Escherichia coli

Mol Biochem Parasitol, 2003 Apr 25, 128(1), 51 - 7
Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis; Lopez AB et al.; The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent . GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate . This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli . Each of these expressed enzymes had the predicted activity and was used to generate antibodies . Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment . Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.

Ann Fr Anesth Reanim, 2003 Feb, 22(2), 130 - 2
{Aortobronchial fistula developing from an infectious aneurysm of the thoracic aorta}; Elkettani C et al.; Aortobronchial fistula presenting as massive haemoptysis is a rapidly fatal process if not promptly diagnosed and repaired . It's an unusual complication of thoracic aneurysm . We report the case of a 61-year-old woman with rupture of a thoracic aortic infectious aneurysm secondary to an Escherichia coli infection . Aortobronchial fistula diagnosis should be considered in patients who have minor or major haemoptysis and correct diagnostic procedures should be performed early . An aggressive surgical approach is often necessary.

J Mol Biol, 2003 May 2, 328(3), 521 - 35
RAD51 is involved in repair of damage associated with DNA replication in mammalian cells; Lundin C et al.; The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells . Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells . Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine . We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea . No DSB was found following treatment with thymidine . Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication . We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster . Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed . This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication . We discuss our results in light of recent models suggested for HR at stalled replication forks.

Vaccine, 2003 May 16, 21(17-18), 2073 - 81
Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL; Dominici S et al.; The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model . HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs) . The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively . Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies . In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat . These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

Vaccine, 2003 May 16, 21(17-18), 1913 - 23
Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3; Rath A et al.; To investigate the immunogenicity of plasmid DNA encoding dog zona pellucida glycoprotein-3 (dZP3), the cDNA corresponding to dZP3, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRdZP3) . In vitro transfection of COS-1 mammalian cells with VRdZP3 plasmid DNA led to its cytosolic expression . The expressed dZP3 has an apparent molecular weight of 45kDa as compared to calculated molecular weight of 38.4 kDa, suggesting possible glycosylation . Immunization of male BALB/cJ mice with VRdZP3 plasmid DNA in saline, by electroporation or adsorbed onto gold microcarriers (delivered by gene gun) generated antibody response against Escherichia coli expressed recombinant dZP3 (r-dZP3) . Administration of r-dZP3 in saline following immunization with plasmid DNA led to boosting of the antibody response . Although mice immunized with gene gun exhibited highest antibody titres, the differences in the antibody titres seen by the three modes of plasmid DNA delivery were not statistically significant (P>0.05) . Interestingly, female mice immunized with VRdZP3 plasmid DNA using gene gun also generated antibodies against r-dZP3 . A dominant IgG1 isotype response was observed in mice immunized with VRdZP3 plasmid DNA using gene gun as compared to a mixed IgG1-IgG2a isotype response when delivered in saline or by electroporation . Immunization with VRdZP3 plasmid DNA also generated cell mediated immune response . The antibodies generated by VRdZP3 plasmid DNA recognized dog native zona pellucida . These studies for the first time, demonstrate the feasibility of generating an immune response to ZP3 by DNA vaccine and that the antibodies thus generated recognize native zona pellucida.

Thromb Res, 2003 Jan 25, 109(2-3), 137 - 44
Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibalpha; Dai K et al.; A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibalpha was developed and characterized . The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbalpha . VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR) . After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained . Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI . For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively . The scFv was arranged in VH-VL orientation, being joined together with a 15-amino-acid (Gly(4)Ser)(3) linker . The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein . Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000 . By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins . The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l . The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry . Analyzed by Western blot, it could bind to platelet GPIb . It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb) . In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation . Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

Biosens Bioelectron, 2003 May, 18(5-6), 547 - 53
Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection; Bontidean I et al.; A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described . Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor . The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>>S(Cd) congruent with S(Pb) . The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.

Res Microbiol, 2003 Apr, 154(3), 191 - 7
A requirement for anaerobically induced redox functions during aerobic growth of Escherichia coli with serine, glycine and leucine as carbon source; Lan J et al.; Escherichia coli strains with mutations in 3 genes coding for redox functions--torA, nuoM and glpC--are able to grow with pyruvate as carbon source, but are not able to use a combination of serine, glycine and leucine as carbon source, unlike the parent strain which uses either . All three mutants are able to produce and activate L-serine deaminase (L-SD) when grown in glucose minimal medium, and thus should be able to convert serine to pyruvate and grow on it . We suggest that activation of L-SD involves specific chemical reactions, perhaps building an Fe-S cluster . Mutant cells can carry out the necessary reaction to activate L-SD when grown in glucose minimal medium but apparently cannot do so when grown in SGL medium.

Arch Biochem Biophys, 2003 May 1, 413(1), 123 - 30
Native uridine 5'-diphosphate-sulfoquinovose synthase, SQD1, from spinach purifies as a 250-kDa complex; Shimojima M et al.; Sulfoquinovosyldiacylglycerol is a polar lipid present in photosynthetic membranes . It contributes to the negative surface charge of the membrane and plays a pivotal role under phosphate stress . The SQD1 protein is the key enzyme involved in the formation of the sulfolipid head group precursor, uridine 5(')-diphosphate (UDP)-sulfoquinovose, from UDP-glucose and sulfite . A cDNA encoding the spinach SQD1 protein was isolated and functionally expressed in Escherichia coli . The recombinant enzyme was compared to the native enzyme purified from isolated spinach chloroplasts . While the K(m) for UDP-glucose was indistinguishable for the two forms, the K(m) for sulfite was more than fourfold lower (< microM) for the native enzyme . Sizing by gel filtration indicated that the native form purified as a large complex of approximately 250 kDa, which is more than twice as large as the calculated size for the homodimer . It is proposed that in vivo SQD1 forms a complex with accessory proteins.

Arch Biochem Biophys, 2003 May 1, 413(1), 91 - 8
Human interferon gamma: significance of the C-terminal flexible domain for its biological activity; Nacheva G et al.; The significance of the C-terminal part of human interferon gamma (hIFNgamma) for its biological activity was studied by 3(')-end gene mutagenesis . A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392 . It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus . To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNgamma proteins was measured in clear cell lysates containing the soluble fractions only . The deletion of the C terminus had a two-step effect on both hIFNgamma antiviral and antiproliferative activities . Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNgamma, the deletion of more than 9 amino acids had an opposite effect . The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNgamma . The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the alpha-helical domain F.

Arch Biochem Biophys, 2003 May 1, 413(1), 23 - 31
Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli; Tesson AR et al.; Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source . In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B . Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely . The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis . The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme . The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.

Arch Biochem Biophys, 2003 May 1, 413(1), 1 - 8
Purification and preliminary characterization of brain aspartoacylase; Moore RA et al.; Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate . An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder . To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli . A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies . A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity . Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group . The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.

Virology, 2003 Mar 30, 308(1), 191 - 205
The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication; Panaviene Z et al.; Two of the five viral-coded proteins of tombusviruses, which are small, nonsegmented, plus-stranded RNA viruses of plants, are required for replication in infected cells . These replicase proteins, namely, p33 and p92, of cucumber necrosis virus are expressed directly from the genomic RNA via a readthrough mechanism . Their overlapping domains contain an arginine/proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP) . Site-directed mutagenesis of p33 expressed in Escherichia coli, followed by a gel shift assay, defined two of the four arginines as required for efficient RNA binding in vitro . In vivo testing of 19 RPR motif mutants revealed that the RPR motif, and therefore the ability to bind RNA, is important for the replication of tombusviruses and their associated defective interfering (DI) RNAs . Mutation within the RPR motif also affected the ratio of subgenomic versus genomic RNAs in infected cells . To test whether the RPR motif is essential for the function of either p33 or p92 in replication, we used a two-component system developed by, J . Virol . 5845-5851), in which p92 was expressed from the genomic RNA of a tombusvirus, while p33 was expressed from a DI RNA . The protoplast experiments with the two-component system revealed that the RPR motif is essential for the replication function of both proteins . Interestingly, mutations within the RPR motif of p33 and p92 had different effects on RNA replication, suggesting different roles for the RNA-binding motifs of these proteins in tombusvirus replication.

Virology, 2003 Mar 30, 308(1), 74 - 82
Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein alpha as an apoptosis inducer; Guo YX et al.; Betanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish and infection by which cause high mortality in larvae and juveniles in a wide range of marine fish species in Asia, Europe, Australia, Martinique, and Tahit . Greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) were investigated for their apoptotic activity in culture cells . GGNNV infection of sea bass (SB) cells appeared to induce a typical cytopathic effect (CPE), i.e., cytoplasmic vacuolation, thinning, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death . The infected SB cells underwent DNA fragmentation and stained positive in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, suggesting that GGNNV infection induced apoptosis in SB cells . In addition, GGNNV-infected SB cells showed an increased activity of caspase-8-like proteases (IETDase) and caspase-3-like proteases (IETDase), whereas inhibitor of caspase-8 and caspase-3 reduced GGNNV-induced apoptosis . This suggests that GGNNV may promote apoptosis via the extrinsic pathway in SB cells . Protein alpha, the precursor of GGNNV capsid proteins, was transiently expressed in SB and Cos-7 cells . The DNA fragmentation and TUNEL positive signal were apparent in SB and Cos-7 cells expressing protein alpha, suggesting that protein alpha may serve as an apoptotic inducer in these cells . Moreover, expression of protein alpha resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease specific inhibitor DEVD-CHO peptide . These results suggest that fish caspases are important elements in GGNNV-meditated apoptosis.

Virology, 2003 Apr 10, 308(2), 216 - 24
Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase; Blakeney SJ et al.; Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites . The cleavage products were similar to those previously identified for Southampton virus, a highly related virus . We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor . NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography . Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein . trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency . NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family . We propose that the proteinase may play a regulatory role in viral replication.

Int J Parasitol, 2003 Apr, 33(4), 445 - 54
Cloning and characterisation of a cysteine proteinase gene expressed in amastigotes of Leishmania (L.) amazonensis; Lasakosvitsch F et al.; The present study describes the cloning and characterisation of a gene encoding a cysteine proteinase isoform, Llacys1, expressed in amastigote forms of Leishmania (L.) amazonensis . Recombinant clones containing the Llacys1 gene were isolated from genomic DNA by PCR amplification and screening of an amastigote cDNA library . Sequence analysis of the Llacys1 gene showed a high identity to sequence of Leishmania (L.) pifanoi Lpcys1, Leishmania (L.) major cpa, Leishmania (L.) mexicana LCPa, and Leishmania (L.) chagasi Ldccys2 . The Llacys1 gene is present in a single copy per L . (L.) amazonensis haploid genome and was mapped on a chromosome of approximately 700 kb . Two transcripts of the Llacys1 gene were identified, one of 2.4 kb transcribed in both forms of L . (L.) amazonensis, and another of 1.6 kb weakly expressed in amastigotes . Related forms of Llacys1 gene exist in other species of Leishmania genus, including L . (L.) major, L . (L.) mexicana, L . (L.) chagasi and Leishmania (V.) braziliensis . The Llacys1 expression in Escherichia coli was obtained when the nucleotide sequence corresponding to the signal sequence was deleted, suggesting that this signal sequence was recognised by Escherichia coli and cleaved, generating a truncated protein.

Biochemistry, 2003 Apr 29, 42(16), 4613 - 25
Escherichia coli uracil- and ethenocytosine-initiated base excision DNA repair: rate-limiting step and patch size distribution; Sung JS et al.; The rate, extent, and DNA synthesis patch size of base excision repair (BER) were measured using Escherichia coli GM31 cell-free extracts and a pGEM (form I) DNA substrate containing a site-specific uracil or ethenocytosine target . The rate of complete BER was stimulated (approximately 3-fold) by adding exogenous E . coli DNA ligase to the cell-free extract, whereas addition of E . coli Ung, Nfo, Fpg, or Pol I did not stimulate BER . Hence, DNA ligation was identified as the rate-limiting step in the E . coli BER pathway . The addition of exogenous DNA polymerase I caused modest inhibition of BER, which was overcome by concomitant addition of DNA ligase . Repair patch size determinations were performed to assess the distribution of DNA synthesis associated with both uracil- and ethenocytosine-initiated BER . During the early phase (0-5 min) of the BER reaction, the large majority of repair events resulted from short patch (1-nucleotide) DNA synthesis . However, during the late phase (>10 min) both short and long (2-20 nucleotide) patches were observed, with long patch BER progressively dominating the repair process . In addition, the patch size distribution was influenced by the ratio of DNA polymerase I to DNA ligase activity in the reaction . A novel mode of BER was identified that involved DNA synthesis tracts of >205 nucleotides in length and termed very-long patch BER . This BER process was dependent upon DNA polymerase I since very-long patch BER was inhibited by DNA polymerase I antibody and addition of excess DNA polymerase I reversed this inhibition.

J Inherit Metab Dis, 2002 Dec, 25(8), 661 - 71
Methionine adenosyltransferase I/III deficiency: two Korean compound heterozygous siblings with a novel mutation; Kim SZ et al.; Two Korean sisters, one detected during neonatal screening, the other ascertained at age 3 years during family screening, have persistent hypermethioninaemia without elevation of plasma tyrosine or severe liver disease . Plasma total homocysteine (tHcy) is mildly elevated, but not so markedly as to establish a diagnosis of homocystinuria due to cystathionine beta-synthase (CBS) deficiency . CBS deficiency was ruled out by the presence of slightly elevated concentrations of plasma cystathionine . Although the plasma concentrations of methionine were markedly elevated, plasma S-adenosylmethionine (AdoMet) was not . This pattern of metabolic abnormalities suggested that the patients have deficient activity of methionine adenosyltransferase (MAT) in their livers (MAT I/III deficiency) . Molecular genetic studies demonstrate that each patient is a compound heterozygote for two mutations in MAT1A, the gene that encodes the catalytic subunit that composes MAT I and MAT III: a previously known inactivating G378S point mutation, and a novel W387X truncating mutation . W387X mutant protein, expressed in E . coli and purified, has about 75% of wild-type activity . Negative subunit interaction between the mutant subunits is suggested to explain the hypermethioninaemia of these sisters . They have had normal growth and development and have no mental retardation, neurological abnormalities, or other clinical problems . They are the first individuals of Korean descent proven to have MAT I/III deficiency.

EMBO Rep, 2003 May, 4(5), 517 - 22
Otubains: a new family of cysteine proteases in the ubiquitin pathway; Balakirev MY et al.; The modification of cellular proteins by ubiquitin (Ub) is an important event that underlies protein stability and function in eukaryotes . Protein ubiquitylation is a dynamic and reversible process; attached Ub can be removed by deubiquitylating enzymes (DUBs), a heterogeneous group of cysteine proteases that cleave proteins precisely at the Ub-protein bond . Two families of DUBs have been identified previously . Here, we describe new, highly specific Ub iso-peptidases, that have no sequence homology to known DUBs, but which belong to the OTU (ovarian tumour) superfamily of proteins . Two novel proteins were isolated from HeLa cells by affinity purification using the DUB-specific inhibitor, Ub aldehyde (Ubal) . We have named these proteins otubain 1 and otubain 2, for OTU-domain Ubal-binding protein . Functional analysis of otubains shows that the OTU domain contains an active cysteine protease site.

Infect Immun, 2003 May, 71(5), 2960 - 5
Binding of intimin from enteropathogenic Escherichia coli to lymphocytes and its functional consequences; Goncalves NS et al.; Intimin-conjugated fluorescent beads bind to spleen CD4 T cells and Peyer's patch, mesenteric lymph node, and cecal follicle lymphocytes, with less binding to lamina propria T cells and intraepithelial lymphocytes . Intimin costimulates proliferation of spleen CD4 T cells and cells from organized lymphoid tissues but does not costimulate cells from the lamina propria of normal or inflamed colon.

Infect Immun, 2003 May, 71(5), 2911 - 5
fhuA of Actinobacillus pleuropneumoniae encodes a ferrichrome receptor but is not regulated by iron; Mikael LG et al.; The swine pathogen Actinobacillus pleuropneumoniae possesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore . Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A . pleuropneumoniae under conditions of iron repletion and restriction . Reverse transcription-PCR confirmed that A . pleuropneumoniae fhuA expression is not upregulated in response to low iron levels . An A . pleuropneumoniae fhuA deletion mutant was generated and showed abolishment of ferrichrome uptake.

Infect Immun, 2003 May, 71(5), 2892 - 6
Expression of a luxS gene is not required for Borrelia burgdorferi infection of mice via needle inoculation; Hubner A et al.; The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria . A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities . Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5 alpha, AI-2-like activity could not be detected within B . burgdorferi culture supernatants or concentrated cell lysates . Finally, a luxS-deficient mutant of B . burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation . Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B . burgdorferi.

Infect Immun, 2003 May, 71(5), 2787 - 97
Modulation of myosin light-chain phosphorylation by p21-activated kinase 1 in Escherichia coli invasion of human brain microvascular endothelial cells; Rudrabhatla RS et al.; Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) . Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the E . coli invasion of HBMEC . The invasive E . coli induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria . We also show that invading E . coli downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK) . Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits E . coli invasion significantly with a concomitant decrease in MLC phosphorylation . The inhibition of E . coli invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points . In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK . Taken together, these results suggest that E . coli invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of E . coli into HBMEC.

Genes Dev, 2003 Apr 15, 17(8), 1030 - 42
Functional interactions between the transcription and mRNA 3' end processing machineries mediated by Ssu72 and Sub1; He X et al.; Transcription and processing of pre-mRNA are coupled events . By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae . Our results demonstrate that Ssu72 is required for 3' end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination . The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72 . Ssu72 interacts physically and genetically with the Pta1 subunit of CPF . Overexpression of PTA1 causes synthetic lethality in an ssu72-3 mutant . Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression of SUB1 suppresses the growth and processing defect of a pta1 mutation . Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive . Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3' end processing machineries might facilitate transitions in the RNAP II transcription cycle.

Genes Dev, 2003 Apr 15, 17(8), 971 - 6
Crystal structure of the Drosophila Mago nashi-Y14 complex; Shi H et al.; Pre-mRNA splicing is essential for generating mature mRNA and is also important for subsequent mRNA export and quality control . The splicing history is imprinted on spliced mRNA through the deposition of a splicing-dependent multiprotein complex, the exon junction complex (EJC), at approximately 20 nucleotides upstream of exon-exon junctions . The EJC is a dynamic structure containing proteins functioning in the nuclear export and nonsense-mediated decay of spliced mRNAs . Mago nashi (Mago) and Y14 are core components of the EJC, and they form a stable heterodimer that strongly associates with spliced mRNA . Here we report a 1.85 A-resolution structure of the Drosophila Mago-Y14 complex . Surprisingly, the structure shows that the canonical RNA-binding surface of the Y14 RNA recognition motif (RRM) is involved in extensive protein-protein interactions with Mago . This unexpected finding provides important insights for understanding the molecular mechanisms of EJC assembly and RRM-mediated protein-protein interactions.

Chem Res Toxicol, 2003 Apr, 16(4), 487 - 92
Carbon dioxide modulation of peroxynitrite-induced mutagenesis of the supF gene in pSP189; Pamir B et al.; Peroxynitrite (ONOO(-)), a potent oxidant formed by the reaction of nitric oxide with superoxide anion, may play a significant role as an intermediate in NO(*)-related cytotoxicity and genotoxicity . In addition to causing other types of toxicity, peroxynitrite damages DNA and induces mutations in genetic targets following in vitro or in vivo exposure . It has recently been established that the reaction of CO(2) with ONOO(-) significantly changes its chemistry in biological media . The objective of this investigation was to characterize impacts of CO(2) on peroxynitrite-induced mutagenesis of the supF gene in the shuttle vector pSP189 . The dose-response relationship between ONOO(-) concentration and mutation frequency was determined following bolus exposure of the plasmid suspended in 150 mM phosphate buffer at pH 7.4, with and without addition of 25 mM sodium bicarbonate . After treatment, plasmids were replicated in Escherichia coli MBL50 cells for mutant identification . CO(2) significantly reduced the mutagenic potency of peroxynitrite, in that increases in mutant fraction were reduced by 47-77% at ONOO(-) doses ranging from 0 to 4 mM . We also characterized the spectrum of mutations induced under these conditions and found mutational hotspots at positions 110, 113, 116, 141, 156, 168, and 172 in mutants induced in the presence of CO(2) and at positions 113, 124, 126, 141, and 156 in those induced in its absence . Among the 22 guanines present in the 84 nucleotide supF sequence, 18 were sites of mutation, including four mutational hotspots (G113, G116, G141, and G156), in plasmids exposed to ONOO(-) in the presence of bicarbonate . After exposure in the absence of bicarbonate, 14 of the 22 guanines were mutation sites, with hotspots located at five (G113, G124, G126, G141, and G156) . Remaining mutations were located almost exclusively at cytosine residues . It is evident from these data that reactive intermediates formed through reaction of ONOO(-) with CO(2) play an important role in the mutagenicity of ONOO(-).

Hum Cell, 2002 Sep, 15(3), 130 - 7
Gene therapy using tissue-specific replication competent HSV; Miyatake S; Tissue- or cell-specific targeting of vectors is critical to the success of gene therapy . I describe a novel approach to viral-mediated gene therapy, where viral replication and associated cytotoxicity are limited to a specific cell-type by the regulated expression of an essential immediate-early viral gene product . This is illustrated with two herpes simplex virus type 1 vectors (G92A and d12.CALP) whose growth are restricted to albumin- or calponin-expressing cells, respectively . G92A was constructed by inserting an albumin enhancer/promoter--ICP4 transgene into the thymidine kinase gene of mutant herpes simplex virus type 1 d120, deleted for both copies of the ICP4 gene . This vector also contains the Escherichia coli lacZ gene under control of the thymidine kinase promoter, a viral early promoter, to permit easy detection of infected cells containing replicating vector . In the adult, albumin is expressed uniquely in the liver and in hepatocellular carcinoma and is transcriptionally regulated . G92A efficiently replicated in vitro in two human hepatoma cell lines expressing albumin, but not in three human non-hepatoma, albumin-non-expressing tumor cell lines, while all cell lines were equally susceptible to a tissue non-specific HSV recombinant, hrR3 . In vivo, G92A replicated well in subcutaneous xenografts of human hepatoma cells (Hep3B) in athymic mice, but not in non-hepatoma subcutaneous tumors (PC3 and HeLa), whereas, hrR3 replicated well in both tumor types . Intratumoral inoculation of G92A inhibited the growth of established subcutaneous hepatoma tumors in nude mice, but not prostate tumors . D12CALP also revealed the cell-specific replication to leiomyosarcoma in which calponin expression was augmented . Using hrR3, we demonstrated inhibition of re-stenosis of rat carotid arteries caused by balloon injury . The antiproliferative effects of this virus was marked in the proliferating smooth muscle cells, however, there still remained the fear for the injury of the endothelial cells . Confining a productive, cytotoxic viral infection to a specific cell-type should be useful for tumor therapy and the ablation of specific cell-types for the generation of animal models of disease . Further experiments using d12CALP will be focused on the arteriosclerosis due to balloon angioplasty or organ transplantation.

Am Nat, 2003 Mar, 161(3), 367 - 79
Assessing the cost of mounting an immune response; Bonneaud C et al.; The evolution of parasite resistance has often been assumed to be governed by antagonistic selection pressures . Defense against pathogens, by mounting an immune response, confers evident benefits but may also incur costs, so that the optimal level of defense is expected to depend on the balance between benefits and costs . Although the benefits of immune surveillance are well known, estimates of costs are still equivocal . Here we studied the behavioral and physiological modifications associated with exposure to a nonreplicating antigen (lipopolysaccharide {LPS} of Escherichia coli) in a passerine species, the house sparrow (Passer domesticus) . We further investigated whether the behavioral and physiological changes provoked by LPS induced measurable repercussions on life-history traits, such as the breeding effort and reproductive success . Finally, we tested whether the trade-off between immune activation and breeding effort was modulated by the workload required to feed the brood . Exposure to LPS reduced activity and increased body mass loss of captive individuals; similarly, LPS injection induced a dramatic drop in feeding rate and reproductive success of breeding females . However, this reduction depended on brood size, suggesting that the strength of the trade-off between immune activation and reproduction was affected by the workload required to feed the brood . Overall, this study stresses the magnitude of costs associated with mounting immune responses and the ecological and evolutionary consequences for natural populations.

RNA, 2003 May, 9(5), 574 - 85
Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9; Jackman JE et al.; Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature . It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae . m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown . We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10 . Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity . Yeast Trm10p purified from E . coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion . Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method . There is no obvious growth defect of trm10-Delta/trm10-Delta strains . Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs . Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

RNA, 2003 May, 9(5), 566 - 73
The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome; Gu SQ et al.; The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence . SRP binds at the peptide exit of the large ribosomal subunit . Structural details of the interaction are not known . Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh . Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP . Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit . The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome . These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.

RNA, 2003 May, 9(5), 518 - 32
The effect of a single, temperature-sensitive mutation on global gene expression in Escherichia coli; Li Y et al.; High-density DNA microarrays have been used to explore the genomic profiling of gene expression of a defective Escherichia coli strain with a temperature-sensitive mutation in the protein component of RNase P . A novel gene cluster was discovered in which two of the genes are known substrates of RNase P . The expression pattern of essential genes and gene discovery from intergenic regions, for which other new transcripts are found, are also discussed.

Protein Eng, 2003 Mar, 16(3), 229 - 40
The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity; Birdsall DL et al.; Cysteine is the only variant of D169, a cofactor-binding residue in thymidylate synthase, that shows in vivo activity . The 2.4 A crystal structure of Escherichia coli thymidylate synthase D169C in a complex with dUMP and the antifolate CB3717 shows it to be an asymmetric dimer, with only one active site covalently bonded to dUMP . At the active site with covalently bound substrate, C169 S gamma adopts the roles of both carboxyl oxygens of D169, making a 3.6 A S...H{bond}N hydrogen bond to 3-NH of CB3717 and a 3.4 A water-mediated hydrogen bond to H212 . Analogous hydrogen bonds formed during the enzyme reaction are important for cofactor binding and are postulated to contribute to catalysis . The C169 side chain is likely to be ionized, making it a better hydrogen bond acceptor than a neutral sulfhydryl group . At the second active site, C169 S gamma makes a shorter (3 A) hydrogen bond to the 3-NH of CB3717, CB3717 is approximately 1.5 A out of its binding site and there is no covalent bond between dUMP and the catalytic cysteine . Changes to partitioning among productive and non-productive conformations of reaction intermediates may contribute as much, if not more, to the diminished activity of this mutant than reduced stabilization of transition states.

