Escherichia coli

J Pharmacol Exp Ther, 2000 Sep, 294(3), 810 - 21
Cloning and cellular localization of the rat mast cell 78-kDa protein phosphorylated in response to the mast cell "stabilizer" cromolyn; Theoharides TC et al.; Disodium cromoglycate (cromolyn) inhibits mast cell secretion, but its mechanism has not been elucidated . One possibility is the phosphorylation of a 78-kDa mast cell protein, two fragments of which are homologous to moesin, a member of the ezrin, radixin, moesin family . These proteins appear to be involved in signal transduction by regulating functional associations between the cell surface and the cytoskeleton . Moesin cDNA was cloned from rat basophil leukemia cells, which are similar to mucosal mast cells, and polyclonal antiserum was prepared against recombinant moesin expressed in Escherichia coli . Moesin phosphorylated in mast cells treated with cromolyn shifted from the soluble to the precipitable fraction and associated with Sepharose-linked beta-actin . Recombinant moesin also associated with Sepharose-linked beta-actin, and so did purified RBL moesin, but only if the latter was first denatured . Moesin thus appears to have actin binding sites that are not exposed under normal conditions but may become available by in vivo phosphorylation or by denaturation . Immunocytochemistry using confocal microscopy showed moesin to be primarily localized on the inner aspect of the plasma membrane and around secretory granules . Double immunocytochemistry for moesin and actin colocalized them in most areas . Ultracryoimmunoelectron microscopy to preserve the antigenicity of moesin identified the protein close to the plasma and secretory granule membranes . Cromolyn appeared to induce clustering of moesin around secretory granules . It is hypothesized that conformational changes of moesin, regulated by phosphorylation/dephosphorylation, may lead to positional rearrangements with respect to the membrane/cytoskeleton that could possibly regulate mast cell secretion.

Hum Gene Ther, 2000 Jul 20, 11(11), 1521 - 8
Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo; Manome Y et al.; One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of the transgene, involves utilization of mechanical forces to facilitate transduction of DNA into cells . In this study, we evaluated the feasibility of mechanical insonation in gene transfers with naked DNA plasmid loading both in vitro and in vivo . We used an ultrasound probe, which can focus the ultrasonic beam in the exit zone of the probe . The reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and the neomycin 3'-phosphotransferase gene (neo), was used for evaluation of transfer efficiency . Expression of beta-gal in MC38 murine colon carcinoma cells was measured after insonation of 20 W/cm2 with continuous 1.0-MHz wave exposure . In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene . After cells were treated with geneticin, we also observed a difference in colonogenicity between noninsonated and insonated groups . When MC38 cells were implanted in syngeneic mice and plasmid was injected, the insonation that followed facilitated beta-galactosidase expression . These results indicate that insonation represents a potential approach for gene therapy when combined with naked DNA plasmid injection.

Cancer Res, 2000 Aug 1, 60(15), 4098 - 104
Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells; Levine RL et al.; To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector . The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations . The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct . Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand . EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells . In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.

J Protein Chem, 2000 Feb, 19(2), 123 - 8
Photoreversible modulators of Escherichia coli beta-galactosidase . 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene; Golan R et al.; Beta-galactosidase (EC 3.2.1.23) is known to be inhibited by some thiol reagents . 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (1) was shown to be an irreversible inhibitor, while 1, 1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2) was demonstrated as a positive irreversible modulator causing a rise of up to 186% in beta-galactosidase activity . Compound 2 is, however, an irreversible inhibitor of the cysteine proteinase papain (preceding paper) . Kinetic values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and for compounds 1 and 2 were determined and in view of model experiments, it was assumed that both compounds possibly reacted with the thiol side chain of Cys in the active site inducing allosteric changes in the enzyme . Since the enzyme, modified by compound 1 or 2, was a 2-nitrobenzyl derivative, near-UV irradiation resulted in a recovery of up to 91% and a reduction of the enzyme's activity to 90%, respectively.

