ADP, the binding affinity decreased approximately fifty-fold . Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch . Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function . Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein. Biomacromolecules, 2004 Jan-Feb, 5(1), 40 - 8 Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase; Zhang S et al.; Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth . The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation . PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization . Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization . The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA) . The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E . coli cells . The PHA synthase expressed in the recombinant E . coli cells was purified to homogeneity . Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases . Two major complexes were identified in preparations of purified PHA synthase . The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer) . In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction . The percentage of dodecameric complex increases with increasing salt concentration . The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period . The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E . shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex. Biotech Histochem, 2003 Jun-Aug, 78(3-4), 201 - 5 Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins; Wang P et al.; Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms . With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly . We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a "library of antibodies," by using an E . coli lysate of the IIM cDNA clone . This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens. Mol Plant Microbe Interact, 2004 Jan, 17(1), 81 - 9 Analysis of the involvement of hydroxyanthranilate hydroxycinnamoyltransferase and caffeoyl-CoA 3-O-methyltransferase in phytoalexin biosynthesis in oat; Yang Q et al.; Two oat genes encoding hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT) and S-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), both of which are possibly involved in the biosynthesis of oat avenanthramide phytoalexins, were cloned and their expression profiles in response to biological stress were studied . Four distinct cDNAs of oat HHT (AsHHT1-4) were isolated with the degenerative polymerase chain reaction method . The enzymatic activity of AsHHT1 expressed in E . coli was found using hydroxyanthranilate and hydroxycinnamoyl-CoAs as cosubstrates . Cloned oat CCoAOMT (AsCCoAOMT) encoded a polypeptide of 130 amino acid residues with 77.7 to 80.8% identities to the CCoAOMT sequences from other plant species . The accumulation of AsHHT1 and AsCCoAOMT transcripts increased concomitantly with phytoalexin accumulation by the treatment of victorin, a specific elicitor in oat lines carrying the Pc-2/Vb gene . Pharmacological approaches indicated the involvement of Ca2+, NO, and protein kinases in the signaling pathways of AsHHT1 and AsCCoAOMT mRNA induction . When oat leaves were inoculated with Puccinia coronata, the mRNA expression of AsHHT1 and AsCCOAOMT increased in both incompatible and compatible interactions but more rapidly in incompatible interaction . Interestingly, however, significant phytoalexin accumulation was observed only in incompatible interaction during the experimental period, suggesting that phytoalexin accumulation may be inhibited in one or more posttranscriptional processes in the compatible interaction. J Protein Chem, 2003 Nov, 22(7-8), 663 - 8 Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537; Roth NJ et al.; Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed . Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins . Circular dichroism studies showed that the structures of the enzymes were intact . E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner . This indicated that Glu-461 and Tyr-503 act independently in catalysis . Because these residues are at opposite sides of the active site and act in different steps, this is expected . E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases . This showed that Glu-537 and Tyr-503 interact cooperatively on the same step . This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose. Exp Dermatol, 2003 Dec, 12(6), 761 - 71 Identification of differentially expressed genes in models of melanoma progression by cDNA array analysis: SPARC, MIF and a novel cathepsin protease characterize aggressive phenotypes; Rumpler G et al.; Currently, the scale and consistency of changes of gene expression profiles in models of melanoma progression are largely unknown . Therefore, we investigated siblings of cell lines or malignant melanomas (MM), which have been selected by nude mouse passages for (a) . increased tumorigenicity (local ECM-independent growth), (b) . metastatic potential, or (c) . selected for increase invasiveness using the Boyden chamber . cDNA array analysis surveying more than 27.000 transcripts per cell line showed that 1.5-2.8% of all detectable transcripts were consistently differentially regulated during selection process in those models . Using array analysis, we identified 33 individual transcripts that exhibited significant differential hybridization paralleling the increased aggressiveness of the selected progeny . Because some of those genes could play a significant functional role in the progression of MM, we additionally proved their regulative pattern using Northern blotting . Among others, progressive overexpression of osteonectin/SPARC, a angiogenesis, was found in the selected offspring from all three experimental models and may therefore be considered as a potential marker for aggressive MM as well a promising therapeutic target . We further show that the selection of MM cells for increased ECM-independent local growth was accompanied by overexpresssion of macrophage migration inhibiting factor (MIF), an important modulator of both cell cycle progression and angiogenesis, and cathepsin Z, a novel member of the family of matrix degrading proteinases. J Biol Inorg Chem, 2004 Mar, 9(2), 171 - 82 Epub 2004 Jan 09. Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction; Rocklin AM et al.; The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) . ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters . Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial . Here these issues are addressed by development of the first functional single turnover system for ACCO . It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate . Thus, ascorbate is not required for O(2) activation or product formation . Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent . By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover . This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate . Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent . This role can be partially mimicked by a non-redox-active ascorbate analog . A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate . Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC. Bull Exp Biol Med, 2003 Oct, 136(4), 380 - 4 Structural and functional study of the receptor binding site for FimH adhesin in uropathogenic strains of Escherichia coli; Trinchina EV; We evaluated binding capacity of FimH-FocH hybrid adhesins during their interaction with model 1M and 3M substrates and epithelial cells . Introduction of the Glu89Lys point mutation into the fimH gene induced a new 1M-specific phenotype of adhesin . The role of a new pathoadaptive sign in the population of E . coli is discussed. J Pharm Pharmacol, 2003 Nov, 55(11), 1539 - 45 Non-genomic effects of tamoxifen on the activation of membrane-bound guanylate cyclase GC-A; Chen ZJ et al.; Oestrogen is known to exert both genomic and non-genomic effects on target tissues . Unlike the genomic effects, the identity of receptors mediating the non-genomic effects of oestrogen remains controversial . 17beta-estradiol has been shown to activate membrane-bound guanylate cyclase GC-A in PC12 cells in a non-genomic manner . To examine whether 17beta-estradiol exerts a similar effect in other cell types, we measured the effect of 17beta-estradiol and tamoxifen, an anti-oestrogen, on guanylate cyclase activity in porcine kidney proximal tubular LLC-PK1 cells . 17beta-estradiol increased cGMP levels in LLC-PK1 cells . Interestingly, addition of tamoxifen also increased cGMP levels in a concentration-dependent manner in LLC-PK1 cells . The effects of both 17beta-estradiol and tamoxifen on guanylate cyclase activity were not additive, suggesting that oestrogen and tamoxifen activate the same enzyme . Similar phenomena were also observed in LLC-PK1 cell membrane preparation . LLC-PK1 cells do not express membrane-bound guanylate cyclase GC-B and express low levels of membrane-bound guanylate cyclase GC-C . Tamoxifen inhibited the activation of GC-A by atrial natriuretic factor (ANF) . However, it did not affect membrane-bound guanylate cyclase GC-C stimulated by guanylin or Escherichia coli heat-stable toxin STa . These results indicate that 17beta-estradiol and tamoxifen activate GC-A in LLC-PK1 cells . Thus, tamoxifen functions as an agonist rather than an antagonist for the membrane oestrogen receptor coupled to the activation of GC-A. Antioxid Redox Signal, 2004 Feb, 6(1), 63 - 74 Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system; Fernandes AP et al.; Most cells contain high levels of glutathione and multiple glutaredoxins, which utilize the reducing power of glutathione to catalyze disulfide reductions in the presence of NADPH and glutathione reductase (the glutaredoxin system) . Glutaredoxins, like thioredoxins, may operate as dithiol reductants and are involved as alternative pathways in cellular functions such as formation of deoxyribonucleotides for DNA synthesis (by reducing the essential enzyme ribonucleotide reductase), the generation of reduced sulfur (via 3'-phosphoadenylylsulfate reductase), signal transduction, and the defense against oxidative stress . The three dithiol glutaredoxins of E . coli with the active-site sequence CPYC and a glutathione binding site in a thioredoxin/glutaredoxin fold display surprisingly different properties . These include the inducible OxyR-regulated 10-kDa Grx1 or the highly abundant 24-kDa glutathione S-transferase-like Grx2 (with Grx3 it accounts for 1% of total protein) . Glutaredoxins uniquely reduce mixed disulfides with glutathione via a monothiol mechanism where only an N-terminal low pKa Cys residue is required, by using their glutathione binding site . Glutaredoxins also catalyze formation of mixed disulfides (glutathionylation), which is an important redox regulatory mechanism, particularly in mammalian cells under oxidative stress conditions, to sense cellular redox potential. Eur J Endocrinol, 2004 Jan, 150(1), 49 - 56 Autoantibodies against 21-hydroxylase and side-chain cleavage enzyme in autoimmune Addison's disease are mainly immunoglobulin G1; Boe AS et al.; OBJECTIVE: Immunoglobulin G (IgG) antibodies to the steroidogenic enzymes 21-hydroxylase (21OH) and side-chain cleavage enzyme (SCC) are important diagnostic markers for autoimmune Addison's disease and autoimmune polyendocrine syndromes (APS) types I and II . The characterization of autoantibody (IgG) subclasses may reveal information on how tIssue destruction takes place; therefore, IgG subtypes of anti-21OH and anti-SCC antibodies from sera of patients with Addison's disease, APS I and APS II were determined using recombinant 21OH and SCC . METHODS: SCC(51-521) and his-SCC(51-521) were expressed by pET-scc in the Escherichia coli strain BL21 Star (DE3) and inclusion bodies were purified . Full-length, human 21OH fused to an N-terminal 6x histidine affinity tag was expressed in insect cells by using the baculovirus expression system bac-to-bac . Western blots were used to investigate the IgG subtype(s) of the autoantibodies against 21OH and SCC in patients and healthy blood donors . RESULTS: All anti-SCC positive sera (n=10) contained autoantibodies of the IgG1 subclass, while