Genetics, 2003 Apr, 163(4), 1483 - 96
Regulating general mutation rates: examination of the hypermutable state model for Cairnsian adaptive mutation; Roth JR et al.; In the lac adaptive mutation system of Cairns, selected mutant colonies but not unselected mutant types appear to arise from a nongrowing population of Escherichia coli . The general mutagenesis suffered by the selected mutants has been interpreted as support for the idea that E . coli possesses an evolved (and therefore beneficial) mechanism that increases the mutation rate in response to stress (the hypermutable state model, HSM) . This mechanism is proposed to allow faster genetic adaptation to stressful conditions and to explain why mutations appear directed to useful sites . Analysis of the HSM reveals that it requires implausibly intense mutagenesis (10(5) times the unselected rate) and even then cannot account for the behavior of the Cairns system . The assumptions of the HSM predict that selected revertants will carry an average of eight deleterious null mutations and thus seem unlikely to be successful in long-term evolution . The experimentally observed 35-fold increase in the level of general mutagenesis cannot account for even one Lac(+) revertant from a mutagenized subpopulation of 10(5) cells (the number proposed to enter the hypermutable state) . We conclude that temporary general mutagenesis during stress is unlikely to provide a long-term selective advantage in this or any similar genetic system.

Genetics, 2003 Apr, 163(4), 1255 - 71
Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation; Miranda A et al.; RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E . coli . The low viability of recA recBC mutants vs . recA mutants indicates the existence of RecA-independent roles for RecBCD . To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a DeltarecA DeltarecBCD mutant and determined the viability of resulting strains . Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions . We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of sigma-replicating chromosomes could be one of the RecBCD's roles . However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the DeltarecA DeltarecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.

Genetics, 2003 Apr, 163(4), 1243 - 54
Conjugational hyperrecombination achieved by derepressing the LexA regulon, altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12; Lanzov VA et al.; The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions . Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line . Using these conditions we showed a cumulative effect of various mutations on FRE . Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE . The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE . Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE . Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold . We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system . We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.

Blood, 2003 Aug 15, 102(4), 1323 - 32 Epub 2003 Apr 17.
Molecular and functional analysis of Shiga toxin-induced response patterns in human vascular endothelial cells; Matussek A et al.; Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure . EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis . However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs . In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS . We found that Stxs elicited few, but reproducible, changes in gene expression . The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease . In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.

Intensive Care Med, 2003 Jun, 29(6), 904 - 14 Epub 2003 Apr 08.
Effects of exogenous recombinant human granulocyte colony-stimulating factor (filgrastim, rhG-CSF) on neutrophils of critically ill patients with systemic inflammatory response syndrome depend on endogenous G-CSF plasma concentrations on admission; Weiss M et al.; OBJECTIVE: To investigate the effects of exogenous recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) application on the neutrophils of patients at risk of sepsis following major trauma or operation . DESIGN: Randomized controlled trial . SETTING: Surgical intensive care unit and research laboratory of a university hospital . PATIENTS: Twenty-seven patients with systemic inflammatory response syndrome (SIRS) . INTERVENTIONS: Thirteen patients were treated with filgrastim (1 micro g.kg.24 h) for 10 days as a continuous infusion . Fourteen patients served as controls . MEASUREMENTS AND RESULTS: Surface expression of FcgammaR type I (CD64), phagocytosis of E . coli, and the E . coli-induced oxidative burst of neutrophils were tested by flow cytometry . On the first postoperative/posttraumatic day, endogenous G-CSF plasma concentrations were <300 pg/ml in seven controls (subgroup 1) and nine filgrastim patients (subgroup 3), and were already elevated with >500 pg/ml in seven controls (subgroup 2) and four filgrastim patients (subgroup 4) . G-CSF values ( P=0.0026, subgroup 1/3; P=0.0167, 2/4), neutrophil counts ( P=0.0026, 1/3; P=0.0167, 2/4), and CD64 expression ( P=0.0013, 1/3) were higher in filgrastim-treated than non-treated subgroups, but not phagocytic and burst activities . From day zero to day 1, phagocytosis decreased in subgroups 1 (5/7 patients) and 3 (5/9), but increased in subgroups 2 (5/7) and 4 (3/4), and respiratory burst activity decreased in subgroup 3 (8/9) . CONCLUSIONS: Besides activation of neutrophil maturation, low-dose rhG-CSF application in postoperative patients with SIRS has different effects on neutrophil functions, in part depending on already endogenously produced G-CSF.

J Clin Microbiol, 2003 Apr, 41(4), 1775 - 8
Distribution of the saa gene in strains of Shiga toxin-producing Escherichia coli of human and bovine origins; Jenkins C et al.; Certain strains of Shiga toxin-producing Escherichia coli (STEC) which do not have the locus of enterocyte effacement pathogenicity island carry the STEC autoagglutinating adhesin (saa) gene . The distribution of the saa gene in STEC isolates from patients with hemolytic-uremic syndrome (HUS), patients with less severe diarrheal disease, asymptomatic individuals, and healthy cattle was examined . saa-positive strains were detected more frequently (P < 0.001) in STEC strains from bovines (32 of 56 strains) than in those from humans (8 of 91 strains) . No significant association (P = 0.135) was found between the saa gene and STEC isolated from patients with HUS (6 of 46 strains) or diarrhea (2 of 29 strains) and from healthy controls (0 of 16 strains).

J Biol Chem, 2003 Jun 13, 278(24), 21592 - 600 Epub 2003 Apr 07.
Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression; McMahon M et al.; Keap1 is a negative regulator of Nrf2, a bZIP transcription factor that mediates adaptation to oxidative stress . Previous studies suggested this negative regulation is a consequence of Keap1 controlling the subcellular distribution of Nrf2 . We now report that Keap1 also controls the total cellular level of Nrf2 protein . In the RL34 non-transformed rat liver cell line, Nrf2 was found to accumulate rapidly in response to oxidative stress caused by treatment with sulforaphane, and the accumulation resulted from inhibition of proteasomal-mediated degradation of the bZIP protein . By heterologously expressing in COS1 cells epitope-tagged Nrf2 and an Nrf2DeltaETGE mutant lacking the Keap1-binding site, in both the presence and absence of Keap1 we demonstrate that Nrf2 is subject to ubiquitination and proteasomal degradation independently of both Keap1 and the redox environment of the cell . In oxidatively stressed cells, this is the sole mechanism responsible for Nrf2 degradation . However, under homeostatic conditions Nrf2 is subject to a substantially more rapid mode of proteasomal degradation than it is in oxidatively stressed cells, and this rapid turnover of Nrf2 requires it to interact with Keap1 . Within Nrf2, the N-terminal Neh2 domain is identified as the redox-sensitive degron . These data suggest that Keap1 negatively regulates Nrf2 by both enhancing its rate of proteasomal degradation and altering its subcellular distribution.

J Biol Chem, 2003 Jun 27, 278(26), 23706 - 13 Epub 2003 Apr 07.
Insight into the role of Escherichia coli MobB in molybdenum cofactor biosynthesis based on the high resolution crystal structure; McLuskey K et al.; Two proteins, which are co-transcribed in Escherichia coli (MobA and MobB), are involved in the attachment of a nucleotide moiety to the molybdenum cofactor to form active molybdopterin guanine dinucleotide . Although not essential for this process, the dimeric MobB increases the activation of molybdoenzymes, incorporating this cofactor by a mechanism that is not understood . The structure of MobB has been elucidated in two crystal forms, one of which has provided a model at 1.9-A resolution with Rwork and Rfree values of 21.5 and 28.7%, respectively . The MobB subunit displays an alpha/beta-fold arranged into a major and minor domain, the latter of which is inserted between the major and minor domains of the partner subunit, creating an elongated dimer constructed around a 16-stranded beta-sheet . Structural homologues have been identified, and they include a number of nucleotide-binding proteins . Comparisons indicate that although the phosphate-binding regions are highly conserved, MobB lacks the elements of structure required to interact with and efficiently bind a nucleotide base . In the present structure, a sulfate is bound to the Walker A phosphate-binding motif of MobB . The possibility that MobB forms a complex with the nucleotide-binding MobA, the protein with which it is co-transcribed, is explored, and modeling suggests that such a MobA:MobB complex is feasible . This hypothesis is supported by recent biochemical evidence indicating that MobB interacts with several proteins involved in various stages of molybdenum cofactor biosynthesis including MobA . We propose therefore that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of molybdopterin guanine dinucleotide.

J Biol Chem, 2003 Jun 20, 278(25), 22325 - 30 Epub 2003 Apr 07.
Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins; Lillig CH et al.; Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase . Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate . We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239 . A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism . This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S . However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2 . Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.

Biotechnol Bioeng, 2003 Jun 30, 82(7), 809 - 17
Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer; Menzella HG et al.; An important characteristic of promoters used in recombinant protein production in Escherichi coli is their inducibility in a simple and cost-effective manner . The IPTG inducible promoters lac, tac, and trc are powerful and widely used for basic research . However, the use of IPTG in large-scale production is undesirable due to its high cost and toxicity . The promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source . Nevertheless, the use of this sugar in industrial processes has several drawbacks, which result in low volumetric yields and difficulties in process control . We have genetically engineered a BL21 strain to allow the efficient use of lactose as inducer in fed-batch cultures . Two modifications were introduced, the exchange of the wild-type lac operator by a constitutive one (lacO(c)) and the replacement of the gal alleles to recover the Gal(+) phenotype . The constitutive expression of the lac operon overcame the negative effects that the Lac nongenetic heterogeneity of wild-type E . coli introduces when lactose is used as inducer . The gal(+) genotype allowed the complete use of the lactose as carbon and energy source . The relevance of these two modifications in the efficient utilization of lactose as inducer was demonstrated in fed-batch cultures of the novel recombinant strain MP101 harboring expression vectors containing the calf prochymosin gene or the pccB gene, which encodes for the carboxyltransferase component of a propionyl-CoA carboxylase complex from Streptomyces coelicolor . Similar levels of recombinant protein production (up to 16 g/L) were obtained by using either lactose or IPTG as inducers, which confirmed the success of the genetics modifications introduced .

Zentralbl Chir, 2003 Apr, 128(4), 291 - 7
{Functions of circulating and intra-abdominal polymorphonuclear leukocytes during human secondary peritonitis}; Holzer K et al.; INTRODUCTION: Aim of the study was to characterize different functions of circulating and emigrated, intra-abdominal polymorphonuclear leukocytes (cPMNs, ePMNs) during human secondary peritonitis . METHODS: In patients (n=25) with diffuse secondary peritonitis circulating and emigrated PMNs were characterized intra- and until 96 h postoperatively . Patients were allocated to two different groups, e . g . patients with septic complications (shock, organ failure, n=11) and patients without complications (n=14) during peritonitis . In addition a control group of patients (n=10) with abdominal surgery but without peritonitis was investigated . The lucigenin- and luminol-enhanced chemiluminescence was used to determine extra- and intracellular oxygen radical generation of PMNs . Besides spontaneous oxygen radical generation of PMNs, stimulated radical production was investigated after the addition of ionophores A23 187 and C3-coated zymosan . Phagocytosis by PMNs was characterized with opsonized E . coli bacteria and fluorescence-activated cell analysis . RESULTS: Especially patients with complicated peritonitis had strong and long-lasting changes of PMNs functions . The toxic and tissue-destroying production of extracellular oxygen radicals by circulating PMNs was enhanced (e . g., A23 187 - stimulated oxygen radical generation 433 +/- 89 cpm/cPMNs (peritonitis with complications) versus 90 +/- 30 cpm/cPMNs (peritonitis without complications) versus 110 +/- 44 cpm/cPMNs (controls), p < 0.05) . Phagocytosis (58 +/- 9 % (ePMNs, peritonitis with complications) versus 81 +/- 6 % (ePMNs, peritonitis without complications) versus 82.2 +/- 1.6 % (ePMNs, controls), p < 0.05) and phagocytosis-associated intracellular oxygen radical generation (8.23 +/- 1.6 x 10(3) cpm/ePMNs (peritonitis with complications) versus 25.2 +/- 5.2 x 10(3) cpm/ePMNs (peritonitis without complications) versus 11.7 +/- 2.8 cpm x 10(3) cpm/ePMNs (controls) p < 0.05) were suppressed . CONCLUSION: Not for all patients with peritonitis does it seem favourable to modulate PMNs-functions . If immunomodulation would be able to down-regulate exaggerated functions of circulating PMNs and to up-regulate the suppressed functions of emigrated PMNs patients with complicated peritonitis might benefit from this therapy.

Toxicol Sci, 2003 May, 73(1), 60 - 5 Epub 2003 Apr 15.
Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen; Cheng Q et al.; Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation . Despite the importance of this chemical, little is known about its mode of action . We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C . elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain . Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air . All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism . We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism . Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3% . This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing . Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5091 - 6 Epub 2003 Apr 16.
Identifying residue-residue clashes in protein hybrids by using a second-order mean-field approach; Moore GL et al.; In this article, a second-order mean-field-based approach is introduced for characterizing the complete set of residue-residue couplings consistent with a given protein structure . This information is subsequently used to classify protein hybrids with respect to their potential to be functional based on the presenceabsence and severity of clashing residue-residue interactions . First, atomistic representations of both the native and denatured states are used to calculate rotamer-backbone, rotamer-intrinsic, and rotamer-rotamer conformational energies . Next, this complete conformational energy table is coupled with a second-order mean-field description to elucidate the probabilities of all possible rotamer-rotamer combinations in a minimum Helmholtz free-energy ensemble . Computational results for the dihydrofolate reductase family reveal correlation in substitution patterns between not only contacting but also distal second-order structural elements . Residue-residue clashes in hybrid proteins are quantified by contrasting the ensemble probabilities of protein hybrids against the ones of the original parental sequences . Good agreement with experimental data is demonstrated by superimposing these clashes against the functional crossover profiles of bidirectional incremental truncation libraries for Escherichia coli and human glycinamide ribonucleotide transformylases.

J Bacteriol, 2003 May, 185(9), 2967 - 71
Isolation of a new hemimethylated DNA binding protein which regulates dnaA gene expression; d'Alencon E et al.; In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.

J Bacteriol, 2003 May, 185(9), 2835 - 47
Identification of the DNA binding sites of PerA, the transcriptional activator of the bfp and per operons in enteropathogenic Escherichia coli; Ibarra JA et al.; The bundle-forming pilus (BFP) is an important virulence factor for enteropathogenic Escherichia coli (EPEC) . Genes involved in its biogenesis and regulation are tightly regulated by PerA (BfpT), a member of the AraC/XylS family of transcriptional regulators . The aim of this work was to purify PerA and determine its association with bfpA and perA (bfpT) regulatory regions by electrophoretic mobility shift and DNase I footprinting assays . PerA was purified as a maltose-binding protein (MBP) fusion, which was capable of complementing bfpA expression and which was able to restore the localized adherence phenotype of an EPEC perA mutant strain . Upstream of bfpA and perA, MBP-PerA recognized with similar affinity asymmetric nucleotide sequences in which a 29-bp-long AT-rich consensus motif was identified . These DNA motifs share 66% identity and were previously shown, by deletion analysis, to be involved in the PerA-dependent expression of both genes . Interestingly, in perA, this motif spans the sequence between positions -75 and -47, approximately one helix turn upstream of the -35 promoter sequence, while in bfpA, it spans the sequence between positions -83 and -55, approximately two helix turns upstream from the promoter . An additional PerA binding site was identified at the 5' end of the bfpA structural gene, which was not required for its activation . Experiments with LexA-PerA fusions suggested that PerA acts as a monomer to activate the transcription of both perA and bfpA, in contrast to what has been documented for other members of this family of transcriptional regulators.

J Bacteriol, 2003 May, 185(9), 2811 - 9
Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane; Blaudeck N et al.; In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane . Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein . The TorA signal peptide selectively and specifically directed MalE into the Tat pathway . The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway . Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked . These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity . Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

J Bacteriol, 2003 May, 185(9), 2793 - 801
Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways; Hove-Jensen B et al.; An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli . This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B . Hove-Jensen, J . Bacteriol . 178:714-722, 1996) . The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase . The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously . The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP . Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate . The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase . The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end . PhnN was purified almost to homogeneity and characterized . The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor . The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.

J Bacteriol, 2003 May, 185(9), 2731 - 8
Structure-function studies of Escherichia coli RpoH (sigma32) by in vitro linker insertion mutagenesis; Narberhaus F et al.; The sigma factor RpoH (sigma(32)) is the key regulator of the heat shock response in Escherichia coli . Many structural and functional properties of the sigma factor are poorly understood . To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique . Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background . Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity . Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.

Front Biosci, 2003 May 01, 8, s265 - 74
Maltodextrin transport through lamb; Charbit A; The trimeric protein LamB of E . coli K12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane and acts as a non-specific porin for small hydrophilic molecules . LamB serves also as a specific cell surface receptor for phages, including phage lambda . Each monomer consists of an eighteen-stranded antiparallel beta-barrel with nine surface loops (L1 to L9) . Three loops fold into the beta-barrel, with loop L3 constricting the channel about half way . Monomers bind sugars independently of each other . Structural studies of maltoporin in complex with maltodextrin showed that the binding site, located at the channel constriction, was composed of : i) a "greasy slide", a left-handed helical arrangement of aromatic residues extending along the channel providing a hydrophobic path to the glycosyl moieties; and ii) an "ionic track", found on both sides of the channel constriction zone, providing residues available for forming hydrogen bonds with the sugars . The participation of the surface loops that cover the entry of the pore to phage binding and to sugar binding and transport has also been thoroughly investigated . Genetic and biochemical analyses suggest that some of the surface loops participate directly in the orientation and entry of maltooligosaccharides into the channel and, thus, control access to the binding site.

Bioelectrochemistry, 2003 Apr, 59(1-2), 41 - 7
Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode; Johnson D et al.; Adrenodoxin (Adx) functions as a redox protein in the delivery of electrons to all mitochondrial cytochromes P450 . In order to further characterize the human form of this protein, direct electrochemistry of human adrenodoxin (Hadx) has been observed for the first time on a pyrolytic graphite electrode (PGE) modified with poly-L-lysine . A single well-defined redox wave was observed with a midpoint potential of -448+/-3 mV vs . Ag/AgCl (sat . KCl) at scan rates of 10 mV/s and over the pH range 4.0-8.0 . At slow scan rates, the reduction process was close to being electrochemically reversible whereas, at faster scan rates, only quasi-reversibility was observed . A correlation was observed between the peak separation (DeltaE) for the cyclic voltammograms and pH over a wide range of scan rates . The variation of DeltaE with pH was at a minimum (optimum reversibility) at pH 7.0 for all scan rates tested . This correlation may suggest that the direct electrochemistry method could possibly provide a means for determining protein or enzyme activity . The electron transfer rate constant, k(s), was determined to be 0.28 s(-1) at pH 7.0 and a small pH dependence was observed . The results obtained in this study demonstrate the facile nature of direct electron transfer for human adrenodoxin, and provide an estimate of the midpoint reduction potential at a pyrolytic graphite electrode via electrostatic immobilisation.

Bioorg Med Chem Lett, 2003 May 5, 13(9), 1509 - 12
Effects of 8-chlorodeoxyadenosine on DNA synthesis by the Klenow fragment of DNA polymerase I; Chen LS et al.; 8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into synthetic DNA oligonucleotides to determine its effects on DNA synthesis by the 3'-5' exonuclease-free Klenow fragment of Escherichia coli DNA Polymerase I (KF-) . Single nucleotide insertion experiments were used to determine the coding potential of 8-Cl-dAdo in a DNA template . KF- inserted TTP opposite 8-Cl-dAdo in the template, but with decreased efficiency relative to natural deoxyadenosine . Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis . The 2'-deoxyribonucleoside triphosphate analogue, 8-Cl-dATP, was incorporated opposite thymidine (T) approximately two-fold less efficiently than dATP.

Protein Expr Purif, 2003 Apr, 28(2), 362 - 7
A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process; Kohli BM et al.; Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine(4) center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E . coli . Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process . Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs . Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein . Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols . Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm . We propose the "RubyTag" as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties . Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.

Protein Expr Purif, 2003 Apr, 28(2), 357 - 61
Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm; Szurmak B et al.; A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells . The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter . The E1alpha sequence was engineered to include an N-terminal His-tag . When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association . When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer . Recombinant E1 was able to decarboxylate {1-14C}pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.

Protein Expr Purif, 2003 Apr, 28(2), 287 - 92
One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis; Oliveira JS et al.; Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030 . The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents . The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis . We have previously reported the cloning and overexpression of M . tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction . Here, we describe the purification of M . tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells . Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column . The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins . A total of 53 mg of homogeneous enzyme could be obtained from 1L of LB cell culture, with a specific activity value of approximately 18 Umg(-1) . The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

Protein Expr Purif, 2003 Apr, 28(2), 280 - 6
Expression and purification of glycine N-methyltransferases in Escherichia coli; Luka Z et al.; Expression and purification of recombinant mouse, rat, and human glycine N-methyltransferases (GNMTs) in pTYB and pET expression vectors was done in order to prepare the proteins for structure studies of the enzymes from different sources . When human and mouse GNMTs were expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, an unusual cleavage of intein was found . This cleavage takes place at two sites near the N-terminus of intein . This resulted in the appearance of an abnormal GNMT protein after on-column cleavage of the fusion protein, which could not be separated from normal GNMT . For this reason expression of mouse, rat, and human GNMTs was done in the pET-17b expression vector, resulting in the expression of soluble protein at about 20-40mg/L of culture . A new procedure for GNMT isolation after expression in the pET vector was developed . This included only two steps, ammonium sulfate precipitation and ion-exchange chromatography, and resulted in preparations containing 95-97% pure protein . All expressed proteins were tetrameric with molecular weights of 130kDa as determined by size-exclusion chromatography . Activity in Tris buffer at pH 9 of mouse, rat, and human GNMTs was found to be 255, 260, and 540U/mg, respectively . This implies that expressed and purified GNMT proteins are biologically active and suitable for biochemical and structural studies.

Protein Expr Purif, 2003 Apr, 28(2), 252 - 8
Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells; Galfione M et al.; The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity . Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies . These problems limited the use of recombinant 16-kDa PRL . To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system . The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct . Recombinant 16-kDa PRL was detected in both the cell pellet and the medium . About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography . Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE . The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL . N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL . Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli . This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor.

Protein Expr Purif, 2003 Apr, 28(2), 246 - 51
Preparation of uniformly labeled NMR samples in Escherichia coli under the tight control of the araBAD promoter: expression of an archaeal homolog of the RNase P Rpp29 protein; Boomershine WP et al.; We report the first use of the tightly regulated araBAD promoter for generating uniformly labeled samples for NMR . The araBAD promoter provides a distinct advantage over that of the most commonly used protein overexpression systems in bacteria (e.g., in pET vectors: T7lac), in that it provides much tighter control over basal expression . However, use of araBAD-regulated expression for preparation of uniformly labeled protein samples for NMR is complicated by the fact that glucose (the most commonly used carbon source in defined minimal media) indirectly represses transcription, and thus, cannot be used . After experimenting with alternative media, we found that uniformly labeled NMR samples can be prepared using the highly regulated arabinose-inducible promoter and that suitable yields can be obtained in defined minimal media containing glycerol, not glucose, as the carbon source.

Protein Expr Purif, 2003 Apr, 28(2), 241 - 5
Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase; Kriek M et al.; Lipoic Acid Synthase (LipA) can accommodate a {4Fe-4S} cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid . With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon . Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster . This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.

Clin Exp Immunol, 2003 May, 132(2), 309 - 15
A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease; Virginio VG et al.; Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid . Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD . Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity . A cut-off value was determined by receiver-operating-characteristic plots for each antigen . A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test . The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value . In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens . There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.

Cell Mol Biol (Noisy-le-grand), 2002 Dec, 48(8), 861 - 6
Residual activity of human porphobilinogen deaminase with R167Q or R167W mutations: an explanation for survival of homozygous and compound heterozygous acute intermittent porphyrics; Edixhoven-Bosdijk A et al.; To find an explanation for survival of homozygous or compound heterozygous variants of acute intermittent porphyria, we studied the three mutant forms of porphobilinogen deaminase (PBG-d) described in the four reported patients with homozygous acute intermittent porphyria . Wild-type human PBG-d and the PBG-d R167W, R167Q and R173Q mutants were expressed in Escherichia coli and the recombinant mutant human enzyme were examined for enzyme activity . Specific antibodies against human PBG-d detected the three human PBG-d mutants . All three had less than 2% of wild-type enzyme activity when examined under customary assay conditions (pH 8.0), but the R167W and R167Q mutants were found to have about 25% of normal activity when assayed at pH 7.0 . This residual activity at a more physiological pH provides an explanation for survival when these mutations are inherited in a homozygous or compound heterozygous fashion.

J Gastroenterol, 2003 Mar, 38 Suppl 15, 36 - 42
Introduction and overview: recent advances in the immunotherapy of inflammatory bowel disease; Hibi T et al.; Ulcerative colitis (UC) and Crohn's disease (CD) comprise a series of inflammatory bowel disease (IBD) resulting from chronic upregulation of the mucosal immune system . Although the pathogenesis of IBD remains elusive, it appears that there is chronic activation of the immune and inflammatory cascade in genetically susceptible individuals . Current disease management guidelines have therefore focused on the use of antiinflammatory agents, aminosalicylates and corticosteroids . However, some patients are still refractory to these therapies . Recent advances in the understanding of the pathophysiological conditions of IBD have provided new immune system modulators as therapeutic tools . Cytapheresis has demonstrated effectiveness against UC and has practical use in Japan . Immunosuppressive agents including cyclosporin A and tacrolimus (FK506) have expanded the choice of medical therapies available for certain subgroups of patients . Furthermore, biological therapies have begun to assume a prominent role . Studies with chimeric monoclonal anti-TNF-alpha antibody treatment of CD have been reported with dramatic success . Another antiinflammatory cytokine therapy includes anti-IL-6 receptor, anti-IL-12, or toxin-conjugated anti-IL-7 receptor . Given the diversity of proinflammatory products under its control, NF-kappa B may be viewed as a master switch in lymphocytes and macrophages, regulating inflammation and immunity . In the murine 2,4,6-trinitrobenzen sulfonic acid (TNBS) colitis model, an antisense oligonucleotide to NF-kappa B p65 ameliorated inflammation even after induction of colitis . Recently, a clinical pilot trial of this agent demonstrated promising results . Accumulating evidence suggests that luminal bacterial flora is a requisite and central factor in the development of IBD . Probiotic therapies such as a nonpathogenic Escherichia coli strain have been well tolerated, but larger clinical trials are needed . In addition, novel therapeutic strategies targeting adhesion molecules and costimulatory molecules, or enhancing tissue repair, are under investigation . Although some still need more confirmatory studies, these immune therapies will provide new insights into cell-based and gene-based treatment against IBD in the near future.