Intensive Care Med, 2000 Jun, 26(6), 770 - 5
The effect of CVVHD and endotoxin on the oxidative burst, adhesion molecules and distribution in tissues of granulocytes; Toft P et al.; OBJECTIVE: Extracorporeal circulation, such as cardiopulmonary bypass and haemodialysis, has been associated with an activation of the immune system, especially the granulocytes . Continuous veno-venous haemodiafiltration (CVVHD) is used in critically ill septic patients . During CVVHD cytokines are excreted in the ultrafiltrate . But when the membranes used in CVVHD are cultured with granulocytes, the granulocytes are slightly activated . This effect is potentiated by endotoxin . We therefore, in vivo, compared the effect on granulocyte activation of CVVHD with an endotoxin group and a control group . METHODS: Thirty-one pigs were anaesthetized and mechanically ventilated . In ten pigs CVVHD was performed . Eleven pigs received an infusion of Escherichia coli endotoxin 30 mu/kg(-1) and ten pigs served as a control group . The adhesion molecules CD18 and CD62L were measured using monoclonal antibodies . The oxidative burst activity was assayed as superoxide dismutase-inhibitory reduction of cytochrome c . The number of granulocytes in peripheral blood and in the lungs and liver were counted . RESULTS: The infusion of endotoxin was followed by granulocytopenia, reduced oxidative burst activity, increased expression of CD18 and decreased expression of CD62L on granulocytes . Accumulation of granulocytes in liver and lung tissue was also noted in this group . CVVHD was only associated with a non-significant decrease in CD62L expression on granulocytes . It did not affect any of the other measured immunological parameters . CONCLUSION: In contrast to endotoxin-induced sepsis, the granulocytes were not activated during CVVHD.

Plant Cell Physiol, 2000 Jun, 41(6), 785 - 90
Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K+ channel KAT1 alters current parameters in Xenopus laevis oocytes; Berkowitz G et al.; Increased guard cell cytosolic {Ca2+} is known to be involved in signal transduction pathways leading to stomatal closure, and inhibit the inward rectifying guard cell K+ channel KAT1 . Guard cell calcium-dependent protein kinase (CDPK) has been shown to phosphorylate KAT1; such phosphorylation is known to modulate other K+ channels involved in signal transduction cascades . The work reported here focused on demonstrating CDPK-dependent inhibition of KAT1 currents . A cDNA encoding soybean CDPK was generated and it's translation product was shown to be functional; demonstrating Ca2+-dependent autophosphorylation and phosphorylation of a target protein . Ion currents were monitored using voltage clamp techniques upon expression of KAT1 in Xenopus laevis oocytes . Coexpression of recombinant CDPK with KAT1 in oocytes altered the kinetics and magnitude of induced K+ currents; at a given hyperpolarizing command voltage, the magnitude of KAT1 currents was reduced and the half-time for channel activation was increased . This finding supports a model of Ca2+-dependent ABA inhibition of inward K+ currents in guard cells as being mediated by CDPK phosphorylation of KAT1.

Plant Cell Physiol, 2000 Jun, 41(6), 666 - 75
Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli; Yabuta Y et al.; A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves . The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues . In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells . The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively . Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme . Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities . We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli . The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.

J Vet Med Sci, 2000 Jul, 62(7), 717 - 23
Immunization with recombinant surface antigens p26 with Freund's adjuvants against Babesia rodhaini infection; Igarashi I et al.; The surface proteins of Babesia rodhaini have previously been shown to induce a high degree of protective immunity . In the present study, one of those proteins, B . rodhaini antigen p26 was expressed in Escherichia coli and in insect cells infected with a recombinant baculovirus . These proteins were recognized by immune serum from a drug-cured BALB/c mouse . While BALB/c mice immunized with both recombinant antigens and Freund's adjuvants showed 40-100% survival rate against challenge infection with B . rodhaini, saponin failed to induce protection, although significant levels of B . rodhaini-specific antibodies were produced in both immunized mice (1:1,000-2,000 by indirect immunofluorescent antibody test) . The immunization of IFN-gamma-deficient mice with the recombinant proteins was not protective against B . rodhaini infection, indicating that IFN-gamma is one of the important factors for the survival against lethal B . rodhaini infection.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1547 - 51
A flexible single-step detection of blotted antigen using a fusion protein between protein A and green fluorescent protein; Aoki T et al.; A green fluorescent protein mutant (S147P GFP) was fused with protein A and expressed in Escherichia coli . This fusion protein (PA-GFP147) was used in immunoblotting studies as a new detection system, designated as "flexible single-step detection (FSSD)" . In FSSD, the detection of blotted antigen was done in one step, and the antigen-antibody reaction can be monitored by UV-irradiation in real time . The reaction time, therefore, can be flexibly controlled by monitoring the green fluorescence.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1430 - 6
Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate; Yasohara Y et al.; An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals . The gene encoding the enzyme was cloned and sequenced . The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da . The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily . The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter . The enzyme expressed in E . coli was purified to homogeneity and had the same catalytic properties as the enzyme from C . magnoliae did . An E . coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1394 - 401
Cloning, sequencing, and expression of the tulip bulb chitinase-1 cDNA; Yamagami T et al.; A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a lambda ZAP cDNA library with anti-TBC-1 antiserum and the 5' rapid amplification of cDNA end (RACE) method, and sequenced . The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids . Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N- and C-termini, respectively . The deduced sequence of TBC-1 had 10-20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively . The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3) . The recombinant TBC-1 (rTBC-1) expressed in E . coli was purified by gel filtration followed by ion-exchange chromatography . Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin . This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.

