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 t