Vopr Med Khim, 2002 Nov-Dec, 48(6), 577 - 85
{Characterization of S1' subsite specificity of Thermoactinomyces vulgaris carboxypeptidase T by site-directed mutagenesis}; Trachuk LA et al.; The site-directed mutagenesis in the S1'-pocket of Thermoactinomyces vulgaris carboxypeptidase T was performed and two variants containing double D253S, T255D and single T255D mutations were obtained . Precursors of the wild-type carboxypeptidase T and its mutant derivatives were expressed in Escherichia coli as inclusion bodies, refolded, activated by subtilisin, purified by gel efiltration on Superdex G-75 . The catalytic activity with tripeptide substrates DNPAAR and ZAAL was analysed . The introduction of the aspartic residue in 255 position (like to mammalian carboxypeptidase B), insigniticantly enzymatic activity of the double mutant towards both substrates, as measured by Km and Kcat . An addition of the aspartic residue into S1'-binding pocket did not affect single mutant binding with the basic substrate while the Km value for the hydrophobic substrate increased approximately 40 times as compared with wild-type carboxypeptidase T and attained a level comparable with carboxypeptidase B.

Vopr Med Khim, 2002 Nov-Dec, 48(6), 553 - 60
{Ulysses retrotransposon aspartate proteinase (Drosophila virilis)}; Volkov DA et al.; Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals . Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases . Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis . We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones . The appropriate cDNA fragment has been cloned and expressed in E . coli . The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose . The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

Infez Med, 2001 Jun, 9(2), 98 - 100
Expression of P fimbriae of uropathogenic Escherichia coli strains; Cosar G et al.; The occurrence of P fimbriae in a total of 222 uropathogenic Escherichia coli (UPEC) strains was investigated . Out of the total, 31 (14%) were P fimbriated . Of 24 pyelonephritogenic E . coli strains, three (13%) with P fimbriae occurred in children with clinical pyelonephritis, and of 198 E . coli strains 29 (15%) occurred in children with cystitis . Prevalence of P fimbriae of E . coli strains was found to be quite similar in patients with cystitis and pyelonephritis

Technol Health Care, 2003, 11(2), 143 - 7
Inhibition of foreign-body sarcoma by mammalian RNA?
Lavelle SM, MhicIomhair M.
Long-implanted foreign bodies can provoke sarcoma in several species, although rarely in man . In neoplasia, RNA metabolism is highly active . Several kinds of RNA from 5 species were tested for prevention of foreign-body sarcoma in 8 experiments . Nitrocellulose filters or dialysis bags were the implants used . RNA, usually 1 g/dl, was applied to saturate the filters . They were implanted in groups of 30--50 mice, while concomitant controls received saline-saturated implants . The filters surfaces were 9.8 or 19.6 sq cm with pore diameters (roughness) of 0.05, 0.1 and 0.22 mu . The dialysis bags had 6.3 sq cm surface area and RNA content . Tumour yield was logged weekly . It was compared to that of the controls in each experiment and in the series of trials . Mammalian RNA decreased tumour yield in 9 of 11 trials (1 only at p<0.05), ribosomal RNA (85% reduction) being best . Yeast RNA was inactive . RNA from E.Coli increased tumour yield . Two of three samples of bovine RNase also increased tumour yield, an effect counteracted by admixed RNA . The results would indicate an antitumour effect of mammalian RNA, or a species or concomitant of it, which was irregularly present in the fractions tested, perhaps of ribosomal origin and dialysable.

Mol Cell Biol, 2003 May, 23(9), 3320 - 8
Dimerization of MLH1 and PMS2 limits nuclear localization of MutLalpha; Wu X et al.; DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails . The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation . Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer) . We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha . Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS . Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions . This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.

Mol Cell Biol, 2003 May, 23(9), 3152 - 62
Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells; Meservy JL et al.; Expansion of CTG triplet repeats in the 3' untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy . Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures . How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells . To examine the stability of (CTG)(17), (CTG)(98), and (CTG)(183) repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes . Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change . Among recombinants from cells with (CTG)(98) and (CTG)(183), 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels . Analysis of individual rearrangements confirmed the involvement of the CTG repeats . Similar changes were not observed in proliferating (CTG)(98) and (CTG)(183) cells that were not recombinant at APRT . Instead, they displayed high frequencies of small changes in repeat number . The (CTG)(17) repeats were stable in all assays . These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.

J Biol Chem, 2003 Jun 27, 278(26), 23545 - 52 Epub 2003 Apr 15.
The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities; Colin C et al.; Different haloperoxidases, one specific for the oxidation of iodide and another that can oxidize both iodide and bromide, were separated from the sporophytes of the brown alga Laminaria digitata and purified to electrophoretic homogeneity . The iodoperoxidase activity was approximately seven times more efficient than the bromoperoxidase fraction in the oxidation of iodide . The two enzymes were markedly different in their molecular masses, trypsin digestion profiles, and immunological characteristics . Also, in contrast to the iodoperoxidase, bromoperoxidases were present in the form of multimeric aggregates of near-identical proteins . Two full-length haloperoxidase cDNAs were isolated from L . digitata, using haloperoxidase partial cDNAs that had been identified previously in an Expressed Sequence Tag analysis of the life cycle of this species (1) . Sequence comparisons, mass spectrometry, and immunological analyses of the purified bromoperoxidase, as well as the activity of the protein expressed in Escherichia coli, all indicate that these almost identical cDNAs encode bromoperoxidases . Haloperoxidases form a large multigenic family in L . digitata, and the potential functions of haloperoxidases in this kelp are discussed.

J Biol Chem, 2003 May 30, 278(22), 19583 - 6 Epub 2003 Apr 14.
In vitro deamination of cytosine to uracil in single-stranded DNA by apolipoprotein B editing complex catalytic subunit 1 (APOBEC1); Petersen-Mahrt SK et al.; Apolipoprotein B-editing complex catalytic subunit 1 (APOBEC1) is the catalytic component of an RNA-editing complex that deaminates C6666 --> U in apolipoprotein B RNA in gastrointestinal tissue, thereby generating a premature stop codon . Whereas RNA is the physiological substrate of APOBEC1, recent experiments have strongly indicated that, when expressed in bacteria, APOBEC1 and some of its homologues can deaminate cytosine in DNA . Indeed, genetic evidence demonstrates that the physiological function of activation-induced deaminase, a B lymphocyte-specific APOBEC1 homologue, is to perform targeted deamination of cytosine within the immunoglobulin locus, thereby triggering antibody gene diversification . However, biochemical evidence of in vitro DNA deamination by members of the APOBEC family is still needed . Here, we show that deamination of cytosine to uracil in DNA can be achieved in vitro using partially purified APOBEC1 from extracts of transformed Escherichia coli . Thus, APOBEC1 can deaminate cytosine in both RNA and DNA . Strikingly, its activity on DNA is specific for single-stranded DNA and exhibits dependence on local sequence context.

J Biotechnol, 2003 Apr 24, 102(2), 177 - 89
Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli; Guo JQ et al.; A functional single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was produced at high level in Escherichia coli . The variable regions of heavy chain (V(H)) and light chain (V(L)) from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction . The construct of V(H)-linker-V(L) was inserted into a phagemid pCANTAB 5 E followed by selection with the Recombinant Phage Antibody System (RPAS) . Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E . coli BL21(DE3) . With an on-column refolding procedure based on Ni-chelating chromatography, the active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75% confirmed by spectrophotometer . The activity of refolded scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay . The results showed anti-AIF scFv retains the specific binding activity to AIF with an affinity constant of 7.29 x 10(-8) mol l(-1) . The overall yield of anti-AIF scFv with bioactivity in E . coli flask culture was more than 60 mg l(-1).

Biochim Biophys Acta, 2003 Apr 15, 1626(1-3), 102 - 5
A heme-deficient strain of Escherichia coli has a three-base pair deletion in a "hotspot" in hemA; Li J et al.; The key regulatory step in heme biosynthesis in Escherichia coli is at the level of glutamyl-tRNA reductase (GTR), an enzyme which is encoded by hemA . A strain, HU227, with a spontaneous in-frame mutation in hemA has no GTR activity . The mutation is shown to be a three-base deletion at a "hotspot" in the gene . The amino acid sequence in this region is highly conserved.

Biochim Biophys Acta, 2003 Apr 15, 1626(1-3), 75 - 82
tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons; Yamada Y et al.; The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli . Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon . The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons . This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical . The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm) . The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.

J Agric Food Chem, 2003 Apr 23, 51(9), 2569 - 75
Novel broccoli 1-aminocyclopropane-1-carboxylate oxidase gene (Bo-ACO3) associated with the late stage of postharvest floret senescence; Yang CY et al.; A novel 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (Bo-ACO3) was first isolated from senescing broccoli florets by subtractive hybridization . The cDNA clone comprised a 963 bp open reading frame encoding a protein of 321 amino acids . The predicted molecular mass and pI were 36 kDa and 5.42, respectively . Bo-ACO3 shares 68% identity in the coding region with Bo-ACO1 (ACC Ox1) and Bo-ACO2 (ACC Ox2) and is quite divergent from the 3' untranslated regions . Bo-ACO3 transcript was accumulated to high levels only at the late stage of senescence after harvest . Southern blot hybridization using full-length cDNA as a probe suggested that the Bo-ACO3 gene is a single-copy gene in the broccoli genome . The deduced 321 amino acid sequence of Bo-ACO3 shares 70% identity with either Bo-ACO1 or Bo-ACO2 . The BO-ACO3 gene was expressed in Escherichia coli as a 38 kDa active ACO enzyme . It was concluded that Bo-ACO3 is a senescence-associated gene involved in the late-phase senescence of postharvest broccoli.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4672 - 7 Epub 2003 Apr 07.
Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray analysis; Lobner-Olesen A et al.; High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins . Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon . Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase . The latter two strains differed from WT in two ways . First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage . Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction . We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.

Proteins, 2003 May 15, 51(3), 321 - 6
NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor; Wilkinson A et al.; Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase . We have cloned both open reading frames and overexpressed the protein products in Escherichia coli . In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor . Expression of either protein is able to complement E . coli GR501, which carries a temperature-sensitive mutation in ligA . Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M . tuberculosis and S . coelicolor are NAD(+)-dependent DNA ligases .

Am J Hum Genet, 2003 May, 72(5), 1300 - 7 Epub 2003 Apr 14.
2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene; Ofman R et al.; 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency is a novel inborn error of isoleucine degradation . In this article, we report the elucidation of the molecular basis of MHBD deficiency . To this end, we purified the enzyme from bovine liver . MALDI-TOF mass spectrometry analysis revealed that the purified protein was identical to bovine 3-hydroxyacyl-CoA dehydrogenase type II . The human homolog of this bovine enzyme is a short-chain 3-hydroxyacyl-CoA dehydrogenase, also known as the "endoplasmic reticulum-associated amyloid-beta binding protein" (ERAB) . This led to the identification of the X-chromosomal gene involved, which previously had been denoted "HADH2." Sequence analysis of the HADH2 gene from patients with MHBD deficiency revealed the presence of two missense mutations (R130C and L122V) . Heterologous expression of the mutant cDNAs in Escherichia coli showed that both mutations almost completely abolish enzyme activity . This confirms that MHBD deficiency is caused by mutations in the HADH2 gene.

Curr Genet, 2003 May, 43(2), 112 - 20 Epub 2003 Feb 21.
Expression of the carG gene, encoding geranylgeranyl pyrophosphate synthase, is up-regulated by blue light in Mucor circinelloides; Velayos A et al.; A new structural gene, carG, involved in the biosynthesis of carotenoids in the fungus Mucor circinelloides was isolated by heterologous hybridisation, using a probe derived from the Gibberella fujikuroi ggs1 gene . Functional analyses in Escherichia coli showed that the encoded protein has geranylgeranyl pyrophosphate (GGPP) synthase activity . A comparison of the deduced protein with other GGPP synthases suggested that the carG gene might have evolved from other larger genes present in some fungi . The analysis of carG mRNA accumulation after blue light irradiation showed that the expression of this gene is up-regulated by blue light, as happens with the other structural genes involved in carotenogenesis in M . circinelloides . Analysis of the promoter region revealed the presence of several APE-like sequences, which participate in the blue-light regulation of the expression of different fungal genes . These sequences are also present in the above-mentioned Mucor genes and strongly support the idea that this gene plays an important role in the regulation of carotenoid synthesis, despite belonging to a more general metabolic pathway.

Lab Invest, 2003 Apr, 83(4), 561 - 70
Recombinant adenovirus vector bearing antisense macrophage migration inhibitory factor cDNA prevents acute lipopolysaccharide-induced liver failure in mice; Iwaki T et al.; Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in delayed hypersensitivity and cellular immunity . MIF also acts as a proinflammatory cytokine and counterregulates the anti-inflammatory effects of glucocorticoids . Exogenous gene transfer mediated by adenovirus is useful to study a particular molecular function as well as to develop gene therapy strategies . A recombinant adenovirus containing sense and antisense murine MIF (mMIF) cDNA inserts was constructed using a cosmid-terminal protein complex method . The sense mMIF adenovirus (AxCA-mMIFS) efficiently induced mMIF in COS-7 cells that endogenously lack mMIF in a dose-dependent manner . In contrast, the antisense mMIF adenovirus (AxCA-mMIFAS) inhibited the expression of mMIF in NIH3T3 cells in a dose-dependent manner . To assess the pathophysiologic role of MIF in acute liver failure, we induced acute onset of liver damage in mice (male Jcl:ICR) by a combined treatment of Bacille Calmette-Guerin (BCG) and lipopolysaccharide (LPS) . mMIF level in the liver of mice infected with AxCA-mMIFAS showed a significant reduction in MIF production in response to BCG-LPS compared with mice treated without viral infection and with AxCA-mMIFS . In addition, the immunohistochemical staining demonstrated that F4/80 antigen on macrophage was enhanced in liver infected with AxCA-mMIFS but reduced in liver infected with AxCA-mMIFAS . The staining intensity is correlated with the mMIF antigen level in liver tissue . The survival rate of mice infected with AxCA-mMIFAS was significantly higher than that of mice treated with PBS and infected with AxCA-LacZ in BCG-LPS . These results suggest that inhibition of MIF production, using recombinant adenovirus bearing the antisense MIF gene, reduced the mortality rate in BCG-LPS-induced liver failure in mice . This finding might aid in the further development of gene therapy targeting MIF.

Mol Pharmacol, 2003 May, 63(5), 1137 - 47
Characterization of pulmonary CYP4B2, specific catalyst of methyl oxidation of 3-methylindole; Carr BA et al.; The selective toxicity of chemicals to lung tissues is predominantly mediated by the selective expression of certain pulmonary cytochrome P450 enzymes . This report describes the purification, cloning, and characterization of a unique enzyme, CYP4B2, from goat lung . The purified P450 enzyme was isolated by multistep ion exchange chromatography to electrophoretic homogeneity with an apparent molecular mass of 55,000 Da . Western blotting studies demonstrated that CYP4B enzymes were selectively expressed in lung tissues of rabbits, rats, and mice . Two cDNAs, CYP4B2 and CYP4B2v, were cloned from goat lung tissue . CYP4B2 was predicted to be 511 amino acids and approximately 82% similar to the four known CYP4B1 proteins . Concurrently, a variant of the known human CYP4B1 cDNA, that contained a S207 insertion, was cloned from human lung tissue . The modified recombinant goat CYP4B2 was expressed in Escherichia coli and the enzyme catalyzed the N-hydroxylation of the prototypical substrate 2AF . CYP4B2 preferentially dehydrogenated, rather than hydroxylated, the pneumotoxicant 3-methylindole (3MI) (V(max) = 4.61 versus 0.83 nmol/nmol of P450/min, respectively) . To investigate the relevance of covalent heme binding of CYP4 enzymes in CYP4B2-mediated metabolism of 3MI, a site-directed mutant (CYP4B2/A315E) was evaluated . The mutation had little effect on the V(max) of either dehydrogenation or hydroxylation but increased the K(m), which decreased the catalytic efficiency (V/K) for 3MI . The A315E mutation shifted the absorbance maximum of the enzyme from 448 to 451 nm, suggesting that the electron density of the heme was altered . These results demonstrate that CYP4B2 is highly specific for methyl group oxidation of 3MI, without formation of ring-oxidized metabolites, and seems to be predominately responsible for the highly organ-specific toxicity of 3MI in goats.

Genes Dev, 2003 May 1, 17(9), 1101 - 14 Epub 2003 Apr 14.
Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network; Orian A et al.; The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites . We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins . Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells . Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes . This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding . Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs . The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome . Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae . These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression.

Genome Res, 2003 May, 13(5), 905 - 15 Epub 2003 Apr 14.
Tn7-based genome-wide random insertional mutagenesis of Candida glabrata; Castano I et al.; We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7 . Tn7 was used to mutagenize a C . glabrata genomic fosmid library . Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli . Subsequently, these were introduced by recombination into the C . glabrata genome . We found that C . glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement . We were able to generate a highly representative set of approximately 10(4) allele replacements in C . glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis . Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen . In addition, the method might be broadly applicable to mutational analysis of other organisms.

Mol Immunol, 2003 May, 39(15), 965 - 75
Molecular cloning and immunological characterisation of potential allergens from the mould Fusarium culmorum; Hoff M et al.; BACKGROUND: High quality and stability are essential requirements of commercial allergen preparations . Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum.OBJECTIVE: The present study was performed to identify, isolate and characterise allergens of F . culmorum as a basis for a stable allergenic reference material . In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated.METHODS: Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F . culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST) . Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection . A complementary (c)DNA expression library of F . culmorum was prepared and screened for IgE-binding clones using sera from F . culmorum-sensitive individuals . Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E . coli . The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST.RESULTS: Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F . culmorum-specific IgE antibodies . Compared to the in-house extract, nearly all IgE-reactivties in the range of 15-30kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F . culmorum-specific IgE by EAST . IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts . Three IgE binding clones were isolated from the cDNA-library . One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds . The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2) . The third clone (Fus c 3) was not related to known proteins . With sera from 26 individuals sensitised to F . culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively.CONCLUSIONS: F . culmorum may represent an underestimated source of aeroallergens . In contrast to highly labile and poorly standardised F . culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material . A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F . culmorum-sensitised subjects . IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.

Biochem Pharmacol, 2003 Apr 15, 65(8), 1373 - 82
Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock; Veres B et al.; The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc . in critically ill patients has shifted interest towards developing alternative treatments . Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role . In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice . We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities . We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine . Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice . Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.

Micron, 2003, 34(1), 25 - 37
Terminal deletion mutants of myelin basic protein: new insights into self-association and phospholipid interactions; Hill CM et al.; The 18.5kDa isoform of myelin basic protein (MBP) has strong and probably specific interactions with phosphoinositides that are of interest regarding this protein's function, and in effecting its two-dimensional crystallization for structural determination . We have designed and constructed truncation mutants of recombinant 18.5kDa murine myelin basic protein (rmMBP) lacking either the N- or C-terminal third, i.e . rmMBPDeltaN and rmMBPDeltaC, respectively . Both variants rmMBPDeltaC and rmMBPDeltaN generally had a reduced ability to aggregate lipid vesicles, compared to the whole protein, especially at lower protein/lipid ratios . Lipid vesicle cosedimentation showed that both truncated variants exhibited altered binding with phosphatidylinositol (PI) . Incubation of these proteins under monolayers comprising PI and a nickel-chelating lipid yielded crystalline arrays of rmMBPDeltaC (but not rmMBPDeltaN) in the absence of high salt or osmolytes, which are required for crystallization of whole protein . This result suggests that the C-terminal segment of MBP is a significant source of conformational heterogeneity, and its removal will facilitate future planar or three-dimensional crystallization attempts . Incubation of rmMBPDeltaN and rmMBPDeltaC under monolayers comprising phosphatidylinositol-4-phosphate and a nickel-chelating lipid yielded tubular structures of opposite chirality, suggesting a synergistic effect of both termini of MBP in organizing myelin lipids.

Micron, 2003, 34(1), 9 - 18
Direct observations of freeze-etching processes of ice-embedded biomembranes by atomic force microscopy; Yamamoto D et al.; We have fabricated a cryogenic atomic force microscope that is designed for structural investigation of freeze-fractured biological specimens . The apparatus is operated in liquid nitrogen gas at atmospheric pressure . Freeze-fracturing, freeze-etching and subsequent imaging are carried out in the same chamber, so that the surface topography of a fractured plane is easily visualized without ice contamination . A controlled superficial sublimation of volatile molecules allows us to obtain three-dimensional views of ultrastructures of biological membranes.

Anal Biochem, 2003 May 1, 316(1), 66 - 73
Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process; Eon-Duval A et al.; Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA . Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process . Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance . Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant . Calcium chloride is a potent precipitant of high-molecular-weight RNA . The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.

Mol Microbiol, 2003 May, 48(3), 845 - 54
Replication restart in gyrB Escherichia coli mutants; Grompone G et al.; Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases . Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected . We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants . The helicase function of PriA is not essential . The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains . The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation . Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate . We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.

Mol Microbiol, 2003 May, 48(3), 781 - 94
Regulatory and functional co-operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease; Barnich N et al.; Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease . Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon . An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili . Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon . This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly . Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant . Thus, we showed that flagella play a direct role in the adhesion process via active motility . In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.

Mol Microbiol, 2003 May, 48(3), 699 - 712
Regulatory network of acid resistance genes in Escherichia coli; Masuda N et al.; Overexpression of the response regulator EvgA confers an acid-resistant phenotype to exponentially growing Escherichia coli . This acid resistance is partially abolished by deletion of ydeP, yhiE or ydeO, genes induced by EvgA overexpression . Microarray analysis identified two classes of operons (genes) . The first class contains seven operons induced by EvgA overexpression in the absence of ydeO, an AraC/XylS regulator gene . The second class contains 12 operons induced by YdeO overexpression . Operons in the second class were induced by EvgA overexpression only in the presence of ydeO . EvgA is likely to directly upregulate operons in the first class, and indirectly upregulate operons in the second class via YdeO . Analysis using the motif-finding program alignace identified an 18 bp inverted repeat motif in six upstream regions of all seven operons directly regulated by EvgA . Gel mobility shift assays showed the specific binding of EvgA to the six sequences . Introduction of mutations into the inverted repeats upstream of ydeP and b1500-ydeO resulted in reduction in EvgA-induced ydeP and ydeO expression and acid resistance . These results suggest that EvgA binds to the inverted repeats and upregulates the downstream genes . Overexpression of YdeP, YdeO and YhiE conferred acid resistance to exponentially growing cells, whereas GadX overexpression did not . Microarray analysis also identified several GadX-activated genes . Several genes induced by overexpression of YdeO and GadX overlapped; however, yhiE was induced only by YdeO . The acid resistance induced by YdeO overexpression was abolished by deletion of yhiE, gadC, slp-yhiF, hdeA or hdeD, genes induced by YdeO overexpression, suggesting that several genes orchestrate YdeO-induced acid resistance . We propose a model of the regulatory network of the acid resistance genes.

Mol Microbiol, 2003 May, 48(3), 631 - 7
Aminoacyl-tRNA synthesis in archaea: different but not unique; Praetorius-Ibba M et al.; Accurate aminoacyl-tRNA synthesis is essential for correct translation of the genetic code in all organisms . Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system . These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine . Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria . Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis . Most recently a new co-translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria . Thus, whereas archaea contain a preponderance of non-canonical aminoacyl-tRNA synthesis systems most are also found elsewhere albeit less frequently.

Genes Cells, 2003 May, 8(5), 437 - 49
Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro; Higuchi K et al.; BACKGROUND: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability . However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication . Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA . RESULTS: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis . In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis . However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced . CONCLUSIONS: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.

Eur J Biochem, 2003 Apr, 270(8), 1838 - 49
The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase; Miertzschke M et al.; A thermostable enzyme from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus, was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli . Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III . The 774 bp long gene was isolated from a culture sample and cloned into different vectors . Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU, ileY and leuW tRNA genes as a host strain . The lack of thioredoxin reductase (trxB) and glutathione reductase (gor) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol . Purification was performed by heat treatment of the soluble fraction at 80 degrees C for 30 min followed by a two-step ion exchange chromatography . The activity of the enzyme could be maintained . Optimal activity was achieved at 80 degrees C and at a pH of 7 . Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg2+ as described {Ankenbauer, W., Laue, F., Sobek, H., & Greif, M . (2000), patent number WO2001023583} . Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed . In contrast to exonuclease III from Escherichia coli, the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3'-termini of linear double stranded DNA . The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg2+ with Ca2+ ions.

Eur J Biochem, 2003 Apr, 270(8), 1775 - 83
Isocitrate dehydrogenase of Plasmodium falciparum; Wrenger C et al.; Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages . Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell . The gene encoding P . falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence . The protein is very similar to NADP+-dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli . Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion . Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 micro m and 22 micro m, respectively . Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 micro m and that of d-isocitrate was determined to be 40 micro m . Incubation of P . falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites.

Eur J Biochem, 2003 Apr, 270(8), 1767 - 74
Functional analysis of disease-causing mutations in human galactokinase; Timson DJ et al.; Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose . The sugar is phosphorylated at position 1 at the expense of ATP . Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts . Human galactokinase (GALK1) was expressed in and purified from Escherichia coli . The recombinant enzyme was both soluble and active . Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind . The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis . Ten mutations that are known to cause galactosemia were constructed and expressed in E . coli . Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further . Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme . One mutant (A198V) had kinetic properties that were essentially wild-type . These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia.

Eur J Biochem, 2003 Apr, 270(8), 1724 - 34
The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC; Facey SJ et al.; KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions . The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop . The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD . To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region . The results show that also the first loop was translocated independently of YidC and the Sec translocase . We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases . When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC . To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments . Both constructed fragments, which contained KdpD-N (residues 1-448 of KdpD) and the KdpD-C (residues 444-894 of KdpD), readily inserted into the membrane . Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translocase and YidC . This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.