Planta, 2000 Jul, 211(2), 191 - 9
Clonal analysis of the Arabidopsis root confirms that position, not lineage, determines cell fate; Kidner C et al.; The cellular organization of the Arabidopsis thaliana (L.) Heynh . root meristem suggests that a regular pattern of cell divisions occurs in the root tip . Deviations from this pattern of division might be expected to disrupt the organization of cells and tissues in the root . A clonal analysis of the 3-d-old primary root meristem was carried out to determine if there is variability in division patterns, and if so to discover their effect on cellular organization in the root . Clones induced in the seedling meristem largely confirmed the predicted pattern of cell divisions . However, the cellular initials that normally give rise to the different cell files in the root were shown to exhibit some instability . For example, it was calculated that a lateral root cap/epidermal initial is displaced every 13 d . Furthermore, the existence of large marked clones that included more than two adjacent cell layers suggests that intrusive growth followed by cell division may occur at low frequency, perhaps in response to local cell deaths in the meristem . These findings support the view that even in plant organs with stereotypical cell division patterns, positional information is still the key determinant of cell fate.

Planta, 2000 Jul, 211(2), 173 - 81
The H1 histone variant of tomato, H1-S, is targeted to the nucleus and accumulates in chromatin in response to water-deficit stress; Scippa GS et al.; Water deficit has a significant impact on patterns of gene expression . Based on the deduced amino acid sequence, it has been proposed that the drought and abscisic acid-induced gene (his1-s) of tomato (Lycopersicon esculentum Mill.) encodes an H1 histone variant . To study the role of H1-S it is important to understand the expression characteristics of the protein . To identify the his1-s product in vivo the his1-s cDNA was fused to a (His)6 tag and overexpressed in Escherichia coli . The H1-S fusion protein was used to generate an antibody that recognized a protein with an apparent molecular weight of 31 kDa that accumulates in response to water deficit in the whole plant and detached leaves . A time course of his1-s expression showed that protein accumulation is delayed compared to the mRNA accumulation in both the whole plant and detached leaves . Cellular fractionation, immunofluorescence and H1-S::beta-glucuronidase fusion analyses in transgenic tissues were used to determine the cellular localization of H1-S . The results showed that H1-S accumulates in nuclei and is associated with chromatin of wilted tomato leaves . The drought- and abscisic acid-induced gene his1-s encodes a linker-histone subtype specifically accumulated in the nuclei and chromatin of tomato leaves subjected to water-deficit conditions . Although the molecular mechanism of H1-S function is still unclear, the expression characteristics of H1-S are consistent with a potential role of this protein in the regulation of gene expression in response to water deficit.

Biochem Biophys Res Commun, 2000 Aug 18, 275(1), 169 - 73
Suppressive mechanism of salmosin, a novel disintegrin in B16 melanoma cell metastasis; Kang IC et al.; We have previously reported that salmosin, a novel disintegrin, was isolated from Korean snake (Agkistrodon halys brevicaudus) venom and significantly inhibited solid tumor growth in mice by perturbation of tumor-specific angiogenesis via blocking alphavbeta3 integrin expressed on vascular endothelial cells . In this study, we investigated the functional specificity of salmosin in tumor cell metastasis . Recombinant salmosin expressed in E . coli that has the RGD sequence markedly inhibited both B16F10 melanoma cell adhesion to the extracellular matrix proteins as well as B16F10 melanoma cell invasion through Matrigel-coated filter . The inhibition by salmosin can be caused by blocking integrins expressed on the surface of B16F10 melanoma cells . Salmosin significantly inhibited the proliferation of B16F10 melanoma cells on the plate coated with collagen I in a dose-dependent manner . In vivo B16F10 melanoma experimental metastasis, salmosin showed remarkable significant inhibitory effect on lung tumor colonization in a concentration-dependent manner . These results clearly demonstrate that antimetastatic activity of salmosin resulted from blocking the integrin-mediated adherence and alphavbeta3 integrin-mediated proliferation of the melanoma cells .