four out of ten also contained IgG3 . All anti-21OH positive sera (n=16) had autoantibodies exclusively against IgG1 . Sera from 20 healthy subjects did not show any reactivity against 21OH or SCC . CONCLUSIONS: The finding of a predominating IgG1 response against 21OH and SCC may suggest that T helper (Th) cells of the Th1 subclass are involved in destruction of the adrenal cortex in patients with autoimmune Addison's disease. Methods Enzymol, 2003, 370, 300 - 12 Techniques for studying the oxygen-sensitive transcription factor FNR from Escherichia coli; Sutton VR et al.; A large variety of techniques can be adapted for use with oxygen-sensitive samples . The growth of cells and in vivo analyses, as well as protein purification and in vitro assays, can be executed either by performing necessary steps in anaerobic environments (ranging from simple closed containers to the anaerobic chamber) or by circumventing the need for anaerobiosis with the use of oxygen-resistant protein variants. Cell Cycle, 2004 Feb, 3(2), 116 - 8 When pol I goes into high gear: processive DNA synthesis by pol I in the cell; Camps M et al.; Pol I is the most abundant polymerase in E . coli and plays an important role in short patch repair . In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro . Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication . In a previous publication, we described the generation of a highly error-prone DNA polymerase I . Expression of this mutant Pol I results in errors during the replication of a ColE1 plasmid . The distribution and spectrum of mutations in the ColE1 plasmid sequence downstream the ori indicates that Pol I is capable of more processive replication in vivo than previously accepted . Here, we review evidence suggesting that Pol I may be recruited into a replisome-like holoenzyme and speculate that processive DNA replication by Pol I may play a role in recombination-dependent DNA replication in the cell. J Biol Chem, 2004 Mar 26, 279(13), 13102 - 9 Epub 2004 Jan 07. Structural determinants of conformationally selective, prion-binding aptamers; Sayer NM et al.; We have recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind selectively to disease-associated beta-sheet-rich forms of the prion protein, PrP, from a number of mammalian species . These aptamers inhibit the accumulation of protease-resistant forms of PrP in a prion-seeded, in vitro conversion assay . Here we identify the minimal portions of two of these aptamers that retain binding specificity . We determine their secondary structures by a combination of modeling and solution probing . Finally, we identify an internal site for biotinylation of a minimized, synthetic aptamer and use the resultant reagent in the detection of abnormal forms of PrP in vitro. J Biol Chem, 2004 Mar 19, 279(12), 11163 - 9 Epub 2004 Jan 07. Rat long chain acyl-CoA synthetase 5, but not 1, 2, 3, or 4, complements Escherichia coli fadD; Caviglia JM et al.; Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (fatty acid CoA ligase: AMP forming, E.C . 6.2.1.3; ACS) . Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source . Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways . We expressed rat ACS isoforms 1-5 in an E . coli strain that lacked FadD . All rat ACS isoforms were expressed in E . coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD . In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized {(14)C}oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source . To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation . In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5 . Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate . These studies show that although ACS isoforms 1-4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth . Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function. J Biol Chem, 2004 Mar 26, 279(13), 12588 - 97 Epub 2004 Jan 07. Probing the ArcA-P modulon of Escherichia coli by whole genome transcriptional analysis and sequence recognition profiling; Liu X et al.; The ArcB/ArcA two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth . Over the years, genetic screens have lead to the identification of about 30 ArcA-P-controlled operons that are involved in redox metabolism . However, the discovery of 3 targets that are not implicated in respiratory metabolism (the tra operon for plasmid conjugation, psi site for Xer-based recombination, and oriC site for chromosome replication) suggests that the Arc modulon may comprise additional operons that are involved in a myriad of functions . To identify these operons, we derived the ArcA-P-dependent transcription profile of E . coli using oligonucleotide-based microarray analysis . The findings indicated that 9% of all open reading frames in E . coli are affected either directly or indirectly by ArcA-P . To identify which operons are under the direct control of ArcA-P, we developed the ArcA-P recognition weight matrix from footprinting data and used it to scan the genome, yielding an ArcA-P sequence affinity map . By overlaying both methods, we identified 55 new Arc-regulated operons that are implicated in energy metabolism, transport, survival, catabolism, and transcriptional regulation . The data also suggest that the Arc response pathway, which translates into a net global downscaling of gene expression, overlaps partly with the FNR regulatory network . A conservative but reasonable assessment is that the Arc pathway recruits 100-150 operons to mediate a role in cellular adaptation that is more extensive than hitherto anticipated. J Biol Chem, 2004 Mar 5, 279(10), 8648 - 54 Epub 2004 Jan 07. Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I; Cheng B et al.; The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA . The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures . Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change. Appl Environ Microbiol, 2004 Jan, 70(1), 625 - 30 Molecular cloning, purification, and biochemical characterization of hydantoin racemase from the legume symbiont Sinorhizobium meliloti CECT 4114; Martinez-Rodriguez S et al.; Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli . The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa . The optimum temperature and pH for the enzyme were 40 degrees C and 8.5, respectively . The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings . Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition. Appl Environ Microbiol, 2004 Jan, 70(1), 356 - 62 Occurrence of genes associated with enterotoxigenic and enterohemorrhagic Escherichia coli in agricultural waste lagoons; Chern EC et al.; The prevalence among all Escherichia coli bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic E . coli, and of three genes (stxI, stxII, and eaeA) associated with enterohemorrhagic E . coli was determined in farm waste disposal systems seasonally for 1 year . Single- and nested-PCR results for the number of E . coli isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method . The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested . Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the stxI, stxII, and eaeA genes . The geometric mean prevalence of each toxin gene within the E . coli community in waste disposal site waters after nested MPN-PCR was 1:8.5 E . coli isolates (1:8.5 E . coli) for the LTIIa toxin gene and 1:4 E . coli for the STII toxin gene . The geometric mean prevalence for the simultaneous occurrence of toxin genes stxI, stxII, and eaeA, was 1:182 E . coli . These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates . With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans. Appl Environ Microbiol, 2004 Jan, 70(1), 76 - 86 Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins; Okuno K et al.; Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins . However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide . The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity . Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1') . The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus . The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins . Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture . Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins. Int J Parasitol, 2004 Jan, 34(1), 95 - 100 Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine; Berglez J et al.; Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate . In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P . falciparum dihydropteroate synthase gene in Escherichia coli . When each construct was produced in a dihydropteroate synthase disrupted E . coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred . In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E . coli . When tested against twelve additional 'sulfa' drugs a variety of responses were obtained . All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs . Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa . All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide . These results suggest new drugs that could be tried for effective malaria treatment. Protein Expr Purif, 2004 Feb, 33(2), 332 - 8 Purification and refolding of a novel cancer/testis antigen BJ-HCC-2 expressed in the inclusion bodies of Escherichia coli; Yang XA et al.; BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy . To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli . The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer . Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97% . The yield of the purified protein was about 78% . The purified recombinant protein was refolded in a simple way . The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra . The recovery rate of refolded protein was 92.1% . The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting . This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods. Protein Expr Purif, 2004 Feb, 33(2), 311 - 25 Membrane protein expression and production: effects of polyhistidine tag length and position; Mohanty AK et al.; Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC) . Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp . suitable crystals for X-ray crystallography) . We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins . Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C-termini of the subcloned gene . The E . coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized . These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein . In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production . We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5A resolution have been obtained. Protein Expr Purif, 2004 Feb, 33(2), 297 - 303 Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa; Azzoni AR et al.; The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain . Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC) . The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3) . The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF) . The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy . Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity. Protein Expr Purif, 2004 Feb, 33(2), 256 - 64 Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase; Gillet SM et al.; Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease . Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues . Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography . Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol . Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme . We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase. Protein Expr Purif, 2004 Feb, 33(2), 246 - 55 Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts; Larsen AK et al.; The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues . They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear . The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k) . Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success . Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit . Rat m-calpain (m-80k/21k) is well expressed in E . coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical . It had also been reported that human m-calpai