Eur J Biochem, 2003 Apr, 270(8), 1680 - 8
Mutational analysis of plasminogen activator inhibitor-1; Wind T et al.; The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis . PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in beta-sheet A, thereby converting to the so-called latent state . We have investigated the importance of the amino acid sequence of alpha-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition . We grafted regions of the hF/s3A-loop from antithrombin III and alpha1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition . We prepared 28 PAI-1 variants with individual residues in hF and beta-sheet A replaced by an alanine . We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case . Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases . The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins.

Eur J Biochem, 2003 Apr, 270(8), 1672 - 9
Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents; Bodker JS et al.; Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors . Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin . Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A . We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region . Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region . By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule . We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.

Eur J Biochem, 2003 Apr, 270(8), 1662 - 71
A dimer of the FeS cluster biosynthesis protein IscA from cyanobacteria binds a {2Fe2S} cluster between two protomers and transfers it to {2Fe2S} and {4Fe4S} apo proteins; Wollenberg M et al.; Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803 . One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E . coli and purified . The purified protein was found to be mainly dimeric and did not contain any cofactor . Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one {2Fe2S} cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and Mossbauer spectroscopy . Mossbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues . Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding . It was therefore concluded that the {2Fe2S} cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers . The cluster could be transferred to apo ferredoxin, a {2Fe2S} protein, with a half-time of 10 min . Surprisingly, incubation of cluster-containing IscA with apo adenosine 5'-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a {4Fe4S} cluster . This demonstrates that it is possible to build {4Fe4S} clusters from {2Fe2S} units.

Biochem J, 2003 Jul 15, 373(Pt 2), 403 - 8
A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants; Gurskaya NG et al.; We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL) . The A . coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae . Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration . Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222) . Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein . This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form . Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines . Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

Biochemistry (Mosc), 2003 Jan, 68(1), 121 - 8
Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation; Zhang L et al.; Myristoylated G(o)alpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G(o)alpha) and pBB131 (N-myristoyltransferase, NMT) . Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G(o)alpha, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5;-3-O-(thio)triphosphate (GTPgammaS) activation induced disaggregation of the G(o)alpha oligomers to monomers . The G(o)alpha was crosslinked by a cross-linker, N,N-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products . In contrast, no obvious cross-linking occurred when G(o)alpha was pretreated with GTPgammaS . Immunoblot analysis also demonstrated oligomerization of the purified G(o)alpha proteins and its disaggregation triggered by GTPgammaS . These results provided direct evidence for the "disaggregation-coupling" theory and the disaggregation action of GTPgammaS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.

Biochemistry (Mosc), 2003 Feb, 68(2), 188 - 95
Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain; Potekhina ES et al.; LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis . LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis . To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E . coli . Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable . H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides . The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain . The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity . Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone . Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect . Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase . These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

Biochemistry, 2003 Apr 22, 42(15), 4552 - 9
Stability, homodimerization, and calcium-binding properties of a single, variant betagamma-crystallin domain of the protein absent in melanoma 1 (AIM1); Rajini B et al.; AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains . The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus . To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein . The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure . The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine . AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a . Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods . AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane . AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM) . However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence . The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding . These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.

Biochemistry, 2003 Apr 22, 42(15), 4544 - 51
Modification of Escherichia coli thymidylate synthase at tyrosine-94 by 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate; Saxl RL et al.; Evidence is presented that 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of Escherichia coli thymidylate synthase (TS), which covalently modifies Tyr94 at the active site of the enzyme . The inactivation of TS was time and concentration dependent and did not require the folate cofactor . Due to the rapidity of the inactivation process, accurate kinetic parameters could be determined only in the presence of saturating concentrations (1000K(M)) of the competing substrate, dUMP . Under these conditions, a K(I) of 0.36 +/- 0.09 microM and an inactivation rate constant (k(inact)) of 0.53 +/- 0.15 min(-1) were obtained from Kitz-Wilson plots . Electrospray ionization-mass spectrometry (ESI-MS) determined a 412 amu mass increase of TS after inhibition by IP-dUMP with no mass difference being detected for the TS mutants Tyr94Phe or Cys146Ala, thus indicating the importance of these residues for complex formation . The change in WT-TS mass was consistent with covalent modification by IP-dUMP, which was confirmed by proteolytic digestion of the modified protein followed by ESI-MS . By these means, a 43-residue trypsin peptide (residues 54-96), a 16-residue endoAspN peptide (residues 89-104), and an 8-residue endoAspN/endoLysC peptide (residues 89-96), each containing the IP-dUMP adduct, were observed . MS/MS analysis of the IP-dUMP-endoAspN peptide identified a modified 3-residue daughter ion, YGK (residues 94-96) . A mechanistic scheme requiring the participation of Cys146 is proposed for the covalent modification of IP-dUMP by Tyr94, which, unlike an earlier proposal {Kalman, T . I., Nie, Z., and Kamat, A . (2001) Nucleosides Nucleotides Nucleic Acids 20, 869-871}, does not require the release of imidazole for the activation of the inhibitor.

Biochemistry, 2003 Apr 22, 42(15), 4414 - 20
Galactose mutarotase: pH dependence of enzymatic mutarotation; Beebe JA et al.; Here we report pH dependence of kinetic parameters for the mutarotation of alpha-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli . The values of k(cat) and k(cat)/K(m) for the mutarotation of alpha-D-galactose were found to be 1.84 x 10(4) s(-1) and 4.6 x 10(6) M(-1) s(-1), respectively, at pH 7.0 and 27 degrees C . The corresponding values for alpha-D-glucose were 1.9 x 10(4) s(-1) and 5.0 x 10(5) M(-1) s(-1) . Inasmuch as the value of k(cat)/K(m) for the reaction of alpha-D-galactose is 10 times that for alpha-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of alpha-D-glucose should not be diffusion controlled . Therefore, pH-rate profiles should not be distorted by diffusion . The k(cat) for the mutarotation of alpha-D-glucose is independent of pH . Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion . The profile of log k(cat)/K(m) versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and -2 on the alkaline side . The values of pK(a) are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated . On the acid side, ring opening of alpha-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate . A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms . In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation . The groups are postulated to be His 104, His 175, and Glu 309 . Mutations of these residues grossly impair catalytic activity . Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k(cat)/K(m) versus pH to be constructed . Both profiles show breaks on the acid side corresponding to pK(a) values of 5.8 for H104Q and 6.3 for E309Q . Apparently, ring opening of alpha-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.

Acta Virol, 2002, 46(4), 211 - 7
Chimeric Newcastle disease virus nucleocapsid with parts of viral hemagglutinin-neuraminidase and fusion proteins; Rabu A et al.; The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles . Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer . These chimeric proteins were expressed efficiently in soluble form in E . coli as detected by Western blot analysis . Electron microscopy of the purified products revealed that they self-assembled into ring-like particles . These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles . Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.

Yi Chuan Xue Bao, 2002 Dec, 29(12), 1063 - 7
{The cloning and expression of rat insulin-like growth factor I gene}; Liang DC et al.; In this study an exons-connecting technique was used to amplify the complete DNA sequence encoding the mature peptide of IGF-I . Two pairs of primers were synthesized, primer a (Pa) and primer b (Pb) were used to amplify exon 2 coding fragment while primer c (Pc) and primer d (Pd) for amplifying exon 3 coding fragment . Pb was 40 bp, 18 bp at its 5' end was same with the anti-sense of exon 3's 5' end . Pc consisted of 41 bp with 22 bp at its 5' end consistent with the sense of the exon 2's 3' end . Genome DNA was extracted from rat liver . Using rat genome DNA as template PCR was performed with Pa, Pb and Pc, Pd as primers respectively . Two kinds of PCR products were obtained . One was 90 bp corresponding with the exon 2, another was 160 bp corresponding with the exon 3 . Another PCR was done with these two PCR products used as not only template but also primers for each other . A 210 bp DNA fragment was produced, which encodes the mature peptide of rat IGF-I . Firstly the gene was cloned into plasmid pUC18 . Then the recombinant plasmid pUC-IGF-I was digested with restriction endonuclease BamHI and EcoRI, plasmid pGEX-3X was digested with the same enzyme . IGF-I gene was linked into pGEX-3X and expressing plasmid pGEX-IGF-I was constructed . Plasmid pGEX-IGF-I was transformed into E . coli DH5 alpha and induced to express by IPTG . Expressed protein was analysized by SDS-PAGE . A fusion protein of GST-IGFI about 32.5 kDa could be observed . Western blot was performed with goat anti-human IGF-I IgG as primary antibody and donkey anti-goat IgG HRP as secondary antibody . Its result confirmed that the fusion protein really containing IGF-I . GST-IGFI was purified by affinity-chromatography and carrier-protein GST was cut off by Factor Xa . The bioactivity of purified IGF-I was detected with cultured NIH3T3 cell . The recombinant rat IGF-I can promote cell proliferation, shown by the elevation of 3H-TdR incorporation.

Nat Immunol, 2003 May, 4(5), 452 - 6
Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand; Ramiro AR et al.; Somatic hypermutation and class switch recombination are DNA modification reactions that alter the genes encoding antibodies in B lymphocytes . Both of these distinct reactions require activation-induced deaminase (AID) and transcription . Here we show that in Escherichia coli, as in eukaryotic cells, the mutation frequency is directly proportional to the transcription of target genes . Transcription enhances mutation of the nontemplate DNA strand, which is exposed as single-stranded DNA during the elongation reaction, but not mutation of the template DNA strand, which is protected by E . coli RNA polymerase . Our results establish a direct link between AID and transcription and suggest that the role of transcription in facilitating mutation is to provide AID with access to single-stranded DNA.

J Korean Med Sci, 2003 Apr, 18(2), 236 - 41
Effects of MK-801 (dizocilpine) on brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet; Ko SY et al.; We evaluated the efficacy of non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine) as an adjuvant therapy in experimental neonal bacterial meningitis . Meningitis was induced by injecting 10(6) colony forming units of Escherichia coli into the cisterna magna . MK-801 3 mg/kg was given as a bolus intravenous injection, 30 min before the induction of meningitis . MK-801 did not down-modulate the inflammatory parameters, such as increased intracranial pressure, cerebrospinal fluid (CSF) leukocytosis, increased lactate and TNF-alpha levels in the CSF, and hypoglycorrhachia observed in the meningitis group . MK-801 did not significantly attenuate the elevated glutamate concentration in the CSF . However, MK-801 showed some neuroprotective effects as evidenced by significant attenuation of cerebral lipid peroxidation products (conjugated dienes) and increase of brain high-energy phosphate compounds (ATP and PCr) . Improvement in cerebral cortical cell membrane Na+, K+ -ATPase activity did not reach a statistical significance . These results suggest that MK-801 was effective in ameliorating brain injury in neonatal bacterial meningitis, although it failed to attenuate the inflammatory responses.

Plant Physiol, 2003 Apr, 131(4), 1868 - 76
Volatile ester formation in roses . Identification of an acetyl-coenzyme A . Geraniol/Citronellol acetyltransferase in developing rose petals; Shalit M et al.; The aroma of roses (Rosa hybrida) is due to more than 400 volatile compounds including terpenes, esters, and phenolic derivatives . 2-Phenylethyl acetate, cis-3-hexenyl acetate, geranyl acetate, and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var . "Fragrant Cloud." Cell-free extracts of petals acetylated several alcohols, utilizing acetyl-coenzyme A, to produce the corresponding acetate esters . Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA (RhAAT1) encoding a protein with high similarity to several members of the BAHD family of acyltransferases . This cDNA was functionally expressed in Escherichia coli, and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro . The RhAAT1 protein accepted other alcohols such as citronellol and 1-octanol as substrates, but 2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates, suggesting that additional acetyltransferases are present in rose petals . The RhAAT1 protein is a polypeptide of 458 amino acids, with a calculated molecular mass of 51.8 kD, pI of 5.45, and is active as a monomer . The RhAAT1 gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4 of flower development, where scent emission is at its peak.

Plant Physiol, 2003 Apr, 131(4), 1748 - 55
Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity; Abebe T et al.; Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress . Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity . Wheat (Triticum aestivum L . cv Bobwhite) was transformed with the mtlD gene of Escherichia coli . Tolerance to water stress and salinity was evaluated using calli and T(2) plants transformed with (+mtlD) or without (-mtlD) mtlD . Calli were exposed to -1.0 MPa of polyethylene glycol 8,000 or 100 mM NaCl . T(2) plants were stressed by withholding water or by adding 150 mM NaCl to the nutrient medium . Fresh weight of -mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress . Growth of +mtlD calli was not affected by stress . In -mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants . Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in -mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants . However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 micromol g(-1) fresh weight in the callus and 0.6-2.0 micromol g(-1) fresh weight in the leaf) was too small to protect against stress through osmotic adjustment . We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.

J Gen Virol, 2003 May, 84(Pt 5), 1261 - 8
A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication; Bredenbeek PJ et al.; Yellow fever virus (YF) is the prototype member of the Flavivirus genus . Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D . YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E . coli strains . Transcribed RNAs had a specific infectivity of 10(5)-10(6) p.f.u . ( micro g RNA)(-1), and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture . This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3' stem-loop structure in virus replication . The conserved sequences 5'CS and CS1, as well as the 3' stem-loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable . This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.

J Virol, 2003 May, 77(9), 5401 - 14
Protein-protein interactions between hepatitis C virus nonstructural proteins; Dimitrova M et al.; Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses . The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components . Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum . This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome . The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es) . We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system . In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells . We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins . Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

J Virol, 2003 May, 77(9), 5218 - 25
Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells; Guebre-Xabier M et al.; Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees . Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects . Topical delivery of strong adjuvants such as heat-labile enterotoxin from Escherichia coli (LT) induces potent immune responses . We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines . This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN . We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced . Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production . An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation . Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.

J Biol Chem, 2003 Jun 20, 278(25), 22446 - 52 Epub 2003 Apr 11.
DNA loop repair by Escherichia coli cell extracts; Fang WH et al.; The nick-directed DNA repair efficiency of a set of M13mp18-derived heteroduplexes containing 8-, 12-, 16-, 22-, 27-, 45-, and 429-nucleotide loops was determined by in vitro assay . Unpaired nucleotides of each heteroduplex reside within overlapping recognition sites for two restriction endonucleases, permitting independent evaluation of repair occurring on either DNA strand . Our results show that a strand break located either 3' or 5' to the loop is sufficient to direct heterology repair to the nicked strand in Escherichia coli extracts . Strand-specific repair by this system requires Mg2+ and the four dNTPs and is equally efficient on insertions and deletions . This activity is distinct from the MutHLS mismatch repair pathway . Strand specificity and repair efficiency are largely independent of the GATC methylation state of the DNA and presence of the products of mismatch repair genes mutH, mutL, and mutS . This study provides evidence for a loop repair pathway in E . coli that is distinct from conventional mismatch repair.

J Biol Chem, 2003 Jun 20, 278(25), 22460 - 5 Epub 2003 Apr 12.
The second microtubule-binding site of monomeric kid enhances the microtubule affinity; Shiroguchi K et al.; Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase . To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro . Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric . However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain . This was ascertained by binding of Kid fragments lacking the motor domain to microtubules . The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner . KmMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site . Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules . The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid . Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo.

FASEB J, 2003 Jun, 17(9), 1121 - 3 Epub 2003 Apr 08.
Recombinant modular transporters for cell-specific nuclear delivery of locally acting drugs enhance photosensitizer activity; Rosenkranz AA et al.; The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities . Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced . Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin . These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs . The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.

J Mol Biol, 2003 Apr 25, 328(2), 409 - 18
Probing catalytically essential domain orientation in histidine kinase EnvZ by targeted disulfide crosslinking; Cai SJ et al.; EnvZ, a dimeric transmembrane histidine kinase, belongs to the family of His-Asp phosphorelay signal transduction systems . The cytoplasmic kinase domain of EnvZ can be dissected into two independently functioning domains, A and B, whose NMR solution structures have been individually determined . Here, we examined the topological arrangement of these two domains in the EnvZ dimer, a structure that is key to understanding the mechanism underlying the autophosphorylation activity of the kinase . A series of cysteine substitution mutants were constructed to test the feasibility of chemical crosslinking between the two domains . These crosslinking data demonstrate that helix I of domain A of one subunit in the EnvZc dimer is in close proximity to domain B of the other subunit in the same dimer, while helix II of domain A of one subunit interacts with domain B of the same subunit in the EnvZc dimer . This is the first demonstration of the topological arrangement between the central dimerization domain containing the active center His residues (domain A) and the ATP-binding catalysis assisting domain (domain B) in a class I histidine kinase .

J Mol Biol, 2003 Apr 25, 328(2), 395 - 408
Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants; Bullock TL et al.; The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft . In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain . The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction . Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate . At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule . These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested . Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation . The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases .

J Endotoxin Res, 2003, 9(1), 60 - 4
Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance; Medvedev AE et al.; TLR4 and MD-2 are necessary for conferring cellular responsiveness to LPS . Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS re-stimulation, known as 'endotoxin tolerance' . While induction of LPS tolerance has been reported to correlate with down-regulation of cell surface expression of TLR4/MD-2, other mechanisms of LPS tolerance have been revealed that target intracellular intermediates downstream of the TLR4/MD-2 complex . In this study, we sought to examine whether endotoxin tolerance could be induced under conditions where expression of TLR4 and MD-2 proteins is not affected by LPS . Human HEK 293T cells are completely unresponsive to LPS, but acquire high LPS sensitivity following transient transfection with CD14, TLR4, and MD-2 (293T/CD14/TLR4/MD-2 cells), as judged by NF-kappaB activation, ERK 1/2 phosphorylation, and TNF-alpha gene expression . Prior exposure of 293T/CD14/TLR4/MD-2 cells to LPS resulted in a significant decrease of LPS-mediated responses, yet failed to affect expression levels of TLR4 and MD-2 . Thus, altered expression and/or function of intracellular mediators downstream of the TLR4/MD-2 complex play an important role in mediating LPS tolerance.

J Endotoxin Res, 2003, 9(1), 51 - 4
Synergistic and antagonistic interactions between LPS and superantigens; Dalpke AH et al.; Superantigens trigger polyclonal activation of T lymphocytes with cytokine release that eventually may lead to lethal cytokine syndrome (toxic shock) . In contrast, bacterial components that are recognized by Toll-like receptors (e.g . LPS or CpG DNA) primarily target macrophages and dendritic cells . We have analyzed whether superantigens and TLR ligands interact with each other . We found that superantigens synergize with LPS in an IFN-gamma-dependent pathway . More important, we found compelling evidence that superantigens prime the innate immune cell system to a subsequent challenge with endotoxin . This sensitization was critically dependent on T-cell derived IFN-gamma . When we analyzed the underlying molecular mechanisms, we additionally found that TLR stimulation enhanced IFN-gamma-mediated cellular responses . Moreover, TLR ligands induced proteins of the SOCS family thus shutting off IFN-gamma-mediated cellular activation . Since IFN-gamma is synthesized by T cells after superantigen triggering, these results show that superantigen and TLR pathways are interconnected and regulate each other . They further show that the outcome of this interaction may include activation as well as down-regulation of the respective response pattern.

J Endotoxin Res, 2003, 9(1), 33 - 7
Lipopolysaccharide from Rhodobacter sphaeroides is an agonist in equine cells; Lohmann KL et al.; Endotoxemia is associated with the principal causes of death in adult horses and equine neonates and, therefore, veterinary researchers are expending efforts to identify new therapeutic interventions that might be beneficial in these animals . Endotoxin antagonists inhibit interaction of endotoxin with cellular receptors and may be beneficial in the treatment of endotoxemia and sepsis . Diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA) is a potent antagonist of enteric LPS in human cells, but is an agonist in hamster cells . In this study, the effect of lipopolysaccharide from R . sphaeroides (RsLPS) on equine whole blood and isolated monocyte preparations was investigated by comparing tumor necrosis factor (TNF) production in response to RsLPS and Escherichia coli O55:B5 LPS . Our results indicate that RsLPS is a potent agonist in equine cells, which precludes therapeutic use of this agent in equine patients . In contrast to the results in equine cells, RsLPS did not elicit TNF production by itself, and inhibited the response to E . coli O55:B5 LPS in a human monocytic cell line.

Inorg Chem, 2003 Apr 21, 42(8), 2751 - 8
A density functional evaluation of an Fe(III)-Fe(IV) model diiron cluster: comparisons with ribonucleotide reductase intermediate X; Han WG et al.; Using broken-symmetry density functional theory and spin-projection methods, we have examined the electronic structure and properties of a large mixed-valent Fe(III)-Fe(IV) diiron system that displays two bidentate carboxylates and a single mu-oxo moiety as bridging ligands . Two carboxylates and a single oxygen species have long been implicated as core elements of the elusive intermediate X in ribonucleotide reductase . Spectroscopic studies of X have also identified the presence of an additional terminal or bridging oxygen-based ligand . Introduction of a second oxygen and protonated variants thereof in the core of our structural model is favored as a bridging hydroxide based on the lowest energy structure . Mossbauer measurements indicate clearly that the two iron sites of X are distinct and that there is significant electron delocalization onto the oxygen-based ligands . For several examined spin states of our model cluster, Mossbauer parameters from density functional calculations are neither able to differentiate between the iron sites nor reproduce the strong spin delocalization onto the oxygen-based ligands observed experimentally . The combined comparison of the calculated geometries, spin states, spin densities, and Mossbauer properties for our model clusters with available experimental data for X implies that intermediate X is significantly different from the diiron structural models examined herein.

Cent Eur J Public Health, 2003 Mar, 11(1), 31 - 7
The disinfection of water by microalloyed aluminium-based composite; Bojic A et al.; In the submitted paper the water disinfection capacity of the microalloyed aluminium based composite (MABC) was studied . MABC is material in the form of steel wire, plated with microalloyed aluminium . The effects of the composite are based on the very negative stationary potential of microalloyed aluminium, and its spontaneous dissolution in water with generation of AI(III) ions, and reduction of water with the generation of H2 and OH ions . As a final product of these reactions, a voluminous Al(OH)3 precipitate is formed . Having in mind its great efficacy in purification of different waters from many chemical pollutants we made the following hypothesis: reduction characteristics of the MABC surface, presence of Al(III) and OH ions, and coprecipitation on Al(OH)3, can be also toxic and destructive for bacteria in water . The experiments were carried out with the water model solutions (WMS) based on adapted natural surface water (NSW), inoculated with the Escherichia coli . All treatments were performed in the original semi-flow system (SFS), in which convection increases efficacy . The results show that approximately every 10 min the number of viable bacteria was reduced for about one log10 count, with the complete disinfected water phase as the outcome of the treatment . At the end of the treatment, the Al(OH)3 precipitate still contained a low amount of coprecipitated viable bacteria, which died within a relatively short period.

Naunyn Schmiedebergs Arch Pharmacol, 2003 Apr, 367(4), 372 - 9 Epub 2003 Mar 04.
Selective versus non-selective suppression of nitric oxide synthase on regional hemodynamics in rats with or without LPS-induced endotoxemia; Cheng X et al.; The late phase of severe septic shock is associated with reduced cardiac output (CO) and activation of the inducible isoform of nitric oxide synthase (NOS) . This study examined the effects of 1400 W (N-3-aminomethyl-benzyl-acetamidine), a new selective inhibitor of inducible NOS (iNOS), relative to those of N(G)-nitro-L-arginine (L-NNA, non-selective inhibitor of NOS) and the vehicle, on mean arterial pressure (MAP), CO, total peripheral resistance (TPR) and tissue blood flow (BF) in thiobutabarbital-anesthetized rats with lipopolysaccharide (LPS, 10 mg/kg, i.v.) induced endotoxemia . At 2.5 as well as 4 h after injection of LPS, MAP, CO, and BF of the stomach, skeletal muscle and skin were decreased, but TPR was increased, BF to the heart and kidneys were also decreased at 4 h after injection of LPS . Treatment of endotoxemic rats with 1400 W (3 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h after endotoxin challenge prevented the late phase fall in MAP without exacerbating the decreases in CO and tissue BF . In contrast, treatment with L-NNA (8 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h did not prevent the decline in MAP in the LPS-treated rats . Furthermore, CO drastically decreased, TPR markedly increased, and BF to the heart, brain, intestine and skeletal muscle were decreased at 4 h relative to the readings in saline- or 1400 W-treated endotoxemic rats . Therefore, selective inhibition of iNOS by 1400 W restores MAP without compromising CO, but non-selective inhibition of NOS is detrimental at the late stage of septic shock.

J Biol Chem, 2003 Jun 27, 278(26), 23882 - 9 Epub 2003 Apr 09.
Importance of the broad regional interaction for spectral tuning in Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II); Shimono K et al.; Natronobacterium pharaonis phoborhodopsin (ppR; also called N . pharaonis sensory rhodopsin II, NpsRII) is a photophobic sensor in N . pharaonis, and has a shorter absorption maximum (lambdamax, 500 nm) than those of other archaeal retinal proteins (lambdamax, 560-590 nm) such as bacteriorhodopsin (bR) . We constructed chimeric proteins between bR and ppR to investigate the long range interactions effecting the color regulation among archaeal retinal proteins . The lambdamax of B-DEFG/P-ABC was 545 nm, similar to that of bR expressed in Escherichia coli (lambdamax, 550 nm) . B-DEFG/P-ABC means a chimera composed of helices D, E, F, and G of bR and helices A, B, and C of ppR . This indicates that the major factor(s) determining the difference in lambdamax between bR and ppR exist in helices DEFG . To specify the more minute regions for the color determination between bR and ppR, we constructed 15 chimeric proteins containing helices D, E, F, and G of bR . According to the absorption spectra of the various chimeric proteins, the interaction between helices D and E as well as the effect of the hydroxyl group around protonated Schiff base on helix G (Thr-204 for ppR and Ala-215 for bR) are the main factors for spectral tuning between bR and ppR.