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1051 - 4
Crystallization and preliminary crystallographic investigations of avian 5-aminoimidazole-4-carboxamide ribonucleotide transformylase-inosine monophosphate cyclohydrolase expressed in Escherichia coli; Reyes VM et al.; ATIC {5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase)-inosine monophosphate cyclohydrolase (IMPCH)} is a bifunctional enzyme that catalyzes the penultimate and final steps in the de novo purine biosynthesis pathway and thus is an attractive anticancer target . Recombinant avian ATIC has been purified from an Escherichia coli expression system and crystallized in a binary complex with methotrexate (MTX) . Crystals were obtained from PEG 4000 or MPEG 5000 buffered at pH 7.0-7.2 and data were collected from a single crystal at 96 K to 2.3 A resolution at the Stanford Synchrotron Radiation Laboratory (SSRL) . The crystals are monoclinic and belong to space group P2(1), with unit-cell dimensions a = 65.17, b = 105.93, c = 103.47 A, beta = 108.27 degrees . Assuming two molecules per asymmetric unit, the Matthews coefficient V(m) is 2.63 A(3) Da(-1) and the solvent volume is 52.9%.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1049 - 50
Crystallization and preliminary X-ray crystallographic studies on maltosyltransferase from Thermotoga maritima; Burke J et al.; Thermotoga maritima maltosyltransferase (MTase) is a 73.7 kDa molecular weight amylolytic enzyme which catalyzes the transfer of maltosyl units from maltodextrins or starch to suitable acceptors . Crystals of recombinant MTase have been obtained by the hanging-drop vapour-diffusion method using ammonium phosphate as a precipitating agent . The crystals belong to space group P4(1)22 or its enantiomorph P4(3)22, with unit-cell parameters a = b = 148.7, c = 106.7 A . The asymmetric unit appears to contain one subunit, corresponding to a very low packing density of 4.0 A(3) Da(-1) . The crystals diffract X-rays to at least 2.4 A resolution on a synchrotron-radiation source.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1045 - 8
Crystallization and preliminary X-ray crystallographic analysis of PdxJ, the pyridoxine 5'-phosphate synthesizing enzyme; Garrido Franco M et al.; The enzyme PdxJ catalyzes the condensation of 1-deoxy-D-xylulose-5-phosphate (DXP) and 1-amino-3-oxo-4-(phosphohydroxy)propan-2-one to form pyridoxine 5'-phosphate (PNP) . The protein from Escherichia coli has been crystallized in several forms under different conditions . The best diffracting crystals were obtained by a combination of the hanging-drop vapour-diffusion and microseeding techniques . Using an in-house image plate, the PdxJ crystals diffracted under cryo-conditions to 2.6 A resolution . The space group has been determined as C222(1), with unit-cell parameters a = 132.5, b = 154 . 4, c = 131.4 A, corresponding to four monomers per asymmetric unit . In the search for heavy-atom derivatives, a mercury derivative has been interpreted . The 12 mercury sites located are related by 222 symmetry and, in combination with self-rotation search analyses and gel-filtration experiments, indicate the quaternary assembly of PdxJ into octamers with 422 symmetry.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1042 - 4
Crystallization and preliminary crystallographic analysis of the Rho-binding domain of bovine Rho-kinase; Ihara K et al.; Rho-kinase binds to a small GTPase Rho in a GTP-dependent manner and regulates many cytoskeletal events in the cell . The minimum region of bovine Rho-kinase sufficient for Rho-binding was expressed as a fusion protein with glutathione S-transferase . After removal of the glutathione S-transferase, thin plate crystals were obtained . The selenomethionine-substituted protein was introduced and crystallized, as was the native protein . The crystals of the Rho-binding domain of Rho-kinase belong to the space group C2, with unit-cell parameters a = 148.0 (2), b = 26.1 (1), c = 39.6 (1) A, beta = 90.3 (1) degrees . The crystals diffract to a resolution beyond 1.5 A.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1038 - 41
A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis; Danley DE et al.; The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain . Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity . A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct . Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit . However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize . A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies . The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1017 - 9
Plasmodium falciparum rab6 GTPase: expression, purification, crystallization and preliminary crystallographic studies; Chattopadhyay D et al.; The Plasmodium falciparum rab6 gene encodes a 208 amino-acid polypeptide . Two recombinant versions of P . falciparum Rab6 protein were expressed in Escherichia coli: the full-length protein and a truncated form containing residues 1-175 . Both forms were purified from the soluble fraction of bacterial extract and were purified by ion-exchange chromatography and size-exclusion chromatography . Purified proteins were crystallized at pH 6.5 using the hanging-drop vapor-diffusion technique at room temperature . The full-length protein diffracted to 2.4 A and belongs to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 80 . 6, c = 90.4 A . The crystals of the truncated protein were isomorphous with those of the full-length construct and diffracted X-rays to 2.2 A resolution.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1015 - 6
Crystallization and diffraction to ultrahigh resolution (0.8 A) of a designed variant of the Rop protein; Spyridaki A et al.; The Rop protein is the paradigm of a highly regular four-alpha-helix bundle and as such has been subject to numerous structural and mutagenesis studies . Crystals of a designed Rop variant which establishes a continuous heptad pattern through the bend region have been obtained by a combination of vapour-diffusion and seeding techniques . The crystals diffract to ultrahigh (0.8 A) resolution using synchrotron radiation and cryogenic conditions.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 986 - 95
The high-mosaicity illusion: revealing the true physical characteristics of macromolecular crystals; Bellamy HD et al.; Typical measurements of macromolecular crystal mosaicity are dominated by the characteristics of the X-ray beam and as a result the mosaicity value given during data processing can be an artifact of the instrumentation rather than the sample . For physical characterization of crystals, an experimental system and software have been developed to simultaneously measure the diffraction resolution and mosaic spread of macromolecular crystals . The contributions of the X-ray beam to the reflection angular widths were minimized by using a highly parallel, highly monochromatic synchrotron source . Hundreds of reflection profiles over a wide resolution range were rapidly measured using a charge-coupled device (CCD) area detector in combination with superfine varphi-slicing data collection . The Lorentz effect and beam contributions were evaluated and deconvoluted from the recorded data . Data collection and processing is described . From 1 degrees of superfine varphi-slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured . The average mosaicity was 0.0101 degrees (s.d . 0.0035 degrees ) measured as the full-width at half-maximum (FWHM) and ranged from 0.0011 to 0 . 0188 degrees . Each reflection profile was individually fitted with two Gaussian profiles, with the first Gaussian contributing 55% (s.d . 9%) and the second contributing 35% (s.d . 9%) of the reflection . On average, the deconvoluted width of the first Gaussian was 0.0054 degrees (s.d . 0.0015 degrees ) and the second was 0.0061 degrees (s . d . 0.0023 degrees ) . The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140 and 0.0046 degrees along the a, b and c axes, respectively . The anisotropic mosaicity analysis indicates that the crystal is most perfect in the direction that corresponds to the favored growth direction of the crystal.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9413 - 8
The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme; Dym O et al.; d-Lactate dehydrogenase (d-LDH) of Escherichia coli is a peripheral membrane respiratory enzyme involved in electron transfer, located on the cytoplasmic side of the inner membrane . d-LDH catalyzes the oxidation of d-lactate to pyruvate, which is coupled to transmembrane transport of amino acids and sugars . Here we describe the crystal structure at 1.9 A resolution of the three domains of d-LDH: the flavin adenine dinucleotide (FAD)-binding domain, the cap domain, and the membrane-binding domain . The FAD-binding domain contains the site of d-lactate reduction by a noncovalently bound FAD cofactor and has an overall fold similar to other members of a recently discovered FAD-containing family of proteins . This structural similarity extends to the cap domain as well . The most prominent difference between d-LDH and the other members of the FAD-containing family is the membrane-binding domain, which is either absent in some of these proteins or differs significantly . The d-LDH membrane-binding domain presents an electropositive surface with six Arg and five Lys residues, which presumably interacts with the negatively charged phospholipid head groups of the membrane . Thus, d-LDH appears to bind the membrane through electrostatic rather than hydrophobic forces.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9367 - 72
Site-directed ligand discovery; Erlanson DA et al.; We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether . A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM) . The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly . This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases . The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex . Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 9886 - 91
A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage; Vispe S et al.; DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair . Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase . We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase-RNA complexes . In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD(+), resulting in poly(ADP-ribose) polymerase inactivation . We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription . Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 9925 - 30
Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways; Jurgens C et al.; Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme . We sought to create a precursor-like enzyme for N'-{(5'-phosphoribosyl)formimino}-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure . Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity . A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold . These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