Circ Res, 2003 May 16, 92(9), 1033 - 40 Epub 2003 Apr 10.
Identification of a nuclear orphan receptor (Ear2) as a negative regulator of renin gene transcription; Liu X et al.; A potent transcriptional enhancer was previously identified upstream of the mouse renin gene . Within the enhancer is a TGACCT direct-repeat motif, required for enhancer activity, that is the consensus sequence recognized by members of the thyroid hormone subfamily of steroid hormone receptors . We previously reported that RAR/RXR bind to this sequence and mediate the induction of renin promoter activity by retinoids . However, gel mobility shift assays clearly show that other as yet unidentified factors also bind to this motif . In order to identify some of these TGACCT binding factors, we screened a yeast one-hybrid cDNA library derived from mouse As4.1 cells . One of these encoded the orphan nuclear receptor Ear2 . Recombinant Ear2 was purified from Escherichia coli and an antipeptide antisera was generated . EMSA showed that purified recombinant Ear2 specifically binds the TGACCT direct-repeat motif . Transfection assays showed that Ear2 potently decreases both baseline and retinoid-induced mouse renin promoter activity in a dose-dependent, enhancer-dependent, and sequence-specific manner . Mutations in Ear2, which abolish its binding to the TGACCT motif, also abolish transcriptional repression . Ear2 was identified as a nuclear protein in As4.1 cells, is one of the proteins binding to the TGACCT repeat motif, and its overexpression can repress transcription of the endogenous renin gene in As4.1 cells . These data suggest that Ear2 is a negative modulator of renin gene transcription in As4.1 cells, and that the renin enhancer may actually encode a complex positive and negative regulator of transcription.

Am J Physiol Heart Circ Physiol, 2003 Aug, 285(2), H549 - 61 Epub 2003 Apr 10.
A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter; Bobofchak KM et al.; With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains . Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys . This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood . In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA) . The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time . Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin . The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation . The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation . Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22% . Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb . Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.

Prog Lipid Res, 2003 Jul, 42(4), 289 - 317
Structural and functional organization of the animal fatty acid synthase; Smith S et al.; The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase . Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase . The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product . Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers . Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit . Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein . The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit . In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit . These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between and within subunits.

J Biochem Mol Biol, 2003 Mar 31, 36(2), 196 - 200
The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli; Seo HS et al.; Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997) . The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration . The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1) . Here, we examined the effects of pH and cations on the GTPase activity . The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0 . The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest . It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.

Biotechnol Appl Biochem, 2003 Aug, 38(Pt 1), 1 - 7
Impact of plasmid size on cellular oxygen demand in Escherichia coli; Kay A et al.; Escherichia coli DH5alpha harbouring either pBGS18, a 4.4 kb pUC-based plasmid, or pQR150, a 20 kb derivative of pBGS18, were grown in glucose-limited chemostat culture to investigate the effects of plasmid size and recombinant protein production on oxygen demand . Under non-induced conditions, where no recombinant protein was expressed, the cellular oxygen demand of cells harbouring pBGS18 and pQR150 was not significantly different, mean specific oxygen uptake rate (OUR) being 0.75+/-0.24 and 0.78+/-0.32 mmol/h per mg of plasmid respectively . Under isopropyl beta-D-thiogalactoside-induced conditions, where recombinant protein was expressed, cells harbouring pBGS18 demonstrated a statistically insignificant 37% increase in mean specific OUR while those harbouring pQR150 demonstrated a statistically significant 415% increase . It is concluded that plasmid size does not significantly affect oxygen demand and that increasing oxygen demand observed with increasing plasmid size is due to production of recombinant protein.

Biotechnol Appl Biochem, 2003 Jun, 37(Pt 3), 259 - 66
Evaluation of an ion-exchange membrane for the purification of plasmid DNA; Endres HN et al.; Separation and purification of large quantities of plasmid DNA (pDNA) is a particularly difficult manufacturing issue because of the relatively low capacity, flow rate and purity observed using traditional bead-based chromatography . The objective of the present study was to evaluate the performance of anion-exchange membranes for the purification of pDNA from Escherichia coli lysate solution . The fate of host-cell protein and endotoxin relative to pDNA was measured and used to calculate recoveries, mass balances, dynamic capacities and purification factors as a function of the flow rate and loading volume of the lysate solution . Breakthrough curves were not sigmoidal and symmetric in shape . They rose sharply at first, and then slowly towards, but never reaching, saturation . Conversely, elution curves were independent of flow rate . pDNA bound tightly to the membranes, whereas protein and endotoxin did not . Dynamic binding capacity for pDNA was 20-25 times greater, and the flow rate was 55-550 times greater, than values observed for beads . However, some pDNA bound irreversibly to the membrane surface and was not removed completely during elution . The intrinsic rate of pDNA adsorption to the membrane was found to be rate-limiting, whereas effects of liquid-phase mass transfer and flow non-idealities were negligible . These results were interpreted using models of adsorption that included steric effects using the 'car-parking-problem' model, and surface residence time effects using the spreading model . This work demonstrated the advantages of ion-exchange membranes compared with beads for the purification of large biomolecules such as pDNA.

Biochem J, 2003 Jul 15, 373(Pt 2), 437 - 49
Structural and functional characterization of recombinant mouse annexin A11: influence of calcium binding; Lecona E et al.; Annexin A11 is one of the 12 vertebrate subfamilies in the annexin superfamily of calcium/phospholipid-binding proteins, distinguishable by long, non-homologous N-termini rich in proline, glycine and tyrosine residues . As there is negligible structural information concerning this annexin subfamily apart from primary sequence data, we have cloned, expressed and purified recombinant mouse annexin A11 to investigate its structural and functional properties . CD spectroscopy reveals two main secondary-structure contributions, alpha-helix and random coil (approx . 30% each), corresponding mainly to the annexin C-terminal tetrad and the N-terminus respectively . On calcium binding, an increase in alpha-helix and a decrease in random coil are detected . Fluorescence spectroscopy reveals that its only tryptophan residue, located at the N-terminus, is completely exposed to the solvent; calcium binding promotes a change in tertiary structure, which does not affect this tryptophan residue but involves the movement of approximately four tyrosine residues to a more hydrophobic environment . These calcium-induced structural changes produce a significant thermal stabilization, with an increase of approx . 14 degrees C in the melting temperature . Annexin A11 binds to acidic phospholipids and to phosphatidylethanolamine in the presence of calcium; weaker calcium-independent binding to phosphatidylserine, phosphatidic acid and phosphatidylethanolamine was also observed . The calcium-dependent binding to phosphatidylserine is accompanied by an increase in alpha-helix and a decrease in random-coil contents, with translocation of the tryptophan residue towards a more hydrophobic environment . This protein induces vesicle aggregation but requires non-physiological calcium concentrations in vitro . A three-dimensional model, consistent with these data, was generated to conceptualize annexin A11 structure-function relationships.

Mol Biol Rep, 2003 Mar, 30(1), 27 - 31
A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs; Perbandt M et al.; We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond . A literature search indicated that there is also the case for human initiator tRNA(Met) but not for yeast tRNA(i)Met or E . coli tRNA(f)Met . All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop . It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA(i)Met susceptible to the specific cleavage reaction . Using crystallographic data of tRNA(f)Met of E . coli with U33, we modeled the anticodon loop of this tRNA with C33 . We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond . We conclude that a single nucleotide change in the primary structure of tRNA(i)Met made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.

Crit Care Med, 2003 Apr, 31(4), 1219 - 25
Comparison of polyacrylonitrile (AN69) and polysulphone membrane during hemofiltration in canine endotoxic shock; Rogiers P et al.; OBJECTIVE: This study was designed to compare the effects of continuous venovenous hemofiltration (CVVH) with two different membranes, polysulphone and polyacrylonitrile (AN69), on global and regional hemodynamics, plasma lactate, tumor necrosis factor-alpha levels, and plasma nitrite/nitrate during endotoxic shock in dogs . METHODS: Fifteen pentobarbital anesthetized and mechanically ventilated dogs were randomized into three groups of five dogs each . One group served as an endotoxin alone, time matching group and, 1 hr after endotoxin administration, the two other groups received CVVH at 3 L/hr for 270 mins, with either a polysulphone membrane or an polyacrylonitrile membrane . RESULTS: At 90 mins after endotoxin administration, dogs receiving CVVH with polyacrylonitrile membranes had a higher cardiac output, stroke volume, and left-ventricular stroke work index than the endotoxin alone and the polysulphone groups . CVVH with either polyacrylonitrile or polysulphone membranes prevented the rise in pulmonary artery pressure and pulmonary vascular resistance compared with the endotoxin alone group . Plasma lactate levels were not significantly altered, but the fall in bicarbonate seen in the endotoxin alone group did not occur in the two CVVH groups . Tumor necrosis factor levels in the plasma were not significantly altered by CVVH and remained very low (<50 pg/mL) in the ultrafiltrate fluid . CONCLUSION: In this acute endotoxic shock model, CVVH with the polyacrylonitrile membrane improved cardiac performance when compared with the polysulphone membrane . These effects could be caused by a more effective adsorption of inflammatory mediators other than tumor necrosis factor . Whether the polyacrylonitrile membrane should be preferred over the polysulphone membrane for CVVH in severe sepsis warrants further experimental and clinical study.

Crit Care Med, 2003 Apr, 31(4 Suppl), S258 - 64
Transforming growth factor-beta: a mediator of cell regulation in acute respiratory distress syndrome; Dhainaut JF et al.; OBJECTIVE: To review recent advances in the use of transforming growth factor (TGF)-beta in acute lung injury and to apply this knowledge to understanding the pathophysiology of this syndrome . DATA SOURCES AND STUDY SELECTION: Published research and review articles in the English language related to the role of TGF-beta in acute lung injury . DATA EXTRACTION AND SYNTHESIS: The cytokine TGF-beta plays a critical role in the resolution of tissue injury in multiple organs, including the lung . Following injury, TGF-beta has been most thoroughly evaluated during the late phases of tissue repair, where it plays a critical role in the development of pulmonary fibrosis . In contrast, recent animal studies showed that expression levels of several TGF-beta-inducible genes were dramatically increased as early as 2 days after the induction of injury . The integrin alpha(v)beta(6) activates latent TGF-beta in the lungs . Mice lacking this integrin were completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury . Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin . Similar findings also have been reported in patients in a clinical study evaluating TGF-beta in the bronchoalveolar lavage fluid during the course of acute respiratory distress syndrome (ARDS) . Indeed, the bronchoalveolar lavage concentrations were dramatically increased as early as 1 day after the initiation of ARDS criteria and were correlated with decreases in the Pao(2)/Fio(2) ratio, suggesting an important role for TGF-b1 in the development of ARDS in humans . CONCLUSIONS: These studies suggest that TGF-beta not only participates in the late phase of acute lung injury, but also might be active early in acute lung injury and potentially could contribute to the development of pulmonary edema . Integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ARDS, and blocking TGF-beta or its activation could be an effective treatment for this disorder.

J Biosci, 2003 Feb, 28(1), 71 - 6
Three model systems measure oxidation/nitration damage caused by peroxynitrite; McConnell P et al.; Diseases activate the innate immune response which causes ancillary damage to the human body . Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g . DNA, RNA, protein, lipids and sugars . The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage . In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation . (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants . (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants . We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation . In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2077 - 86
Structural basis of replication origin recognition by the DnaA protein; Fujikawa N et al.; Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication . In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 A resolution . DnaA domain IV contains a helix-turn-helix motif for DNA binding . One helix and a loop of the helix- turn-helix motif are inserted into the major groove and 5 bp (3' two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines . In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5' one-third of the DnaA box sequence) by base-specific hydrogen bonds . DNA bending by approximately 28 degrees was also observed in the complex . These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2045 - 55
A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily; Chung JH et al.; Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage . Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage . To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered . Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG . It efficiently removed uracil from both single- and double-stranded DNA . MjUDG also catalyzes the excision of 8-oxoguanine from DNA . MjUDG has a helix-hairpin-helix motif and a {4Fe-4S}-binding cluster that is considered to be important for the DNA binding and catalytic activity . Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families . Also, a homologous member of MjUDG was identified in Aquifex aeolicus . We report that MjUDG belongs to a novel UDG family that has not been described to date.

J Clin Microbiol, 2003 Apr, 41(4), 1763 - 5
DNase pretreatment of master mix reagents improves the validity of universal 16S rRNA gene PCR results; Heininger A et al.; DNase I pretreatment of 16S rRNA gene PCR reagents was tested . The DNase I requirement for the elimination of false-positive results varied between 0.1 and 70 IU per master mix depending on the applied Taq polymerase . PCR sensitivity was mostly maintained when 0.1 IU of DNase I was used.

FEBS Lett, 2003 Apr 10, 540(1-3), 259 - 63
Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins; Fierens K et al.; Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes . Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa . When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor . Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present . Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions . Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested .

Biochemistry, 2003 Apr 15, 42(14), 4243 - 52
Linkage between fructose 1,6-bisphosphate binding and the dimer-tetramer equilibrium of Escherichia coli glycerol kinase: critical behavior arising from change of ligand stoichiometry; Yu P et al.; Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) is inhibited allosterically by fructose 1,6-bisphosphate (FBP), and this inhibition is a primary mechanism by which glucose controls glycerol utilization in vivo . Earlier work indicates that glycerol kinase displays a dimer-tetramer equilibrium in solution, FBP shifts the equilibrium toward the tetramer, and tetramer formation is required for FBP inhibition . However, equilibrium constants for FBP binding and dimer-tetramer assembly that describe the linkage between these processes are unknown . Here, decreased fluorescence anisotropy of extrinsic fluorophores fluorescein and 2',7'-difluorofluorescein due to homo fluorescence resonance energy transfer (homo-FRET) is used to quantitate tetramer assembly and FBP binding . Glycerol kinase is labeled with extrinsic fluorophores covalently attached to an engineered surface cysteine residue under conditions that prevent labeling of native cysteine residues . Tryptic peptide mapping and MALDI-MS verify labeling at the engineered site only . Initial velocity studies show the labeling does not alter the catalytic properties or FBP inhibition . The steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.1 mol of fluorophore/mol of subunit is not sensitive to increased protein concentration or binding of FBP, indicating the absence of homo-FRET . However, steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.4 mol of fluorophore/mol of subunit decreases with increasing protein concentration, which is consistent with depolarization due to homo-FRET . The protein concentration dependence of the decreased fluorescence anisotropy is described by a dimer-tetramer equilibrium with an apparent dissociation constant of 61 +/- 7 nM (subunits) at pH 7.0 and 25 degrees C . FBP binds to both the dimer and tetramer of glycerol kinase, and the FBP concentration dependence of the apparent dissociation constant for the dimer-tetramer equilibrium shows critical behavior . The apparent dissociation constant decreases and then increases with increasing FBP concentration, reaching a minimum at about 20 mM FBP . Critical behavior is seen also in the FBP dependence of the inhibition . The critical behavior arises because tetramer dissociation increases FBP stoichiometry from two sites per tetramer to four half-sites per two dimers . The phenomenological description of the coupling between tetramer assembly and FBP binding shows antagonistic binding of FBP to the two sites on the tetramer, indicating that the strong positive cooperativity observed for FBP inhibition of catalytic activity (Hill coefficient approximately 1.5) is due to the approximately 4000-fold higher affinity of the tetramer for FBP rather than to positive coupling between the two FBP sites.

Biochemistry, 2003 Apr 15, 42(14), 4101 - 7
Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations; Yoshida T et al.; The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation . RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA . MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale . We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered . At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively . These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.

Biometals, 2003 Sep, 16(3), 479 - 84
The ATPase activity of GroEL is supported at high temperatures by divalent cations that stabilize its structure; Melkani GC et al.; Previously, we reported that the ATPase activity of GroEL that requires potassium and magnesium was highly temperature dependent in the 25-60 degrees C range . Here, we report that the monovalent cations, rubidium and ammonium were able to fully substitute for potassium; while the divalent cations manganese, cobalt, and nickel supported the ATPase activity of GroEL albeit to a lesser degree than magnesium . ATPase activities with manganese, cobalt, and nickel were 64%, 41%, and 29%, respectively, of the maximum activity (100%) when utilizing magnesium . Interestingly, the ability of all the cations to support the GroEL ATPase activity was somewhat consistent over the entire 25-60 degrees C range . Maximum ATPase activities were observed at 49 degrees C . Here, the influence of these cations on the thermal denaturation of GroEL was also monitored using bisANS binding as an indication of the exposure of hydrophobic surfaces during thermal denaturation of GroEL . Maximum exposure of hydrophobic surfaces on GroEL alone or in the presence of each of the monovalent cations was determined to occur at 65 degrees C . However, the maximum exposure of hydrophobic surfaces on GroEL in the presence of magnesium, manganese, cobalt or nickel was found to occur at 71 degrees C indicating that GroEL is significantly stabilized against thermal denaturation by these divalent cations.

Virus Genes, 2003 Jan, 26(1), 5 - 13
Identification of a novel protein associated with envelope of occlusion-derived virus in Spodoptera litura multicapsid nucleopolyhedrovirus; Yin C et al.; Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) ORF137 (Splt137) is one of 29 unique SpltMNPV ORFs . Splt137 has the potential to code for a polypeptide of 231 amino acid residues with predicted molecular weight of 27.5 kDa . Computer-assisted analysis of the predicted amino acid sequences of Splt137 protein showed 1 N-glycosylation site and 11 phosphorylation sites . For identification of Spit137, antibody was prepared by immunization of rabbits with purified Splt137 protein produced in Escherichia coli . This antibody was used to analyse Splt137 protein using Western blot . A 36-kDa protein was found both in the infected cells and envelope fractions of occlusion-derived virus (ODV) but could not be detected in the budded virus (BV) . Tunicamycin treatment of SpltMNPV infected cells suggested that the 36-kDa protein had undergone N-glycosylation . Our data suggested that Splt137 protein was a novel envelope protein of ODV and might exist as a more complex form of 79-kDa protein in intact ODV . Further, transcriptional analysis with RT-PCR and 5' RACE analysis suggested that Splt137 might perform functions early and late in infection.

Pediatr Pulmonol, 2003 May, 35(5), 392 - 9
Respiratory burst activity in bronchopulmonary dysplasia and changes with dexamethasone; Ballabh P et al.; The first objective of this study was to evaluate longitudinal changes in respiratory burst activity in circulating neutrophils and monocytes in infants of less than 30 weeks of gestation with respiratory distress syndrome (RDS), and to examine differences in neonates who subsequently developed bronchopulmonary dysplasia (BPD) compared with those neonates who did not . The second objective was to investigate the effects of dexamethasone on respiratory burst activity in neutrophils and monocytes . We measured burst activity on neutrophils and monocytes in fresh heparinized blood in response to E . coli, N-formyl-met-leu-phe (fMLP), and phorbol 12-myristate 13-acetate stimulation on days 3, 7, 14, and 21 of life, before and 2-3 days after initiating a 6-day course of dexamethasone treatment . Infants with RDS participating in the study were followed until discharge, and were classified as non-BPD and either 1) BPD d28, reflecting their oxygen requirement at day of life 28, or 2) BPD 36 weeks, reflecting oxygen dependence at 36 weeks' corrected gestational age . The diagnosis of BPD was supported by radiological changes of BPD . The percentage of activated neutrophils producing a respiratory burst increased in all premature infants with increasing postnatal days during the first 28 days of life, when the physiological stimulus E . coli was used as an activator (P < 0.02) . There was no significant difference in respiratory burst activity measured either as percent activation or as mean fluorescence intensity between non-BPD and BPD infants after adjusting for the difference in weight and gestational age between the two groups . The treatment of premature infants with dexamethasone was associated with decreased activation of neutrophils (P < 0.005) when E . coli was used as a stimulus . In conclusion, a significant increase in neutrophil respiratory burst activity occurs during the first month of life in very low birth weight infants . Greater pulmonary damage in BPD cannot be attributed to reduced burst activity in either neutrophils or monocytes . Dexamethasone treatment was associated with decreased neutrophil respiratory burst activity .

Microbiology, 2003 Apr, 149(Pt 4), 1001 - 10
Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r; Michelsen O et al.; The C and D cell cycle periods of seven Escherichia coli K-12 strains and three E . coli B/r strains were determined by computer simulation of DNA histograms obtained by flow cytometry of batch cultures grown at several different generation times . To obtain longer generation times two of the K-12 strains were cultivated at several different dilution rates in glucose-limited chemostats . The replication period (C period) was found to be similar in K-12 and B/r strains grown at similar generation times . At generation times below 60 min the C period was constant; above 60 min it increased linearly with increasing generation time . The period from termination of replication to cell division (D period) was more variable . It was much shorter in B/r than in K-12 strains . Like the C period it was relatively constant at generation times below 60 min and it increased with increasing generation times at longer generation times . In glucose-limited chemostats good correlation was found between D periods and generation times, whereas batch cultures exhibited carbon-source-dependent variations . Chemostat cultures showed cell cycle variations very similar to those obtained in batch cultures . These flow cytometric determinations of cell cycle periods confirm earlier determinations of the C period and establish that the D period also varies with generation time in slowly growing cultures . In addition they extend the range of growth rates at which cell cycle periods have been determined in E . coli K-12.

Microbiology, 2003 Apr, 149(Pt 4), 877 - 84
Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli; Yona-Nadler C et al.; The flagellar apparatus consists of components that function as a type III secretion system (TTSS) . Enteropathogenic and enterohaemorrhagic E . coli (EPEC and EHEC, respectively) produce an additional TTSS, which is involved in virulence via the translocation of effector proteins into infected host cells . This system is encoded by the locus of enterocyte effacement (LEE) . The authors observed that EPEC and EHEC grown in Dulbecco's modified Eagle's medium to the mid- and late-exponential growth phase at 37 degrees C are non-motile . At the same time these conditions trigger the expression of the LEE-encoded TTSS . Furthermore, it was found that EPEC with an inactivated ihfA, which encodes the IHFalpha subunit of the integration host factor (IHF), becomes hyperflagellated and motile . Similar hypermotility was seen upon inactivation of the ihfA of EHEC strains . IHF-mediated repression of the EPEC flagella involves down-regulation of flhDC, which encodes a positive regulator of the flagellar regulon . IHF indirectly mediates flhDC repression, via a putative EPEC-unique regulator which is not encoded by LEE.

Mol Biol Cell, 2003 Apr, 14(4), 1448 - 59
Autoinhibition of Jak2 tyrosine kinase is dependent on specific regions in its pseudokinase domain; Saharinen P et al.; Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2) . JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive . We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2 . JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate . Three inhibitory regions (IR1-3) within JH2 were identified . IR3 (residues 758-807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1 . Molecular modeling of JH2 showed that IR3 could form a stable alpha-helical fold, supporting that IR3 could independently inhibit JH1 . IR2 (725-757) in the C-terminal lobe of JH2, and IR1 (619-670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1 . Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-gamma-inducible signaling . Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2.

J Biol Chem, 2003 Jun 13, 278(24), 22056 - 60 Epub 2003 Apr 09.
A transmembrane segment mimic derived from Escherichia coli diacylglycerol kinase inhibits protein activity; Partridge AW et al.; The function of membrane proteins is inextricably linked to the proper packing and assembly of their independently helical transmembrane (TM) segments . Here we examined whether an externally added TM peptide analogue could specifically inhibit the function of the membrane protein from which it is derived by competing for native TM helix packing sites, thereby producing a non-functional peptide-protein complex . This hypothesis was tested using Lys-tagged peptides synthesized with sequences corresponding to the three TM segments of the homotrimeric Escherichia coli diacylglycerol kinase (DGK) . The peptide corresponding to wild-type DGK TM-2 inhibited the protein's enzymatic activity in a dose-dependent manner through formation of an inactive pseudo-complex, whereas peptides derived from TM-1 and TM-3 were benign toward DGK structure/function . Also, substitution of a conserved residue (Glu-69) within the TM-2 peptide abolished these effects, demonstrating the strict sequence requirements for TM-2-mediated association . This strategy, coupled with the practical advantages of the water solubility of Lys-tagged TM peptides, may constitute an attractive approach for the design of therapeutic membrane protein modulators even in the absence of a high resolution structure.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 390 - 4
The structural interpretations of residue Ser297 in catalytic efficiency of Escherichia coli phenylalanine aminotransferase; Wu SP et al.; Escherichia coli phenylalanine aminotransferase (ecPheAT) catalyzes the biosynthesis of phenylalanine and tyrosine . The crystal structure of ecPheAT was determined in our previous study . The comparison of the 3-D structure of several aminotransferases revealed that the residue at position 297 plays an important role in enzyme function . Analysis of activities and kinetic parameters of wild type and mutant ecPheATs suggested that the residue Ser(297) was structurally selected for better catalytic efficiency . Computational modeling of ecPheAT mutants further suggested that Ser in position 297 could make ecPheAT easy with change of conformation from open form to closed form.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 376 - 80
Stability and oligomerization of recombinant GadX, a transcriptional activator of the Escherichia coli glutamate decarboxylase system; Tramonti A et al.; One of the most important strategies that enteric bacteria adopt for maintaining the cytoplasmic pH neutral under acid stress involves the glutamate decarboxylase (Gad) system . The system works by the concerted action of a cytoplasmic, pyridoxal 5'-phosphate-dependent glutamate decarboxylase and a transmembrane antiporter, which imports glutamate and exports gamma-aminobutyrate (GABA), the decarboxylation product, thereby providing local buffering of the extracellular environment . Herein, we provide a preliminary biochemical characterization of GadX, an activator of the Gad system belonging to the AraC/XylS family of bacterial transcriptional regulators . The GadX protein has been purified as a chimeric MalE-GadX with a yield of 15-20 mg/l of bacterial culture . The fusion protein is fairly stable, although a conformational change occurs upon storage, which reduces the binding affinity by a factor of 2, without affecting the binding pattern . Partial removal of the MalE moiety from the fusion protein triggers the formation of a species which is likely to be a heterodimer, or a higher oligomer, of the type GadX/MalE-GadX . This experimental evidence is in line with the well-known tendency of AraC/XylS-like proteins to dimerize via their N-terminal domain.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 321 - 4
Characterization of histidinol phosphate aminotransferase from Escherichia coli; Mizuguchi H et al.; Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype . In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode . Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT . Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6 . The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6 . Mutation of Asn(157) to Ala increased the pK(a) to 9.2 . Replacement of Arg(335) by Leu increased the pK(a) to 8.6 . On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6 . These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT . Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 303 - 9
Assembly of iron-sulfur clusters mediated by cysteine desulfurases, IscS, CsdB and CSD, from Escherichia coli; Kurihara T et al.; Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine . The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters . Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD . We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro . Since IscU, an iron-sulfur protein of E . coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU . By surface plasmon resonance analysis, we found that only IscS interacts with IscU . We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 200 - 5
The metal ion in the active site of the membrane glucose dehydrogenase of Escherichia coli; James PL et al.; All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca(2+) bonded to the PQQ at the active site . However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg(2+) ions (but not Ca(2+)) for activity . To address the question of whether the Mg(2+) replaces the usual active site Ca(2+) in this enzyme, mutant GDHs were produced in which residues proposed to be involved in binding metal ion were modified (D354N-GDH and N355D-GDH and D354N-GDH/N355D-GDH) . The most remarkable observation was that reconstitution with PQQ of the mutant enzymes was not supported by Mg(2+) ions as in the wild-type GDH, but it could be supported by Ca(2+), Sr(2+) or Ba(2+) ions . This was competitively inhibited by Mg(2+) . This result, together with studies on the kinetics of the modified enzymes have led to the conclusion that, although a Ca(2+) ion is able to form part of the active site of the genetically modified GDH, as in all other PQQ-containing quinoproteins, a Mg(2+) ion surprisingly replaces Ca(2+) in the active site of the wild-type GDH.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 92 - 7
Pyridoxine 5'-phosphate synthase: de novo synthesis of vitamin B6 and beyond; Garrido-Franco M; Vitamin B(6) is an essential component in human diet . However, some organisms have the required machinery for its synthesis . There are two independent and autoexclusive groups of genes, pdx and SOR1 . Pyridoxine 5'-phosphate (PNP) synthase is the key enzyme in the pdx group . It catalyses a multistep ring closure reaction yielding PNP and inorganic phosphate (Pi) . This is the last step in the de novo synthetic pathway; afterwards, PNP enters the salvage pathway to be transformed to the pyridoxal 5'-phosphate cofactor . Because PNP synthase is not present in humans but is found in many human pathogens, the enzyme can be regarded as a potential target for the development of novel drugs . We have recently solved the structure of PNP synthase in complex with several ligands . The structural information allowed us to characterise the active site of the enzyme and identify the catalytically important residues . Furthermore, a detailed reaction mechanism could be proposed.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 76 - 82
Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase; di Salvo ML et al.; Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP) . This reaction serves as the terminal step in the de novo biosynthesis of PLP in E . coli and as a part of the salvage pathway of this coenzyme in both E . coli and mammalian cells . Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly . The crystal structures of PNPOx with one and two molecules of PLP bound have been determined . In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN) . A large protein conformational change occurs upon binding of PLP . When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site . A possible tunnel exists between the two sites . Site mutants were made of all residues at the active site that make interactions with the substrate . Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP . The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.

Parasitol Res, 2003 Jul, 90(4), 294 - 300 Epub 2003 Apr 01.
Analysis of codon usage in beta-tubulin sequences of helminths; von Samson-Himmelstjerna G et al.; Codon usage bias has been shown to be correlated with gene expression levels in many organisms, including the nematode Caenorhabditis elegans . Here, the codon usage (cu) characteristics for a set of currently available beta-tubulin coding sequences of helminths were assessed by calculating several indices, including the effective codon number (Nc), the intrinsic codon deviation index (ICDI), the P2 value and the mutational response index (MRI) . The P2 value gives a measure of translational pressure, which has been shown to be correlated to high gene expression levels in some organisms, but it has not yet been analysed in that respect in helminths . For all but two of the C . elegans beta-tubulin coding sequences investigated, the P2 value was the only index that indicated the presence of codon usage bias . Therefore, we propose that in general the helminth beta-tubulin sequences investigated here are not expressed at high levels . Furthermore, we calculated the correlation coefficients for the cu patterns of the helminth beta-tubulin sequences compared with those of highly expressed genes in organisms such as Escherichia coli and C . elegans . It was found that beta-tubulin cu patterns for all sequences of members of the Strongylida were significantly correlated to those for highly expressed C . elegans genes . This approach provides a new measure for comparing the adaptation of cu of a particular coding sequence with that of highly expressed genes in possible expression systems.Finally, using the cu patterns of the sequences studied, a phylogenetic tree was constructed . The topology of this tree was very much in concordance with that of a phylogeny based on small subunit ribosomal DNA sequence alignments.

Curr Genet, 2003 Apr, 43(1), 17 - 23 Epub 2003 Feb 18.
Construction of strains for rapid homokaryon purification after integration of constructs at the histidine-3 ( his-3) locus of Neurospora crassa; Lee DW et al.; We report the construction of histidine-3 (his-3) strains of Neurospora crassa containing the hygromycin B phosphotransferase gene of Escherichia coli (hph(+)) fused in-frame to the herpes simplex virus thymidine kinase gene (tk(+); Lupton et al . 1991), integrated at the his-3 locus . We also report the construction of two ampicillin-resistant and two kanamycin-resistant his-3 gene-replacement vector plasmids . The combined use of these strains and plasmids for his-3-targeted gene integration allows for the rapid identification of homokaryotic transformants containing the expected gene replacement event.

Intervirology, 2003, 46(2), 121 - 6
Expression of HCV E1 protein in baculovirus-infected cells: effects on cell viability and apoptosis induction; Ciccaglione AR et al.; The molecular mechanisms of pathogenesis in hepatitis C virus (HCV) infection are not yet understood . Recently, we reported that the expression of the envelope protein E1 is toxic for Escherichia coli cells . The toxicity is related to the ability of C-terminal transmembrane (TM) domain of E1 to modify membrane permeability . In this study we expressed the E1 protein, complete (a.a . 192-383) or deleted (a.a . 192-340) of the TM region, fused to the C-terminus of glutathione-S-transferase by two recombinant baculoviruses . Infection of Sf9 insect cells by E1 baculovirus induced a rapid decrease in cell viability in the first 18-24 h postinfection . Premature cytopathic changes and low level of E1 protein expression were also reported . The analysis of DNA isolated from cells revealed a typical internucleosomal ladder pattern characteristic of apoptosis . The DNA degradation was first detected at 18 h postinfection by ethidium bromide gel electrophoresis and was confirmed by TUNEL assay . The results indicated that the C-terminal domain of E1 is essential for apoptosis induction as neither cell death nor DNA degradation were observed following infection with the recombinant baculovirus expressing the C-terminal-deleted E1 . These findings support the hypothesis that the TM domain of E1 may play a role in viral pathogenesis .

J Biol Chem, 2003 Jun 13, 278(24), 21952 - 9 Epub 2003 Apr 08.
Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties; Kiss RS et al.; Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family . The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure . To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III) . Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized . Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose . Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III . Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments . Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts . We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.

J Biol Chem, 2003 Jun 20, 278(25), 22210 - 6 Epub 2003 Apr 08.
Altered balance of half-reactions in p-hydroxybenzoate hydroxylase caused by substituting the 2'-carbon of FAD with fluorine; Palfey BA et al.; Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2'-fluoro-2'-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2'-center of the ribityl chain was replaced with fluorine in an inverted configuration . The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate . In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound . Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB . Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B . A., Moran, G . R., Entsch, B., Ballou, D . P., and Massey, V . (1999) Biochemistry 38, 1153-1158) . Thus, whereas the change that induces the out conformation is detrimental to the oxidative half-reaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2'-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis.

J Pharm Biomed Anal, 2003 Apr 1, 31(5), 979 - 87
Finding of an isoleucine derivative of a recombinant protein for pharmaceutical use; Muramatsu R et al.; Protein modification generally occurs by addition to the amino acid side-chains of protein at the post-translational stage, for example, by enzymatic or chemical reactions after polypeptide synthesis . Recently, the recombinant hirudin analog CX-397, a potent thrombin inhibitor, was found to contain methylated Ile residues when it was overproduced by Escherichia coli in the absence of amino acids in the culture medium . The Ile derivatives, deduced to be beta-methylnorleucine {betaMeNle; (2S, 3S)-2-amino-3-methylhexanoic acid} by systematic chromatographic analysis, do not appear to be normal post-translational modifications of the protein because Ile has no functional group in its side-chain . We, therefore, propose that betaMeNle is biosynthesized by E . coli, activated by E . coli isoleucyl-tRNA synthetase (IleRS), then incorporated into the overproduced recombinant hirudin analog . The biosynthesis of betaMeNle in E . coli is thought to occur as follows: alpha-ketovalerate is synthesized from alpha-ketobutyrate by three Leu biosynthetic enzymes, alpha-isopropylmalate synthase (IPMS) (EC 4.1.3.12), alpha-isopropylmalate isomerase (ISOM) (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (IPMD) (EC 1.1.1.85), which have broad substrate specificities . alpha-Ketovalerate is then converted to alpha-keto-beta-methylcaproate by three Ile and Val biosynthetic enzymes, acetohydroxy acid synthase (AS) (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86) and dihydroxy acid dehydratase (DH) (EC 4.2.1.9) . Finally, this is converted to betaMeNle by branched-chain amino acid transaminase (EC 2.6.1.42), one of the Ile and Val biosynthetic enzymes .

J Mol Biol, 2003 Apr 18, 328(1), 273 - 88
Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli; Arai M et al.; A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem . It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable . Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized . In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present . The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events . We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR . The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not . Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events . We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable .

J Mol Biol, 2003 Apr 18, 328(1), 109 - 18
Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors; Zeslawska E et al.; Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility . In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target . We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g . phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain . These potent inhibitors are highly selective for uPA . The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99 . Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA .

J Enzyme Inhib Med Chem, 2002 Oct, 17(5), 333 - 43
Carbonic anhydrase inhibitors: synthesis of water soluble sulfonamides incorporating a 4-sulfamoylphenylmethylthiourea scaffold, with potent intraocular pressure lowering properties; Casini A et al.; Reaction of thiophosgene with 4-aminomethyl-benzenesulfonamide afforded 4-isothiocyanatomethyl-benzenesulfonamide, which by reaction with amines, amino acids and oligopeptides, lead to a series of new sulfonamides incorporating a 4-sulfamoylphenylmethylthiourea scaffold . These new thioureas showed strong affinities towards isozymes I, II and IV of carbonic anhydrase (CA, EC 4.2.1.1) . In vitro inhibitory potency was good (in the low nanomolar range) for the derivatives of: amino-benzoic acids, beta-phenyl-serine, alpha-phenyl-glycine, for those incorporating hydroxy- and mercapto-amino acids (Ser, Thr, Cys and Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr; DOPA); dicarboxylic amino acids as well as di-/tri-/tetrapeptides among others . Such CA inhibitors displayed very good water solubility (in the range of 2-3%) as sodium (carboxylate) salts, with pH values for the solutions obtained of 6.5-7.0 . Furthermore, in normotensive rabbits, some of them showed an effective and prolonged intraocular pressure (IOP) lowering when administered topically, as 2% solutions.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 698 - 702
{Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain)}; Jin YM et al.; A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain) . Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain) . The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum . This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction . SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD . Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

World J Gastroenterol, 2003 Apr, 9(4), 726 - 30
Construction, expression and tumor targeting of a single-chain Fv against human colorectal carcinoma; Fang J et al.; AIM: A single-chain antibody fragment, ND-1scFv, against human colorectal carcinoma was constructed and expressed in E.coli, and its biodistribution and pharmacokinetic properties were studied in mice bearing tumor . METHODS: V(H) and V(L) genes were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR, and connected by linker (Gly(4)Ser) (3) to form scFv gene, which was cloned into expression vector pET 28a(+) and finally expressed in E.coli . The expressed product ND-1scFv was purified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDS-PAGE and ELISA . ND-1scFv was labeled with (99m)Tc, and then injected into mice bearing colorectal carcinoma xenograft for phamacokinetic study in vivo . RESULTS: SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 30kDa with purity of 94 % . ELIAS assay revealed that ND-1scFv retained the immunoactivity of parent mAb, being capable of binding specifically to human colorectal carcinoma cell line expressing associated antigen . Radiolabeled ND-1scFv exhibited rapid tumor targeting, with specific distribution in mice bearing colorectal carcinoma xenograft observed as early as 1 h following injection . In vivo pharmacokinetic studies also demonstrated that ND-1scFv had very rapid plasma clearance (T(1/2)alpha of 5.7 min, T(1/2)beta of 2.6 h) . CONCLUSION: ND-1scFv shows significant immunoactivity, and better pharmacokinetic and biodistribution characteristics compared with intact mAbs, demonstrating the possibility as a carrier for tumor-imaging.

Cancer Gene Ther, 2003 Apr, 10(4), 294 - 301
Heat-directed suicide gene therapy for breast cancer; Brade AM et al.; Adjuvant hyperthermia can improve treatment outcome for locally recurrent breast cancer (LRBC) . Previously, we demonstrated that infection of human breast cancer cells with a recombinant adenovirus expressing beta-galactosidase from the human hsp70b gene promoter (Ad.70b.betagal) results in 50- to 800-fold increases in reporter gene expression following heat treatment (30 minutes at 43 degrees C) . Here, we describe a heat-directed suicide gene therapy strategy based on an adenoviral vector (Ad.70b.CDTK) in which expression of the dual prodrug-activating E . coli cytosine deaminase/herpes simplex virus thymidine kinase (CDTK) fusion gene is under the control of the hsp70b promoter . Treatment of T47D and MCF-7 breast cancer cells with mild hyperthermia (43 degrees C/30 minutes) and prodrugs (100 microg/ml 5-fluorocytosine and 10 microg/ml ganciclovir) following infection with Ad.70b.CDTK (10-100 PFU/cell) resulted in 30- to 60-fold decreases in clonogenic survival relative to control cultures treated with heat or prodrugs alone . Clonogenic survival declined even further (up to 240-fold) following heat treatment at 41.5 degrees C for 120 minutes . A decreased clonogenic survival was accompanied by tumor cell apoptosis . These results demonstrate that this combined treatment strategy can be highly effective against heat- and radiation-resistant breast tumor cells and supports the continued development of heat-directed CDTK suicide gene therapy strategies for LRBC.

Mol Biol Evol, 2003 May, 20(5), 775 - 83 Epub 2003 Apr 02.
Exon-intron structure and evolution of the Lipocalin gene family; Sanchez D et al.; The Lipocalins are an ancient protein family whose expression is currently confirmed in bacteria, protoctists, plants, arthropods, and chordates . The evolution of this protein family has been assessed previously using amino acid sequence phylogenies . In this report we use an independent set of characters derived from the gene structure (exon-intron arrangement) to infer a new lipocalin phylogeny . We also present the novel gene structure of three insect lipocalins . The position and phase of introns are well preserved among lipocalin clades when mapped onto a protein sequence alignment, suggesting the homologous nature of these introns . Because of this homology, we use the intron position and phase of 23 lipocalin genes to reconstruct a phylogeny by maximum parsimony and distance methods . These phylogenies are very similar to the phylogenies derived from protein sequence . This result is confirmed by congruence analysis, and a consensus tree shows the commonalities between the two source trees . Interestingly, the intron arrangement phylogeny shows that metazoan lipocalins have more introns than other eukaryotic lipocalins, and that intron gains have occurred in the C-termini of chordate lipocalins . We also analyze the relationship of intron arrangement and protein tertiary structure, as well as the relationship of lipocalins with members of the proposed structural superfamily of calycins . Our congruence analysis validates the gene structure data as a source of phylogenetic information and helps to further refine our hypothesis on the evolutionary history of lipocalins.

J Biol Chem, 2003 Jun 13, 278(24), 21559 - 65 Epub 2003 Apr 04.
Paramecium bursaria Chlorella virus 1 encodes two enzymes involved in the biosynthesis of GDP-L-fucose and GDP-D-rhamnose; Tonetti M et al.; At least three structural proteins in Paramecium bursaria Chlorella virus (PBCV-1) are glycosylated, including the major capsid protein Vp54 . However, unlike other glycoprotein-containing viruses that use host-encoded enzymes in the endoplasmic reticulum-Golgi to glycosylate their proteins, PBCV-1 encodes at least many, if not all, of the glycosyltransferases used to glycosylate its structural proteins . As described here, PBCV-1 also encodes two open reading frames that resemble bacterial and mammalian enzymes involved in de novo GDP-L-fucose biosynthesis . This pathway, starting from GDP-D-mannose, consists of two sequential steps catalyzed by GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, respectively . The two PBCV-1-encoded genes were expressed in Escherichia coli, and the recombinant proteins had the predicted enzyme activity . However, in addition to the dehydratase activity, PBCV-1 GMD also had a reductase activity, producing GDP-D-rhamnose . In vivo studies established that PBCV-1 GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are expressed after virus infection and that both GDP-L-fucose and GDP-D-rhamnose are produced in virus-infected cells . Thus, PBCV-1 is the first virus known to encode enzymes involved in nucleotide sugar metabolism . Because fucose and rhamnose are components of the glycans attached to Vp54, the pathway could circumvent a limited supply of GDP sugars by the algal host.

J Biol Chem, 2003 Jun 13, 278(24), 21860 - 8 Epub 2003 Apr 04.
Concerted folding and binding of a flexible colicin domain to its periplasmic receptor TolA; Anderluh G et al.; Compared with folded structures, natively unfolded protein domains are over-represented in protein-protein and protein-DNA interactions . Such domains are common features of all colicins and are required for their translocation across the outer membrane of the target Escherichia coli cell . All of these domains bind to at least one periplasmic protein of the Tol or Ton family . Similar domains are found in Ton-dependent outer membrane transporters, indicating they may interact in a related manner . In this article we have studied binding of the colicin N translocation domain to its periplasmic receptor TolA, by fluorescence resonance energy transfer (FRET) using fluorescent probes attached to engineered cysteine residues and NMR techniques . The domain exhibits a random coil circular dichroism spectrum . However, FRET revealed that guanidinium hydrochloride denaturation caused increases in all measured intramolecular distances showing that, although natively unfolded, the domain is not extended . Furthermore NMR reported a compact hydrodynamic radius of 18 A . Nevertheless the FRET-derived distances changed upon binding to TolA indicating a significant structural rearrangement . Using 1H-15N NMR we show that, when bound, the peptide switches from a disordered state to an ordered state . The kinetics of binding and the associated structural change were measured by stopped-flow methods, and both events appear to occur simultaneously . The data therefore suggest that this molecular recognition involves the concerted binding and folding of a flexible but collapsed state.

Eur J Pharmacol, 2003 Apr 11, 466(1-2), 181 - 9
A superoxide dismutase mimetic with catalase activity (EUK-8) reduces the organ injury in endotoxic shock; McDonald MC et al.; Reactive oxygen species contribute to the multiple organ failure in endotoxic shock . Here, we investigate the effects of a salen-manganese complex, which exhibits both superoxide dismutase and catalase activity (EUK-8), on the circulatory failure, renal and liver injury and dysfunction caused by endotoxin in the anaesthetised rat . Endotoxaemia (6 mg/kg i.v., Escherichia coli lipopolysaccharide) for 6 h caused hypotension, renal dysfunction and liver injury . Treatment of rats with EUK-8 (0.3 or 1 mg/kg bolus injection followed by an infusion of 0.3 or 1 mg/kg/h) attenuated the renal and liver injury and dysfunction in a dose-related fashion . In addition, the higher dose of EUK-8 attenuated the delayed hypotension caused by endotoxin in the rat . Thus, an enhanced formation of reactive oxygen species importantly contributes to the circulatory failure, as well as the organ injury and dysfunction associated with endotoxic shock . We propose that small molecules, which have the catalytic activity of both superoxide dismutase and catalase, may represent a novel therapeutic approach for the therapy of endotoxic shock.

Cell, 2003 Apr 4, 113(1), 61 - 71
OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain; Walsh NP et al.; Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis . Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm . This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E) . Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription . These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs . DegS activation involves relief of inhibitory interactions between its PDZ and protease domains . Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

Comb Chem High Throughput Screen, 2003 Mar, 6(2), 155 - 60
Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates; Kakiuchi N et al.; Hepatitis C virus is one of the causative agents of non-A non-B hepatitis . Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation . NS3 serine protease is considered to be a suitable target for anti-HCV reagents . We report an assay of HCV NS3 protease in living cells . We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP . Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease . The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed . As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection . We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro . We studied the effect of RNA aptamers introduced into HeLa cells . The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.

Int J Surg Investig, 2001, 2(5), 359 - 67
Enoximone maintains intestinal villus blood flow during endotoxemia; Schmidt W et al.; BACKGROUND: The objective of this study was to determine the effects of a continuous infusion of the phosphodiesterase inhibitor enoximone on mucosal villus blood flow in a normotensive model of endotoxemia . METHODS: Twenty-four anesthetized and ventilated rats underwent laparotomy and a ileal portion was exteriorized and opened by an antimesenteric incision . The ileal segment was fixed on a plexiglass stage with the mucosal surface upward . Microcirculatory parameters were assessed by intravital videomicroscopy . The animals were randomly assigned to receive one of three treatments: infusion of Escherichia coli lipopolysaccharides (LPS, 2 mg/kg/h) without phosphodiesterase inhibitor pretreatment (LPS group); or infusion of LPS with enoximone pretreatment (10 microg x kg(-1) x min(-1), start 30 min before LPS infusion, enoximone group), or infusion of an eqivalent volume of NaCl 0.9% (control group) . Macrohemodynamic parameters (MAP, HR) and microhemodynamic parameters of ileal mucosa (mean diameter of central arterioles = DA, and mean erythrocyte velocity within the arterioles = VE) were measured 30 min before and at 0, 60, and 120 min after induction of endotoxemia . Mucosal villus blood flow was calculated from DA and VE . RESULTS: In this normotensive endotoxemia model MAP remained stable in the control and the LPS group but significantly decreased in the enoximone group . The endotoxin-induced decrease of VE and DE of central arterioles of mucosal villi could be prevented . Thus, mucosal villus blood flow did not decrease compared to the LPS group . CONCLUSIONS: Our results indicate that enoximone during an early stage of sepsis contributes to systemic hypotension but prevents mucosal hypoperfusion.

J Bioenerg Biomembr, 2002 Dec, 34(6), 455 - 64
pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803; Van Walraven HS et al.; It was shown before (Wooten, D . C., and Dilley, R . A . (1993) J . Bioenerg . Biomembr . 25, 557-567; Zakharov, S . D., Li, X., Red'ko, T . P., and Dilley, R . A . (1996) J . Bioenerg . Biomembr . 28, 483-493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E . coli ATP synthase due to closing a "proton gate" proposed to exist in the F0 H+ channel of the F0F1 ATP synthase . This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803 . Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of deltamuH+ . The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid-base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the deltapH of the acid-base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28 . That is, in the absence of added CaCl2, ATP formation did occur under those conditions . However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present . This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5 . Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0 . Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8 . With both strains it was shown that the Ca2+ binding effect on acid-base induced ATP synthesis occurs above an internal pH of about 5.5 . These results were corroborated by 45Ca2+-ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 5Ca2+ binding as occurs with the pea chloroplast subunit III . The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio . The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4469 - 73 Epub 2003 Apr 03.
High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds; Delaney JC et al.; We report studies testing the importance of Watson-Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells . Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria . Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions . Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity . Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity . The data establish that Watson-Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo . The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

Protein Eng, 2003 Feb, 16(2), 125 - 34
Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2); Andre G et al.; Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein . This suggests that there is a substantial conformational shift in the protein upon substrate binding . Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations . Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure . This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water . In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms . Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site . Based on the results of these various MD simulations, a new catalytic mechanism is proposed . Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.

J Biol Chem, 2003 Jul 18, 278(29), 26327 - 32 Epub 2003 Apr 03.
Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive; Mutalik VK et al.; Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades . Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems . Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient . We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme . This robust response of GS adenylylation is also observed for change in system parameters . From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure . By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status.

J Biol Chem, 2003 Jun 20, 278(25), 22193 - 8 Epub 2003 Apr 03.
Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo; Mileykovskaya E et al.; The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells . By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles . MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form . A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T . H., Rowland, S . L., Rothfield, L . I., and King, G . F . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 15693-15698) . To investigate whether E . coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E . coli that lacks phosphatidylethanolamine . In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content . In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2 . Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes . The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes . The degree of acyl chain unsaturation contributed positively to binding strength . These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.

Appl Environ Microbiol, 2003 Apr, 69(4), 2399 - 404
Release of extracellular transformable plasmid DNA from Escherichia coli cocultivated with algae; Matsui K et al.; We studied the effects of cocultivation with either Euglena gracilis (Euglenophyta), Microcystis aeruginosa (Cyanophyta), Chlamydomonas neglecta (Chlorophyta), or Carteria inversa (Chlorophyta) on the production of extracellular plasmid DNA by Escherichia coli LE392(pKZ105) . Dot blot hybridization analysis showed a significant release of plasmid DNA by cocultivation with all the algae tested . Further analysis by electrotransformation confirmed the release of transformable plasmid DNA by cocultivation with either E . gracilis, M . aeruginosa, or C . inversa . These results suggest algal involvement in bacterial horizontal gene transfer by stimulating the release of transformable DNA into aquatic environments.

Appl Environ Microbiol, 2003 Apr, 69(4), 2298 - 305
Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2; Tralau T et al.; TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2 . Transposon mutagenesis was used to knock out tsaR . The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA) . Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA . The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR(His) . TsaR(His) was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used . Gel filtration showed TsaR(His) to be a monomer in solution . Overexpressed TsaR(His) bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD . The dissociation constant (K(D)) for the whole promoter region was about 0.9 micro M, and the interaction was a function of the concentration of the ligand TSA . A regulatory model for this LysR-type regulator is proposed on the basis of these data.

Appl Environ Microbiol, 2003 Apr, 69(4), 1967 - 72
Overproduction of Thermus sp . Strain T2 beta-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports; Pessela BC et al.; A novel thermostable chimeric beta-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the beta-galactosidase from Thermus sp . strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC) . The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the beta-galactosidase . Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability . To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports . The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.

Neurosci Lett, 2003 Apr 24, 341(1), 69 - 73
Apolipoprotein E (apoE) uptake and distribution in mammalian cell lines is dependent upon source of apoE and can be monitored in living cells; Ljungberg MC et al.; As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor . Comparing recombinant, cellular and native apoE, complexed with different preparations of lipid we find that only cellular and native apoE enter a vesicular compartment . Some, but not all of these apoE containing vesicles are lysosomes . In order to further examine the intracellular fate of apoE we demonstrate that apoE-Enhanced green fluorescent protein chimeric protein can be taken up from medium by recipient cells and tracked within these cells for extended periods.