EMBO J, 2000 Aug 15, 19(16), 4393 - 401
Evaluating the oligomeric state of SecYEG in preprotein translocase; Yahr TL et al.; SecA insertion and deinsertion through SecYEG drive preprotein translocation at the Escherichia coli inner membrane . We present three assessments of the theory that oligomers of SecYEG might form functional translocation sites . (i) Formaldehyde cross- linking of translocase reveals cross-links between SecY, SecE and SecG, but not higher order oligomers . (ii) Cross-linking of membranes containing unmodified SecE and hemagglutinin-tagged SecE (SecE(HA)) reveals cross-links between SecY and SecE and between SecY and SecE(HA) . However, anti-HA immunoprecipitates contain neither untagged SecE nor SecY cross-linked to SecE . (iii) Membranes containing similar amounts of SecE and SecE(HA) were saturated with translocation intermediate (I(29)) and detergent solubilized . Anti-HA immunoprecipitation of I(29) required SecYE(HA)G and SecA, yet untagged SecE was not present in this translocation complex . Likewise, anti-HA immunoprecipitates of membranes containing equal amounts of SecY and SecY(HA) were found to contain SecY(HA) but not SecY . Both immunoprecipitates contain more moles of I(29) than of the untagged subunit, again suggesting that translocation intermediates are not engaged with multiple copies of SecYEG . These studies suggest that the active form of preprotein translocase is monomeric SecYEG.