Mol Microbiol, 2003 Apr, 48(2), 561 - 71
DNA supercoiling contributes to disconnect sigmaS accumulation from sigmaS-dependent transcription in Escherichia coli; Bordes P et al.; The sigmaS subunit of RNA polymerase is a key regulator of Escherichia coli transcription in stress conditions . sigmaS accumulates in cells subjected to stresses such as an osmotic upshift or the entry into stationary phase . We show here that, at elevated osmolarity, sigmaS accumulates long before the beginning of the sigmaS-dependent induction of osmEp, one of its target promoters . A combination of in vivo and in vitro evidence indicates that a high level of DNA negative supercoiling inhibits transcription by EsigmaS . The variations in superhelical densities occurring as a function of growth conditions can modulate transcription of a subset of sigmaS targets and thereby contribute to the temporal disconnection between the accumulation of sigmaS and sigmaS-driven transcription . We propose that, in stress conditions leading to the accumulation of sigmaS without lowering the growth rate, the level of DNA supercoiling acts as a checkpoint that delays the shift from the major (Esigma70) to the general stress (EsigmaS) transcriptional machinery, retarding the induction of a subset of the sigmaS regulon until the conditions become unfavourable enough to cause entry into stationary phase.

Mol Microbiol, 2003 Apr, 48(2), 507 - 21
Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC); Grant AJ et al.; BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants . We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69 . The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE) . BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler . Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE . BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS . In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner . Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E . coli.

Mol Microbiol, 2003 Apr, 48(2), 335 - 48
Binding of the Escherichia coli MelR protein to the melAB promoter: orientation of MelR subunits and investigation of MelR-DNA contacts; Grainger DC et al.; The Escherichia coli MelR protein is a melibiose-triggered transcription factor, belonging to the AraC family, that activates transcription initiation at the melAB promoter . Activation is dependent on the binding of MelR to four 18 bp sites, centred at position -42.5 (site 2'), position -62.5 (site 2), position -100.5 (site 1) and position -120.5 (site 1') relative to the melAB transcription start point . Activation also depends on the binding of CRP to a single site located between MelR binding site 1 and site 2 . All members of the AraC family contain two helix-turn-helix (HTH) motifs that contact two segments of the DNA major groove at target sites on the same DNA face . In this work, we have studied the binding of MelR to different sites at the melAB promoter, focusing on the orientation of binding of the two MelR HTH motifs, and the juxtaposition of the different bound MelR subunits with respect to each other . To do this, MelR was engineered to contain a single cysteine residue adjacent to either one or the other HTH motif . The MelR derivatives were purified, and the cysteine residues were tagged with p-bromoacetamidobenzyl-EDTA-Fe, an inorganic DNA cleavage reagent . Patterns of DNA cleavage after MelR binding were then used to determine the positions of the two HTH motifs at target sites . In order to simplify our analysis, we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2', in the absence of CRP, is sufficient for transcription activation . To assist in the interpretation of our results, we also used a shortened derivative of MelR, MelR173, that is able to bind to site 2 but not to site 2' . Our results show that MelR binds as a direct repeat to site 2 and site 2' with the C-terminal HTH located towards the promoter-proximal end of each site . The orientation in which MelR binds to site 2' appears to be determined by MelR-MelR interactions rather than by MelR-DNA interactions . In complementary experiments, we used genetic analysis to investigate the importance of different residues in the two HTH motifs of MelR . Epistasis experiments provided evidence that supports the proposed orientation of binding of MelR at its target site.

Mol Microbiol, 2003 Apr, 48(2), 295 - 303
MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation; Lutkenhaus J et al.; The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli . It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA) . All members of the subgroup have a deviant Walker A motif which contains a conserved 'signature' lysine that characterizes this subgroup . In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis . ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer . Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient . In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable . Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane . The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.

Physiol Plant, 2003 Apr, 117(4), 467 - 475
Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp . W80; Takeda T et al.; A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 (C . W80) by a simple screening method based on the bacterial expression system . The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da . No potential signal peptide was found . The deduced amino acid sequence of the cDNA showed 40-63% and 37-46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively . The C . W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site . However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX . Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H2O2 or phospholipid hydroperoxide . Transformed E . coli cells expressing the C . W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 mM paraquat treatments . Accession number: The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.

Biochem J, 2003 Jul 1, 373(Pt 1), 49 - 55
Exocyst complex subunit sec8 binds to postsynaptic density protein-95 (PSD-95): a novel interaction regulated by cypin (cytosolic PSD-95 interactor); Riefler GM et al.; The PDZ domains of postsynaptic density (PSD) protein-95 play a role in the localization of PSD-95 and binding partners to neuronal synapses . The identification of binding partners to these PDZ domains can help us in understanding how signalling complexes are assembled . We observed that one of the subunits in the sec6/8 or exocyst complex, sec8, contains a C-terminal consensus sequence for PDZ binding . Sec8 binds to PDZ1-2 of PSD-95, and this binding can be competed with a peptide that binds to PDZ1 and PDZ2 in the peptide-binding site . In addition, binding of sec8 is dependent on its C-terminal-binding sequence namely Thr-Thr-Val (TTV) . Immunoblotting of rat tissue extracts shows that sec8 and PSD-95 are enriched in the same brain regions, and sec8 and PSD-95 have the same subcellular distribution in pheochromocytoma cells, suggesting that these proteins may interact in vivo . Immunoprecipitation studies of sec8 and PSD-95 in brain provide further evidence of a sec8 and PSD-95 interaction . Furthermore, the cytosolic PSD-95 interactor competes with sec8 for interaction with PSD-95 . Taken together, our results suggest that the cytosolic PSD-95 interactor may function to regulate the ability of sec8 to bind to PSD-95.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 440 - 7
Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities; Winters MA et al.; The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes . Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid . As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid . The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions . The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics . The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite . On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite . Conversely, the very weakly hydrated chaotrope I(-) may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 744 - 8
{Structure characteristics of ORF2a gene of potato leafroll virus Chinese isolate}; Zhao GF et al.; According to the genomic sequence of foreign four PLRV isolates, three pairs of specific primer were designed and synthesized . The cDNA of the ORF2a gene of PLRV-Ch was synthesized by reverse transcription and followed by Polymerase Chain Reaction amplication . The synthesized 3' and 5' cDNA fragment of the PLRV-Ch ORF2a gene were inserted into pUC19 and cloned in E . coli JM109 and were sequenced respectively . The middle cDNA fragment were directly sequenced . The homology of nucleotide sequence of PLRV-Ch compared with PLRV-S (Scotland, UK), PLRV-N(Netherlands), PLRV-A(Australia) and PLRV-C(Canada) were 98.96%, 98.70%, 94.79%, 97.5%, the homology of putative amino acid sequence are 97.97%, 97.97%, 89.69%, 95.94% . In 3' region of ORF2a gene a slippery sequence for-1 frameshift and its downstream "stem-loop" or "pseudoknot" and upstream nucleotide sequence repeats were found . Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins . This structure possibly has something to do with-1 frameshift . The amino acid sequence of C terminus region of 70 kD protein translated by motif IV has a protease characteristic motif and a helicase motif IV . The amino acid sequence of polypeptide translated by ORF2a gene undergoing frameshift has a single-stranded nucleic acid binding protein-like characteristic motif.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 693 - 7
{The renaturation and purification of RGD-staphylokinase by gel filtration}; Cheng A et al.; A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E . coli . The expression product accumulates as inclusion bodies . In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured . The renaturation of RGD-Sak was performed by gel filtration . Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation, After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95% . Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra . It was demonstrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 676 - 82
{Minimal functional domain of cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase from Escherichia coli}; Jin CS et al.; In comparison with its counterpart from N . meningitides, all conserved motifs were found in the N-termini of E . coli CMP-NeuAc synthetase . E . coli CMP-NeuAc synthetase seems to have redundant C-termini with a less effect on its activity . To explain this speculation, a series of recombinant DNAs with deletion from 3'-end of CMP-NeuAc synthetase were produced by PCR, ligated into expression vector pET-15b and expressed in BL21(DE3)pLysS . After induction with IPTG, we found that the recombinant enzyme with deletion of 189 amino acids from C0termini retained its activity . This result demonstrates that the 229 amino acids of N-termini was the minimal functional domain of E . coli CMP-NeuAc synthetase . The deletions altered the optimum pH and thermostability of active truncated enzymes, indicating that the truncated C-terminal amino acids of E . coli CMP-NeuAc synthetase could affect the conformation of the enzymatic catalytic domain and therefore affect its catalytic activity and thermostability, although it is not involved in enzymatic activity directly.

Biol Chem, 2003 Jan, 384(1), 125 - 37
The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells; Helbig JH et al.; We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila . Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity . Five of the mAbs recognised epitopes in the C-terminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species . Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro . mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity . All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937 . These data demonstrate for the first time that for the virulence-enhancing property of the L . pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.

Biol Chem, 2003 Jan, 384(1), 59 - 69
Purification of Drosophila melanogaster ultraspiracle protein and analysis of its A/B region-dependent dimerization behavior in vitro; Rymarczyk G et al.; Two members of the nuclear receptor superfamily, EcR (ecdysteroid receptor protein) and Usp (Ultraspiracle), heterodimerize to form a functional receptor for the steroid hormone 20-hydroxyecdysone and thus enable it to coordinate morphogenetic events during insect metamorphosis . N-terminally His-tagged Usp was overexpressed in E . coli cells as a non-truncated protein and purified to homogeneity in two chromatographic steps . It was demonstrated that the recombinant receptor specifically binds the ecdysone response element of the hsp27 gene promoter (hsp27EcRE) . Moreover, a highly synergistically formed heterodimeric complex with the DNA-binding domain of EcR was observed on hsp27EcRE, but not on the native Usp response element from the chorion s15 gene promoter . Recombinant Usp forms homodimers and homotetramers in the absence of DNA, as judged from gel filtration and chemical crosslinking experiments . Truncation of its N-terminal A/B region changes molecular characteristics of Usp, considerably weakening its oligomerization potential under the same experimental conditions . This contrasts with the results obtained previously for the similarly truncated RXR--a vertebrate homolog of Usp.

Biol Chem, 2003 Jan, 384(1), 51 - 8
Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP; Rakus D et al.; AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase) . Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively . Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme . An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme . However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM) . The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

Biol Chem, 2003 Jan, 384(1), 25 - 37
Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D); Moraes KC et al.; The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region . The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs . AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing . The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation . Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1 . We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins . We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1 . Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide . Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro . These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.

Biotechnol Bioeng, 2003 Jun 20, 82(6), 670 - 7
Optimization-based framework for inferring and testing hypothesized metabolic objective functions; Burgard AP et al.; An optimization-based framework is introduced for testing whether experimental flux data are consistent with different hypothesized objective functions . Specifically, we examine whether the maximization of a weighted combination of fluxes can explain a set of observed experimental data . Coefficients of importance (CoIs) are identified that quantify the fraction of the additive contribution of a given flux to a fitness (objective) function with an optimization that can explain the experimental flux data . A high CoI value implies that the experimental flux data are consistent with the hypothesis that the corresponding flux is maximized by the network, whereas a low value implies the converse . This framework (i.e., ObjFind) is applied to both an aerobic and anaerobic set of Escherichia coli flux data derived from isotopomer analysis . Results reveal that the CoIs for both growth conditions are strikingly similar, even though the flux distributions for the two cases are quite different, which is consistent with the presence of a single metabolic objective driving the flux distributions in both cases . Interestingly, the CoI associated with a biomass production flux, complete with energy and reducing power requirements, assumes a value 9 and 15 times higher than the next largest coefficient for the aerobic and anaerobic cases, respectively .

Yeast, 2003 Apr 15, 20(5), 439 - 54
Transcriptional regulation of YML083c under aerobic and anaerobic conditions; Ter Linde JJ et al.; YML083c and DAN1 were among the Saccharomyces cerevisiae ORFs that displayed the strongest increase in transcript abundance during anaerobic growth compared to aerobic growth, as determined by oligonucleotide microarrays . We here report that transcription of YML083c is regulated by at least three different factors . First, repression under aerobic conditions depends on the presence of heme . Second, deletion analysis of the 5'-flanking region of YML083c and DAN1 revealed two regions responsible for anaerobic induction . Each of these regions conferred anoxia-regulated expression to the heterologous, minimal, CYC1-lacZ reporter . Mutations in the AAACGA subelement, common to the positive acting regions of YML083c and DAN1, almost completely abolished the ability to drive anaerobic expression of the reporter gene . This subelement is similar to the AR1 site, which is involved in anaerobic induction of the DAN/TIR genes . Activation through the AR1 site depends on Upc2 . Indeed, transcription from the YML083c promoter was decreased in an upc2 null mutant . Third, expression of Sut1 under aerobic conditions enhanced transcription of YML083c, suggesting that aerobic repression of YML083c is promoted by the general Tup1-Ssn6 co-repressor complex . However, despite the presence of a sequence that matches the consensus for binding of Rox1, YML083c is not controlled by Rox1, since deletion or replacement of the putative binding site did not cause aerobic derepression . Moreover, YML083c expression was undetectable in aerobically grown cells of a rox1 null mutant .

Yeast, 2003 Apr 15, 20(5), 381 - 8
Characterization of an alternative oxidase activity of Histoplasma capsulatum; Johnson CH et al.; Histoplasma capsulatum possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consuming activities . The latter, carried out by a single subunit enzyme termed 'alternative oxidase', is the focus of this report . AOX1 cDNA clones were isolated and direct evidence that the cDNA ORF encodes functional alternative oxidase is reported . Also reported are the generation of an antiserum to the AOX1 protein product, and specific detection in vivo of the mRNA and protein products of the AOX1 gene . Finally, initial studies of regulation of H . capsulatum AOX1 gene expression demonstrated that RNA abundance was increased after hydrogen peroxide-mediated oxidative stress and after inhibition of mitochondrial electron transport enzymes with antimycin A or sodium cyanide . This pattern of regulation is consistent with the hypothesis that alternative oxidase contributes to survival of H . capsulatum after oxidative or metabolic stress and may be important for virulence of this pathogenic organism . The GenBank Accession Nos for the cDNA sequences reported in this paper are AF133236, AF133237 (AOX1) .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Apr, 35(4), 360 - 5
{Cloning and expression of a novel ubiquitin fusion gene Uba256}; Li ZF et al.; Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses . With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR . The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E . coli . The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector . Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37 . The results indicated that the purified ubiquitin and GP37 retained their antigenicity . Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

Acta Biochim Pol, 2003, 50(1), 269 - 78
Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens; Shabaan AM et al.; Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively . Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules . The clone was found to encode an 81 amino-acid protein fragment . It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa) . The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae . Clone 1H is one of 76 expressed sequence tags derived from an adult worm library . It encodes the complete sequence of a tegumental membrane protein, Sm13 . The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa . Clone 1H was expressed in E . coli as an insoluble fusion protein with Sj26 of about 40 kDa . In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera . The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

Acta Biochim Pol, 2003, 50(1), 155 - 67
Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders; Copeland WC et al.; This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases . Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma (polgamma) . We investigated the fidelity of DNA replication by polgamma with and without exonucleolytic proofreading and its p55 accessory subunit . Polgamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides . Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA . Recently, several mutations in the polymerase and exonuclease domains of the human polgamma have been shown to be associated with PEO . We are analyzing the effect of these mutations on the human polgamma enzyme . In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of polgamma . These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E.coli pol I . These PEO mutations have been generated in polgamma to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis . One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families . The Y955C mutant polgamma retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP . The Y955C derivative is also much less accurate than is wild-type polgamma, with error rates for certain mismatches elevated by 10- to 100-fold . The error prone DNA synthesis observed for the Y955C polgamma is consistent with the accumulation of mtDNA mutations in patients with PEO . The effects of other polgamma mutations associated with PEO are discussed.

Acta Biochim Pol, 2003, 50(1), 123 - 30
Selective splitting of 3'-adenylated dinucleoside polyphosphates by specific enzymes degrading dinucleoside polyphosphates; Guranowski A et al.; Several 3'-{(32)P}adenylated dinucleoside polyphosphates (Np(n)N'p*As) were synthesized by the use of poly(A) polymerase (Sillero MAG et al., 2001, Eur J Biochem.; 268: 3605-11) and three of them, ApppA{(32)P}A or ApppAp*A, AppppAp*A and GppppGp*A, were tested as potential substrates of different dinucleoside polyphosphate degrading enzymes . Human (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17) acted almost randomly on both AppppAp*A, yielding approximately equal amounts of pppA + pAp*A and pA + pppAp*A, and GppppGp*, yielding pppG + pGp*A and pG + pppGp*A . Narrow-leafed lupin (Lupinus angustifolius) tetraphosphatase acted preferentially on the dinucleotide unmodified end of both AppppAp*A (yielding 90% of pppA + pAp*A and 10 % of pA + pppAp*A) and GppppGp*A (yielding 89% pppG + pGp*A and 11% of pG + pppGp*A) . (Symmetrical) dinucleoside tetraphosphatase (EC 3.6.1.41) from Escherichia coli hydrolyzed AppppAp*A and GppppGp*A producing equal amounts of ppA + ppAp*A and ppG + ppGp*A, respectively, and, to a lesser extent, ApppAp*A producing pA + ppAp*A . Two dinucleoside triphosphatases (EC 3.6.1.29) (the human Fhit protein and the enzyme from yellow lupin (Lupinus luteus)) and dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53) from Saccharomyces cerevisiae did not degrade the three 3'-adenylated dinucleoside polyphosphates tested.

J Perinatol, 2003 Mar, 23(2), 148 - 53
Umbilical cord blood serum procalcitonin concentration in the diagnosis of early neonatal infection; Kordek A et al.; OBJECTIVE: To evaluate serum procalcitonin concentration in umbilical cord blood for diagnosis of intrauterine bacterial infection . MATERIALS AND METHODS: A prospective study was conducted between 2000 and 2001 . Serum procalcitonin concentrations were evaluated in 187 umbilical cord blood samples . Five groups have been defined: controls A (n=37), full-term noninfected B1 (n=80) and infected neonates B2 (n=8), preterm noninfected C1 (n=38) and infected C2 (n=24) newborns . An immunoluminometric assay was used to determine procalcitonin concentration . The Mann-Whitney U-test and Spearman's correlation ratio were applied . The sensitivity and specificity, the positive and negative predictive values, and the area under receiver operating characteristic curves were calculated . RESULTS: A statistically higher serum procalcitonin concentration was found in the preterm infected group (p<0.005; C2 vs A and C1) . CONCLUSION: Serum procalcitonin concentration in umbilical cord blood may be a useful parameter in the diagnosis of early neonatal infection.

Int Arch Allergy Immunol, 2003 Feb, 130(2), 119 - 24
Oral immunization with a recombinant major grass pollen allergen induces blocking antibodies in mice; de Weerd N et al.; BACKGROUND: Immunotherapy for the treatment of pollen allergies traditionally involves a series of parenteral injections of a crude pollen extract . Successful application of this treatment results in the development of systemic tolerance to the sensitizing allergens, including the induction of blocking antibodies . OBJECTIVE: We sought to investigate whether oral immunization with a recombinant pollen allergen could induce a systemic immune response, and the production of systemic blocking antibodies in mice . METHODS: C57BL/10 mice were orally administered rLol p 5 or sodium chloride solution via gavage . RESULTS: We report that the oral administration of rLol p 5 induced a systemic immune response, including the induction of both blocking and interspecific cross-reactive antibodies . CONCLUSION: Our results suggest that oral administration of a major grass pollen allergen can induce the development of a systemic immune response including the production of systemic blocking and cross-reactive antibodies, a response that may offer immunological protection upon subsequent allergen exposure .

Physiol Genomics, 2003 Jun 24, 14(1), 47 - 58
Evolutionary changes in heat-inducible gene expression in lines of Escherichia coli adapted to high temperature; Riehle MM et al.; The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established . However, their importance in genetic adaptation to long-term temperature stress is less clear . Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved at high temperature (41.5 degrees C) for 2,000 generations . These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT . As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression . Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role for the extracytoplasmic stress response in the adaptation to high temperature . Following acclimation at 41.5 degrees C, two of the three lines exhibited significantly improved survival at 50 degrees C, indicating changes in inducible thermotolerance . Thus evolution at high temperature led to significant changes at the molecular level in heat-inducible gene expression and at the organismal level in inducible thermotolerance and fitness.

J Biol Chem, 2003 Jun 20, 278(25), 22199 - 209 Epub 2003 Apr 02.
Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination; Thoren S et al.; Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli . mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography . The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1) . Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1) . The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation . Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1) . In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol min-1 mg-1) . These activities likely represent the evolutionary relationship to microsomal glutathione transferases . Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e . an organization similar to the microsomal glutathione transferase 1 . Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000 . This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.

J Biol Chem, 2003 Jun 20, 278(25), 22703 - 8 Epub 2003 Apr 02.
Properties of phosphoenolpyruvate mutase, the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi; Sarkar M et al.; Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds . The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi . Using partial sequence information from the T . cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis . Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1 . The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46) . Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell . Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T . cruzi cells . Southern and Western analysis suggests that PEP mutase is unique to T . cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.

J Biol Chem, 2003 Jun 20, 278(25), 22812 - 9 Epub 2003 Apr 02.
Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein, Tez1; Zhu Y et al.; Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker . Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype . Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction . Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype . Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response . None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function . In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation . Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input . In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.

J Neurobiol, 2003 May, 55(2), 115 - 33
fruitless gene is required to maintain neuronal identity in evenskipped-expressing neurons in the embryonic CNS of Drosophila; Song HJ et al.; The fruitless (fru) gene acts sex-nonspecifically in the development of the embryonic central nervous system (CNS) and has sex as well as sex-nonspecific functions in the development of the adult CNS . In the embryo, sex-nonspecific fru mRNAs and proteins are widely expressed during neurogenesis and present in both neurons and glia . To assess whether the fru gene played any role in fate determination of neuronal precursors and neurons, we examined the development of Eve-positive (Eve(+)) GMCs and neurons in fru mutants . In fru mutant embryos in which most or all fru transcripts were eliminated, the normal complement of Eve(+) neurons was present initially, but some neurons were unable to maintain their Eve-expression . Concomitantly, a subset of Eve(+) neurons also showed inappropriate expression of the glial marker, reversed polarity . In addition, neurons that normally do not express Eve became Eve(+) in these fru mutants . These defects were rescued in fru mutant embryos expressing specific fru transgenes under the control of the sca-GAL4 and elav-GAL4 drivers . These phenotypic analyses and rescue experiments provide evidence that one of the sex-nonspecific functions of the fru gene is the maintenance of neuronal identity rather than establishment of a neuron's initial fate .

Nat Rev Genet, 2003 Apr, 4(4), 309 - 14
New technologies to assess genotype-phenotype relationships; Bochner BR; The accelerating pace of the discovery of genes has far surpassed our capabilities to understand their biological function--in other words, the phenotypes they engender . We need efficient and comprehensive large-scale phenotyping technologies . This presents a difficult challenge because phenotypes are numerous and diverse, and they can be observed and annotated at the molecular, cellular and organismal level . New technologies and approaches will therefore be required . Here, I describe recent efforts to develop new and efficient technologies for assessing cellular phenotypes.

Exp Biol Med (Maywood), 2003 Apr, 228(4), 377 - 86
Verotoxin 1 from Escherichia coli affects Gb3/CD77+ bovine lymphocytes independent of interleukin-2, tumor necrosis factor-alpha, and interferon-alpha; Menge C et al.; Verotoxin (VT)-induced immunomodulation has been implicated in the ability of VT-producing Escherichia coli (VTEC) to cause persistent infections in cattle . VT1, also referred to as Shiga toxin 1, is a potent cytotoxin that modulates cytokine secretions and functions . This prompted the current investigation to examine whether the inhibiting effect of VT1 on bovine lymphocytes correlates with the expression of the cellular VT1 receptor Gb3/CD77 or is mediated instead via perturbation of cytokine secretion . Using blood mononuclear cells stimulated by mitogens as a model, VT1 significantly blocked lymphoblast transformation and proliferation in the BoCD8+ T cell and BoCD21+ B cell population . In contrast, VT1 dramatically reduced the number of viable Gb3/CD77+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ {i.e., gammadelta T cells} BoCD21+, and BoCD25+) . Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-alpha-sensitizing endothelial cells against VT1, interferon-alpha (IFN-alpha) as bovine IFN-alpha receptors are partially homologous to the B-subunit of VT1, and interleukin-2 that is critical for lymphocyte proliferation in vitro . The addition of these cytokines was neither able to mimic nor to overcome the effects of VT1 . Therefore, it is concluded that VT1 directly acts on bovine lymphocytes rather than inducing a cytokine-mediated effect . VT1 considerably affects all main bovine lymphocyte subpopulations, implicating that the immune system is a predominant target for VT1 in cattle.

Exp Biol Med (Maywood), 2003 Apr, 228(4), 370 - 6
Description of a novel intimin variant (type zeta) in the bovine O84:NM verotoxin-producing Escherichia coli strain 537/89 and the diagnostic value of intimin typing; Jores J et al.; Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually . These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion) . The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island . A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE . Intimin types alpha, beta, gamma, delta, and epsilon have been described previously . Each intimin represents distinct phylogenetic lineages of LEE-positive strains . A new intimin type zeta was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea . zeta intimin showed the highest similarity (88%) of its amino acid sequence to the alpha intimin . For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins) . This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types . By applying the PCR method, intimin zeta in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified . Because the intimin types represent distinctive phylogenetic E . coli lineages, application of the intimin subtyping PCR offers significant benefits . These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.

J Bacteriol, 2003 Apr, 185(8), 2673 - 9
In vitro properties of RpoS (sigma(S)) mutants of Escherichia coli with postulated N-terminal subregion 1.1 or C-terminal region 4 deleted; Gowrishankar J et al.; Derivatives of the stationary-phase sigma factor sigma(S) of Escherichia coli lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several sigma(S)-dependent promoters on supercoiled DNA templates . Unlike wild-type sigma(S), however, the deletion derivatives were inactive on relaxed templates . The anomalous slow electrophoretic mobility of sigma(S) on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in sigma(S) may be structurally and functionally analogous to subregion 1.1 of sigma(70), substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of sigma(70) (V . Gopal and D . Chatterji, Eur . J . Biochem . 244:614-618, 1997).

J Bacteriol, 2003 Apr, 185(8), 2563 - 70
Repression of Escherichia coli PhoP-PhoQ signaling by acetate reveals a regulatory role for acetyl coenzyme A; Lesley JA et al.; The PhoP-PhoQ two-component system regulates the transcription of numerous genes in response to changes in extracellular divalent cation concentration and pH . Here we demonstrate that the Escherichia coli PhoP-PhoQ two-component system also responds to acetate . Signaling by the E . coli PhoP-PhoQ system was repressed during growth in acetate (> or = 25 mM) in a PhoQ-dependent manner . The periplasmic sensor domain of PhoQ was not required for acetate to repress signaling . Acetate-mediated repression of the PhoP-PhoQ system was not related to changes in the intracellular concentration of acetate metabolites such as acetyl-phosphate or acetyladenylate . Genetic analysis of acetate metabolism pathways suggested that a perturbation of acetyl coenzyme A turnover was the cause of decreased PhoP-PhoQ signaling during growth in acetate . Consistent with this hypothesis, intracellular acetyl coenzyme A levels rose during growth in the presence of exogenous acetate . Acetyl coenzyme A inhibited the autokinase activity of PhoQ in vitro, suggesting that the in vivo repressing effect may be due to a direct inhibition mechanism.