EMBO J, 2000 Aug 15, 19(16), 4383 - 92
The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains; Prodromou C et al.; How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure . Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle . C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated . Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization . The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties . The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association . Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization . These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

EMBO J, 2000 Aug 15, 19(16), 4372 - 82
The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex; Vilela C et al.; Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation . The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1 . We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1 . Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G . Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1 . However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G . Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo . These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.

EMBO J, 2000 Aug 15, 19(16), 4248 - 56
ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase; Hanzawa Y et al.; Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear . Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase . Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity . Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product . We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner . The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.

Proc Natl Sci Counc Repub China B, 2000 Jul, 24(3), 123 - 8
Central pool of serotonin and tail-flick latency during two phases of biphasic fever in rats; Koulchitsky SV et al.; In experiments on male Wistar rats, the acute phase reaction was induced by a bolus intravenous injection of Escherichia coli lipopolysaccharide (10 microg/kg) through a silicon catheter pre-implanted into the jugular vein . The colonic and skin temperature was measured with thermocouples . Changes in nociception were assessed based on tail flick latency (TFL) in response to a noxious heat stimulus . In this work, we observed the development of biphasic fever and phasic changes in TFL, namely, hyperalgesia in the first period of the acute phase reaction and hypoalgesia in its second phase . The catabolism of serotonin increased most considerably in the initial period of the acute phase reaction in the midbrain, striatum, and rostrodorsomedial medulla (on average, by 20-25%, 35-40%, and 95-100%, respectively) . In the second phase of the acute phase reaction, despite a significant increase in the serotonin content in the striatum, midbrain, and cerebellum, there were no significant changes in serotonin catabolism in these parts of the CNS, which coincided with hypoalgesia . Thus, the phasic changes in TFL and colonic temperature after initiation of the acute phase reaction were accompanied by determinate changes in the catabolism of serotonin in different brain parts.

RNA, 2000 Aug, 6(8), 1185 - 93
RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli; Marujo PE et al.; Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles . In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II . Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA . Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA . RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As . The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II . Moreover, the rpsO mRNA is stabilized when RNase II is overproduced . This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active . These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.

RNA, 2000 Aug, 6(8), 1166 - 73
Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene; Koosha H et al.; The translocation stage of protein synthesis is a highly conserved process in all cells . Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined . Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame . In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G) . To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E . coli that encodes EF-G . This analysis was performed using the ts E . coli strain PEM100, which contains a point mutation within fusA . The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA . Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle . We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.