J Bacteriol, 2003 Apr, 185(8), 2512 - 9
Regulation of the alternative sigma factor sigma(E) during initiation, adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli; Ades SE et al.; The alternative sigma factor sigma(E) is activated in response to stress in the extracytoplasmic compartment of Escherichia coli . Here we show that sigma(E) activity increases upon initiation of the stress response by a shift to an elevated temperature (43 degrees C) and remains at that level for the duration of the stress . When the stress is removed by a temperature downshift, sigma(E) activity is strongly repressed and then slowly returns to levels seen in unstressed cells . We provide evidence that information about the state of the cell envelope is communicated to sigma(E) primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in sigma(E) activity throughout the stress response . However, the relationship between sigma(E) activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.

J Bacteriol, 2003 Apr, 185(8), 2493 - 502
Geobacter sulfurreducens has two autoregulated lexA genes whose products do not bind the recA promoter: differing responses of lexA and recA to DNA damage; Jara M et al.; The Escherichia coli LexA protein was used as a query sequence in TBLASTN searches to identify the lexA gene of the delta-proteobacterium Geobacter sulfurreducens from its genome sequence . The results of the search indicated that G . sulfurreducens has two independent lexA genes designated lexA1 and lexA2 . A copy of a dinB gene homologue, which in E . coli encodes DNA polymerase IV, is present downstream of each lexA gene . Reverse transcription-PCR analyses demonstrated that, in both cases, lexA and dinB constitute a single transcriptional unit . Electrophoretic mobility shift assays with purified LexA1 and LexA2 proteins have shown that both proteins bind the imperfect palindrome GGTTN(2)CN(4)GN(3)ACC found in the promoter region of both lexA1 and lexA2 . This sequence is also present upstream of the Geobacter metallireducens lexA gene, indicating that it is the LexA box of this bacterial genus . This palindrome is not found upstream of either the G . sulfurreducens or the G . metallireducens recA genes . Furthermore, DNA damage induces expression of the lexA-dinB transcriptional unit but not that of the recA gene . However, the basal level of recA gene expression is dramatically higher than that of the lexA gene . Likewise, the promoters of the G . sulfurreducens recN, ruvAB, ssb, umuDC, uvrA, and uvrB genes do not contain the LexA box and are not likely to bind to the LexA1 or LexA2 proteins . G . sulfurreducens is the first bacterial species harboring a lexA gene for which a constitutive expression of its recA gene has been described.

J Bacteriol, 2003 Apr, 185(8), 2441 - 50
Transcription-defective soxR mutants of Escherichia coli: isolation and in vivo characterization; Chander M et al.; The soxRS regulon protects Escherichia coli from superoxide and nitric oxide stress . SoxR protein, a transcription factor that senses oxidative stress via its {2Fe-2S} centers, transduces the signal to the soxS promoter to stimulate RNA polymerase . Here we describe 29 mutant alleles of soxR that cause defects in the activation of soxS transcription in response to paraquat, a superoxide stress agent . Owing to the selection and screen used in their isolation, most of these mutant alleles encode proteins that retained specific binding activity for the soxS promoter in vivo . The mutations were found throughout the SoxR polypeptide, although those closer to the N terminus typically exhibited greater defects in DNA binding . The degree of the defect in the transcriptional response to superoxide caused by each mutation was closely paralleled by its impaired response to nitric oxide . This work begins the general identification of the residues in the SoxR polypeptide that are critical for transducing oxidative stress signals into gene activation.

J Bacteriol, 2003 Apr, 185(8), 2432 - 40
Signal detection and target gene induction by the CpxRA two-component system; DiGiuseppe PA et al.; The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors . CpxA resides in the inner membrane and has both kinase and phosphatase activities . CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes . Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification . Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE . Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent . We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA . Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points . Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress . Moreover, the cpxP promoter is more inducible than any other regulon member tested . Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone . Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.

J Bacteriol, 2003 Apr, 185(8), 2410 - 7
Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes; Peng X et al.; Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes . The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters . For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca . 1% of the genome . The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the S . solfataricus chromosome . Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S . solfataricus . A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced . The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function . It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure . It also recognizes both main families of repeat sequences in S . solfataricus . The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one . The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif . Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus . We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters.

J Bacteriol, 2003 Apr, 185(8), 2393 - 401
Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system; Lee YY et al.; The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal beta-subunits of eukaryotes . Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex . The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor . Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates . Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA . No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA . However, ClpY, lacking domain I (ClpY(Delta I)) was able to interact with itself and with intact ClpY . The C-terminal region of ClpY is important for interaction with other ClpY subunits . The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding . Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY . Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

J Biol Chem, 2003 Jun 6, 278(23), 20802 - 11 Epub 2003 Apr 01.
Recombinant Rhodobacter capsulatus xanthine dehydrogenase, a useful model system for the characterization of protein variants leading to xanthinuria I in humans; Leimkuhler S et al.; Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs . The crystal structures of bovine XDH and R . capsulatus XDH showed that the two proteins have highly similar folds . We have developed an efficient system for the recombinant expression of R . capsulatus XDH in Escherichia coli . The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase . However, R . capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form . EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase . X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme . Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R . capsulatus XDH, and the protein variant was purified and characterized . Two different forms of XDH-R135C were purified, an active (alphabeta)2 heterotetrameric form and an inactive (alphabeta) heterodimeric form . The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.

J Biol Chem, 2003 Jun 13, 278(24), 21421 - 8 Epub 2003 Mar 31.
Real-time kinetics of the interaction between the two subunits, Escherichia coli thioredoxin and gene 5 protein of phage T7 DNA polymerase; Singha NC et al.; T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host . The holoenzyme is essential for the replication of the phage . We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance . Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling . Reduced thioredoxin bound g5p but oxidized thioredoxin did not . The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nm for thioredoxin with 4.7 x 104.M-1.s-1 and 10.5 x 10-5.s-1 as the corresponding association (ka) and dissociation (kd) rate constants . The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions . Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nm, whereas mutant W28A had a KD of 12.5 nm . No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A . The effect of temperature on KD and the changes in enthalpy (-DeltaH = 20.2 kcal.m-1) and entropy (TDeltaS =-8.4 kcal.m-1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.

J Biol Chem, 2003 Jun 13, 278(24), 22102 - 11 Epub 2003 Mar 31.
Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site; Phillips C et al.; We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A . U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells . U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites . We therefore probed the structure surrounding the novel U1A binding sites . We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site . The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites . However, long range interactions are responsible for maintaining them in an open configuration . Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5' splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site . These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA . This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.

J Biol Chem, 2003 Jun 6, 278(23), 20667 - 72 Epub 2003 Mar 31.
A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii; Bjornberg O et al.; By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides . In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA . The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli . Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e . hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates . When the reaction was followed by thin layer chromatography using {3H}dCTP as substrate, dUMP and not dUTP was identified as a reaction product . In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from {3H}dCTP to the pool of dUTP . This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme . The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released . The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m) . According to limited proteolysis, the C-terminal residues constitute a flexible region . The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases . Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.

J Biol Chem, 2003 Jun 6, 278(23), 21076 - 82 Epub 2003 Apr 01.
Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL: implications for channel gating and evolutionary design; Maurer JA et al.; Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function . Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified . Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function . The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer . The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels.

J Biol Chem, 2003 Jun 13, 278(24), 21467 - 73 Epub 2003 Apr 01.
Identification of membrane domains of the Na+/H+ antiporter (NhaA) protein from Helicobacter pylori required for ion transport and pH sensing; Tsuboi Y et al.; The Na+/H+ antiporter from Helicobacter pylori (HP NhaA) is normally active within the pH range 6.0-8.5 . In contrast, the NhaA from Escherichia coli (EC NhaA) is active only within the alkaline pH range 7.5-8.5 . We studied structures of HP NhaA involved in ion transport and pH sensing by analyzing mutants with defects in NhaA activity . The 36 mutants were classified into three types . The first type exhibited very low or null activity at all pH levels and had amino acid substitutions in the transmembrane segments (TM) 4, 5, 10, and 11, implicating these TMs in ion transport . The second type, which had amino acid substitutions at Met-138, Phe-144, and Lys-347 in TM 4 and 10, exhibited very low antiporter activity at acidic pH but had significantly higher activity at alkaline pH . These results imply that TM 4 (Met-138 and Phe-144) and 10 (Lys-347) are involved in supporting transport activity at acidic pH, in addition to their essential role in the overall transport mechanism . The third type of mutant exhibited very low antiporter activity at alkaline pH but relatively normal activity at acidic pH and had amino acid substitutions in loop 7 (a hydrophilic region between TM 7 and 8) as well as in TM 8, suggesting that these regions are involved in antiporter activation at alkaline pH . Three revertants that suppress a Lys-347 mutation were identified . Two of three suppressor mutations were located in loops 2 and 4, suggesting a functional interaction between these regions (loops 2 and 4 and TM 10) . Thus, HP NhaA activity may be modulated by two independent factors that are dependent on pH: an activation mechanism at acidic pH, which is regulated by residues within TM 4 and 10 and another mechanism functioning at alkaline pH regulated by residues within loop 7 and TM 8.

J Biol Chem, 2003 Jun 6, 278(23), 20761 - 9 Epub 2003 Apr 01.
Activating omega-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3; Zackova M et al.; UCP2 (the lowest Km values: 20 and 29 microm, respectively) for omega-6 polyunsaturated FAs (PUFAs), all-cis-8,11,14-eicosatrienoic and all-cis-6,9,12-octadecatrienoic acids, which are also the most potent agonists of the nuclear PPARbeta receptor in the activation of UCP2 transcription . omega-3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (Km, 50 microm), although as an omega-6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (Km, approximately 200 microm) . These findings suggest a possible dual role of some PUFAs in activating both UCPn expression and uncoupling activity . UCP2 (UCP3)-dependent H+ translocation activated by all tested FAs was inhibited by purine nucleotides with apparent affinity to UCP2 (reciprocal Ki) decreasing in order: ADP > ATP approximately GTP > GDP >> AMP . Also {3H}GTP ({3H}ATP) binding to isolated Escherichia coli (Kd, approximately 5 microm) or yeast-expressed UCP2 (Kd, approximately 1.5 microm) or UCP3 exhibited high affinity, similar to UCP1 . The estimated number of {3H}GTP high affinity (Kd, <0.4 microm) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and approximately 20 in liver mitochondria . We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.

J Biol Chem, 2003 Jun 20, 278(25), 22846 - 52 Epub 2003 Apr 01.
Polyamines and glutamate decarboxylase-based acid resistance in Escherichia coli; Jung IL et al.; The expression of gadA and gadB, which encode two glutamate decarboxylases (GADs) of Escherichia coli, is induced by an acidic environment and participate in acid resistance . In this study, we constructed a polyamine-deficient mutant and investigated the role of polyamines in acid resistance . The expression of gadA and gadB was shown to be dependent on polyamines . For that reason, the polyamine-deficient mutant was completely devoid of GAD activity and was very susceptible to low pH if large amounts of polyamines were not provided . We also showed that the polyamine-deficient mutant contained higher cAMP levels than the isogenic polyamine-proficient wild type, and cAMP negatively regulated the expression of gadA and gadB . Therefore, introduction of the cya (encoding adenylate cyclase) mutation allele into the polyamine-deficient mutant resulted in the increment of GAD activity and thus restored the reduced acid resistance of the mutant . The positive regulators, H-NS (histone-like protein, encoded by the hns gene) and RpoS (alternative RNA polymerase sigma subunit, encoded by rpoS gene), also significantly governed the expression of gadA and gadB, respectively . However, polyamines did not regulate either the intracellular H-NS level or rpoS expression under these culture conditions . These results strongly suggest that there are at least two different regulatory systems in acid resistance, one is positive regulation via a H-NS/RpoS system and the other is negative regulation via a polyamine/cAMP system.

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 295 - 301
Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement; Jiao Z et al.; The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis . It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP) . The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains . The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase . The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.

Bioorg Med Chem, 2003 May 1, 11(9), 2091 - 8
Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions; Wada M et al.; Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis . Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars . The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO . The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes . We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165 . The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine . Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.

Vet Q, 2003 Mar, 25(1), 31 - 44
Sympathoadrenal and immune system activation during the periparturient period and their association with bovine coliform mastitis . A review; Diez-Fraile A et al.; Increased incidence of clinical mastitis in high-yielding cows during early lactation has been attributed to a depressed functional capacity of the immune system . Sympathoadrenal factors are known to play an important role in modulating the host susceptibility and resistance to infectious diseases . Of primary importance in combating acute intramammary infections are polymorphonuclear leukocytes (PMN), as they represent one of the early lines of immunological defense . The release of stress hormones at parturition and during the first weeks of lactation has been proposed to partly contribute to the impaired function of PMN . Here, we summarize the current understanding of the stress-induced peripheral effectors, i.e . the limbs of the sympathetic system and the hypothalamic-pituitary-adrenal axis, on PMN function around parturition and during coliform mastitis . The questions as to whether and how stress induced secretion of glucocorticoids and catecholamines might affect the lactating dairy cow's udder health will be addressed.

Curr Opin Drug Discov Devel, 2003 Mar, 6(2), 188 - 96
Production of antibodies and antibody fragments in Escherichia coli and a comparison of their functions, uses and modification; Humphreys DP; Antibody-based drugs are now used in the clinic, and their importance as therapeutics is expected to increase in the coming decade, both in terms of diseases treated and market value . However, the proportion of patients that can be treated is to some extent affected by the availability and cost of the therapeutic . Therefore, the means of production and purification of the therapeutic entity, as well as the type of therapeutic entity, are of great concern . Escherichia coli represents a good production host in terms of scale and speed of production, but it also has technical and biological limitations . The aim of this review is to describe and discuss the relative benefits and limitations of E coli as a host for the production of antibodies and engineered antibody fragments.

Genetika, 2003 Feb, 39(2), 286 - 92
{Role of "anti-restriction" motif in functional activity of anti-restriction protein ArdA pKM101 (incN)}; Rastorguev SM et al.; A number of mutant forms of the antirestriction protein ArdA encoded by the ardA gene located in a transmissive IncN plasmid pKM101 have been constructed . Proteins belonging to the Ard family are specific inhibitors of type I restriction--modification enzymes . Single mutational substitutions of negatively charged amino acid residues located in the "antirestriction motif" with hydrophobic alanine, E134A, E137A, D144A, or a double substitution E134A, E137A do not affect the antirestriction activity (Ard) of ArdA but almost completely abolish the antimodification activity (Amd) . Mutational substitutions F107D and A110D in the assumed interface ArdA, which determines contact between monomers in the active dimer (Ard)2, cause an approximately 100-fold decrease in the antirestriction protein activity . It is hypothesized that the ArdA protein forms two complexes with the type I restriction--modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with a nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonucleases . The association of ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.

Genetika, 2003 Feb, 39(2), 259 - 68
{Targeted intracellular site-specific drug delivery: photosensitizer targeting to melanoma cell nuclei}; Rozenkrants AA et al.; A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell . The response of different cellular compartments to these effects is different . Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced . This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting . These constructs include (1) the alpha-melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E . coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin . These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude . In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.

Curr Opin Investig Drugs, 2003 Feb, 4(2), 156 - 61
Recent developments in mucosal immunomodulatory adjuvants; Harandi AM et al.; A large proportion of pathogens either invade through, or cause disease at mucosal surfaces . Many new generation mucosal vaccine candidates lack important immunostimulatory features of the original pathogens and thus often do not elicit sufficiently strong immune responses . Despite numerous efforts, there is a profound lack of available agents with mucosal immunomodulatory and adjuvant activity . Immunomodulatory adjuvants are often derived from pathogens and thus efficiently activate the innate immune system leading to subsequent development of strong, specific acquired immunity . In this review, recent advances in mucosal immunomodulators/adjuvants are described with special emphasis on recently developed detoxified cholera toxin and Escherichia coli heat labile enterotoxin derivatives, and the newly described Toll-like receptor ligands CpG DNA and imidazoquinoline compounds . These agents hold much promise as useful mucosal immunomodulators/adjuvants for induction of strong innate immune response and also for subsequent development of specific acquired immunity against mucosal pathogens.

Plant Cell Physiol, 2003 Mar, 44(3), 334 - 41
A role of the -35 element in the initiation of transcription at psbA promoter in tobacco plastids; Hayashi K et al.; Most plastid promoters recognized by bacteria-like plastid RNA polymerase (PEP) are similar to E . coli sigma(70)-type promoters comprising "-35" and "-10" elements . Among them, psbA promoter is unique in bearing additional elements between the conserved -35 and -10 elements . The psbA promoter activity is differentially maintained in the mature chloroplasts where the activity of most PEP promoters declines . Previously, we identified two types of PEP activities in wheat seedlings {Satoh et al . (1999) Plant J . 18: 407}; PEP present in the mature chloroplasts of the leaf tip (tip-type PEP) can initiate transcription from the -35-destructed psbA promoter, but the -35 element is essential for transcription by PEP present in immature chloroplasts of the leaf base (base-type PEP) . To reveal which type of PEP functions in various types of plastids in tobacco, we analyzed the tobacco psbA promoter by means of a transplastomic approach . The promoter core context (-42 to +9) was sufficient for developmental regulation of the psbA promoter activity . The -35 promoter element was important for transcription initiation at the psbA promoter in all types of plastids, including chloroplasts in mature leaves, leucoplasts in roots, etioplasts in etiolated cotyledons . The conclusion is that the PEP bearing a promoter preference, similar to the wheat base-type PEP, functions dominantly in tobacco chloroplasts.

Plant Cell Physiol, 2003 Mar, 44(3), 269 - 76
Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp . PCC 7942; Kobayashi D et al.; We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp . PCC 7942 . The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK . The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system . However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants . The recombinant PRK of Synechococcus sp . PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli . The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1) . The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast . The oxidized PRK was fully activated by treatment with excessive dithiothreitol . Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2) . These results suggest Synechococcus sp . PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.

J Biol Chem, 2003 Jun 6, 278(23), 20526 - 32 Epub 2003 Mar 31.
Mismatch uracil glycosylase from Escherichia coli: a general mismatch or a specific DNA glycosylase?
O'Neill RJ, Vorob'eva OV, Shahbakhti H, Zmuda E, Bhagwat AS, Baldwin GS.
The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U.G mismatches . Subsequently, 3,N4-ethenocytosine (epsilonC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme . The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme . Our results demonstrate that product release by the enzyme is very slow and hence comparing the "steady-state" parameters of the enzyme for different substrates is of limited use . Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions . Although the enzyme excised both epsilonC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis . Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate . This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism.

J Biol Chem, 2003 Jun 6, 278(23), 20731 - 7 Epub 2003 Mar 31.
The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol; Quinlivan EP et al.; Plants synthesize p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria . It has generally been supposed that pABA exists as the free acid in plant cells and that it moves between organelles in this form . Here we show that fruits and leaves of tomato and leaves of a diverse range of other plants have a high capacity to convert exogenously supplied pABA to its beta-D-glucopyranosyl ester (pABA-Glc), whereas yeast and Escherichia coli do not . High performance liquid chromatography analysis indicated that much of the endogenous pABA in fruit and leaf tissues is esterified and that the total pool of pABA (free plus esterified) varies greatly between tissues (from 0.2 to 11 nmol g-1 of fresh weight) . UDP-glucose:pABA glucosyltransferase activity was readily detected in fruit and leaf extracts, and the reaction was found to be freely reversible . p-Aminobenzoic acid beta-D-glucopyranosyl ester esterase activity was also detected in extracts . Subcellular fractionation indicated that the glucosyltransferase and esterase activities are predominantly if not solely cytosolic . Taken together, these results show that reversible formation of pABA-Glc in the cytosol is interposed between pABA production in chloroplasts and pABA consumption in mitochondria . As pABA is a hydrophobic weak acid, its uncharged form is membrane-permeant, and its anion is consequently prone to distribute itself spontaneously among subcellular compartments according to their pH . Esterification of pABA may eliminate such errant behavior and provide a readily reclaimable storage form of pABA as well as a substrate for membrane transporters.

Biophys J, 2003 Apr, 84(4), 2728 - 33
Kinetics of intramolecular electron transfer in cytochrome bo3 from Escherichia coli; Ching E et al.; We have examined the temperature dependence of the intramolecular electron transfer (ET) between heme b and heme o(3) in CO-mixed valence cytochrome bo(3) (Cbo) from Escherichia coli . Upon photolysis of CO-mixed valence Cbo rapid ET occurs between heme o(3) and heme b with a rate constant of 2.2 x 10(5) s(-1) at room temperature . The corresponding rate of CO recombination is found to be 86 s(-1) . From Eyring plots the activation energies for these two processes are found to be 3.4 kcal/mol and 6.7 kcal/mol for the ligand binding and ET reactions, respectively . Using variants of the Marcus equation the reorganization energy (lambda), electronic coupling factor (H(AB)), and the ET distance were found to be 1.4 +/- 0.2 eV, (2 +/- 1) x 10(-3) eV, and 9 +/- 1 A, respectively . These values are quite distinct from the analogous values previously obtained for bovine heart cytochrome c oxidase (CcO) (0.76 eV, 9.9 x 10(-5) eV, 13.2 A) . The differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET suggested by the Marcus parameters can be attributed to structural changes at the Cu(B) site upon change in oxidation state as well as differences in electronic coupling pathways between Heme b and heme o(3).

J Virol Methods, 2003 Apr, 109(1), 75 - 83
Identification of crucial residues of conformational epitopes on VP2 protein of infectious bursal disease virus by phage display; Cui X et al.; A new method for identifying epitopes in viral proteins expressed by filamentous phage has been developed . Filamentous phage fUSE 1 containing the variable region of the VP2 gene of infectious bursal disease virus (IBDV) strain 002-73 was constructed . Neutralizing monoclonal antibodies 17-82 and 33-10 raised against VP2 protein were used to bind phage containing the original variable region of VP2 . The phage bound to monoclonal antibodies, were removed by protein G Sepharose and the unbound phage (escape mutants) were isolated for sequencing to locate the mutations . The crucial amino acid residues for conformational neutralizing epitopes recognized by the monoclonal antibodies were located in the first main hydrophilic region (amino acids from 210 to 225) and the central region of the variable region of VP2 . The amino acid residues on both ends of the variable region of VP2 affected considerably the binding of monoclonal antibodies . This technique might be useful for selecting escape mutants of phage displaying the original antigenic regions of other viruses to define the crucial amino acid residues of their conformational epitopes, especially viruses that cannot be grown in cell cultures.

Complement Ther Med, 2003 Mar, 11(1), 4 - 10
Effect of acupuncture on the neutrophil respiratory burst: a placebo-controlled single-blinded study; Karst M et al.; OBJECTIVES: Little is known about the influence of acupuncture treatment on the phagocytic immune system . This trial was performed to examine whether multiple acupuncture treatment affects the respiratory burst (RB) of neutrophils, a recognised measure of their cytotoxicity . DESIGN: Placebo-controlled single-blinded study . INTERVENTIONS: Eleven volunteers were treated bilaterally with standard needles (real) at acupoint LI11, 11 volunteers with placebo needles (placebo) at the same point . Treatments were performed for 30 min each twice a week for 4 weeks, eight times in all . The standard needles were manipulated until needle sensation (DEQI) developed . Before the treatment course (baseline), 48 h after the fourth (follow-up 1) and 48 h after the last treatment (follow-up 2) blood samples were drawn . MAIN OUTCOME MEASURES: RB and plasma beta-endorphin at each time point . RESULTS: In the real group there was a highly significant increase in the RB at follow-ups 1 (P=0.004) and 2 (P=0.007) . Beta-endorphin levels decreased, but not significantly . In the placebo group there was a significant increase in the RB at follow-up 2 (P=0.048) . In addition, at follow-up 2 a significant drop in beta-endorphin levels was observed (P=0.015) . CONCLUSIONS: The RB of neutrophils is significantly activated by a course of several acupuncture treatments . In addition, psychological effects and a placebo that was not totally inert may contribute to the findings in the placebo group which may be mediated by the opiate endorphin system.

J Inorg Biochem, 2003 Apr 1, 94(4), 343 - 7
CO binding to the isolated oxygenase domain of neuronal nitric oxide synthase: effects of inhibitors and mutations at the substrate-binding site; Bengea S et al.; In order to understand the heme distal structure of neuronal nitric oxide synthase (nNOS), we studied the binding affinity of CO for the ferrous wild type enzyme and the Glu592Ala and Tyr588His substrate binding-site mutants (generated in the oxygenase domain, nNOSox) in the presence of substrates and inhibitors . The CO binding affinities (K(d) = 10-21 mM) of nNOSox in the presence of the substrates, L-Arg and NHA, or inhibitors such as NAME and agmatine were more than two-fold lower than in their absence (K(d) = 5 mM) . The presence of NIL strongly inhibited CO binding and gave a K(d) of more than 100 mM . These effects were not observed for the Glu592Ala mutant . The trend in CO binding affinities observed for the Tyr588His mutant was similar to that of the wild type enzyme . The presence of the isolated reductase domain did not affect CO binding . We discuss the role of substrate and inhibitor binding in CO complexation as well as in catalysis .

J Immunol Methods, 2003 Apr 1, 275(1-2), 203 - 12
From EST to IHC: human antibody pipeline for target research; Frisch C et al.; We have developed a method for the high-level expression of expressed sequence tags (ESTs) as inclusion bodies in Escherichia coli by C-terminal fusion to the N1-domain of g3p of filamentous phage M13 . Soluble fusion protein is obtained by an efficient refolding procedure . We have applied such protein preparations to the selection of human antibody fragments from phage-displayed HuCAL libraries . For all fusion proteins tested in this study, HuCAL antibodies could be generated which specifically detect, e.g . in immunohistochemistry, the maternal full-length protein corresponding to the protein fragment . This expression technology, in combination with the automated HuCAL antibody generation (AutoCAL), has proven to be useful for the rapid, high-throughput generation of high-quality human antibodies against EST-encoded protein fragments for target research.

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