J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 319 - 25
Epitope tagging analysis of the outer membrane folding of the molecular usher FaeD involved in K88 fimbriae biosynthesis in Escherichia coli; Harms N et al.; To analyse the outer membrane folding of the molecular usher FaeD, tagged derivatives were prepared and their expression, tag-localisation and functioning in K88 fimbriae biosynthesis was studied . A semi-random insertion mutagenesis approach with factor Xa cleavage sites yielded six tagged FaeD derivatives . A site-directed mutagenesis approach in which c-myc epitopes were inserted yielded twenty-one different derivatives . Four tagged FaeD constructs were not expressed in the outer membrane as full-sized proteins to levels that could be detected by using immunoblotting analyses . Two of these had an insertion in the amino-terminal part of FaeD, whereas the other two had a tag inserted in the carboxyl-terminal part . The latter ones yielded stable carboxyl-terminally shortened truncates of about 70 kDa, as did other mutations in this region . Six tagged derivatives were expressed but the location of the tag with respect to the outer membrane could not be determined, possibly due to shielding . Functional analysis showed that insertion of a tag in two regions of FaeD, a central region of approximately 200 amino acid residues (a.a . 200-400) and the carboxyl-terminal region (a.a . 600-end), resulted in a defective K88 fimbriae biosynthesis . In-frame deletions in the amino-terminal region of FaeD abolished fimbriae production . The integrity of these regions is obviously essential for fimbriae biosynthesis . Based on the results and with the aid of a computer analysis programme for the prediction of outer membrane beta-strands, a folding model with 22 membrane spanning beta-strands and two periplasmioc domains has been developed.

J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 193 - 202
Cold shock response in Escherichia coli; Yamanaka K; Sensing a sudden change of the growth temperature, all living organisms produce heat shock proteins or cold shock proteins to adapt to a given temperature . In a heat shock response, the heat shock sigma factor plays a major role in the induction of heat shock proteins including molecular chaperones and proteases, which are well-conserved from bacteria to human . In contrast, no such a sigma factor has been identified for the cold shock response . Instead, RNAs and RNA-binding proteins play a major role in cold shock response . This review describes what happens in the cell upon cold shock, how E . coli responds to cold shock, how the expression of cold shock proteins is regulated, and what their functions are.

Ultrasound Med Biol, 2000 Jun, 26(5), 897 - 903
Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects; Koch S et al.; Cationic liposomes (CL) are widely used vectors for gene transfer . Recently, ultrasound (US) was reported to enhance liposome-mediated gene transfer to eucaryotic cells in culture . The present study was aimed at studying the effects of 2-MHz pulsed Doppler US on malignant brain tumor cells transfection by cationic liposome/plasmid-DNA complexes (lipoplexes) . Cationic liposomes consisting of DOSPA/DOPE were complexed with a plasmid carrying the cDNA encoding green autofluorescent protein (EGFP) . Rodent (9L) and canine (J3T) glioma cells were exposed to pulsed US in the presence of EGFP-lipoplexes . A diagnostic transcranial Doppler device (MultiDop L) was used for insonation for 30, 60, and 90 s at 2 MHz/0.5 W/cm(2) . To eliminate US reflection and cavitation, a custom-made absorption chamber was designed, where US is applied through a water tank before interacting with the cells and is fully absorbed after passing through the cell layer . Expression of the marker gene EGFP was quantified by FACS analysis and intravital fluorescent microscopy . Cell viability was accessed by Trypan Blue staining . US treatment of tumor cells on microplates for 60 s yielded a significant increase in transfection rates without damaging the cells, but 90-s treatment killed most of the cells . In the absorption chamber, no significant effects of US on transfection were noted . Additional experiments employed US contrast agent (Levovist, Schering) which was able to significantly increase tumor cell transfection rate by enhancing cavitation effects, and also severely damaged most cells when applied at a concentration of 200 mg/mL . In conclusion, our results support the assumption that US effects on lipoplex transfection rates in brain tumor cells in culture are mediated by cavitation effects.

Blood, 2000 Aug 15, 96(4), 1596 - 8
Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identical to p36, a protein associated to lupus inclusion formation in response to alpha-interferon; Amici A et al.; Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides . We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia . Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein . PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library . PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme . The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36 . Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I . Molecular properties of the two proteins were essentially the same . We conclude that p36 and PN-I are identical proteins . (Blood . 2000;96:1596-1598)

J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 307 - 16
Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase, in a detergent--polymer aqueous two-phase system containing metal-chelating polymer; Sivars U et al.; A system has been developed for selective partitioning of membrane proteins . For the first time, an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli, has been affinity partitioned in an aqueous two-phase system . The systems used were different detergent/polymer aqueous two-phase systems containing a metal-chelating polymer, such as poly(ethyleneglycol)-iminodiacetic acid-Cu(II) as well as dextran-iminodiacetic acid-Cu(II) . Many non-ionic detergents, such as alkyl(polyethyleneoxide) (CmEOn), Triton, Tween and alkylglucosides, form two-phase systems in mixture with polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enriched phase in equilibrium with a polymer-enriched phase are formed . In general, membrane proteins partition strongly to the micelle phase . We show that it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, with a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold) . The affinity partitioning was characterized and the effects of ligand concentration, pH, time, salts, buffer type, imidazole and charged detergent are discussed . Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer aqueous two-phase systems, and the method is especially suitable for the purification of labile integral membrane proteins, such as receptors.

J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 281 - 6
Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system; Engel S et al.; Extraction in a polyethylene glycol (PEG)-phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E . coli extract . Extraction optimization was achieved by varying the system parameters . Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)-phosphate (6%) and PEG-4000 (14%)-phosphate (5.5%)-KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system . Significant purification was achieved by a single extraction step with 70% recovery of the enzyme . After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.

J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 225 - 9
Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system; Engel S et al.; An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K(dis), of the Escherichia coli isoenzyme AHAS III regulatory subunit, ilvH protein, from the feedback inhibitor valine . The amounts of the bound and free radioactive valine in the system were determined . A Scatchard plot of the data revealed a 1:1 valine-protein binding ratio and K(dis) of 133+/-14 microM . The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding . This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method.

J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 21 - 30
Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction; Selber K et al.; Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E . coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol-phosphate systems . The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems . A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared . The methods differ in the height of the optimum found, the number of experiments and the time required . The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems . The simplex procedure has to be further improved.

Mol Cell Biochem, 2000 Jun, 209(1-2), 69 - 77
Protein kinase C-gamma phorbol-binding domain involved in protein-protein interaction; Pawelczyk T et al.; Protein kinase C-gamma (PKC-gamma) contains two cysteine-rich regions (Cys1, Cys2) responsible for interaction with phospholipids . However, previous experiments suggested that, only Cys1 represents the high affinity site involved in diacylglycerol-dependent activation of PKC-gamma . This raises the question whether Cys2 might participate in other functions of the PKC-gamma regulatory domain . The purpose of our studies was to examine the ability of Cys2 domain to bind cellular proteins . The Cys2 domain (residues 92-173) was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and purified . In order to investigate protein-protein interaction of Cys2 domain we used affinity column and an overlay assay . Our results demonstrate that the Cys2 domain of PKC-gamma binds several proteins from rat brain extracts . In the absence of phospholipids the Cys2 domain binds some proteins in the cytosolic fraction of rat brain, but no binding was detected with the proteins extracted from particulate fraction . Ca2+ at 1 microM concentration potentiated binding of cellular proteins to Cys2 domain . In the absence of Ca2+ the Cys2 domain binds proteins in the cytosolic fraction of rat brain in the presence of phosphatidylserine and to the lesser extend in the presence of phosphatidylinositol but neither phosphatidylcholine nor phosphatidylethanolamine . These results suggest that the Cys2 domain of PKC-gamma has the ability to interact with two classes of proteins . One class binds the Cys2 domain in the phosphatidylserine dependent fashion, and the other proteins bind Cys-2 domain in the Ca2+ dependent and phospholipid independent manner.

Microbiol Immunol, 2000, 44(6), 543 - 50
Hepatitis C virus NS5B RNA replicase specifically binds ribosomes; Tanaka T et al.; Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase . We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B) . MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin . The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP . Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g . The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B . Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin . Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes . We further found that NS5B also bound with human ribosomes . Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.

Microbiol Immunol, 2000, 44(6), 481 - 8
Mutation of aromatic amino acid residues located at the amino- and carboxy-termini of Escherichia coli heat-stable enterotoxin Ip reduces the efficiency of the toxin to cross the outer membrane; Yamanaka H et al.; Heat-stable enterotoxin Ip (STIp) of Escherichia coli is synthesized as a precursor form consisting of pre- (amino acid residues 1 to 19), pro- (amino acid residues 20 to 54) and mature (amino acid residues 55 to 72) regions . Mature STIp (bioactive STIp) is formed in the periplasmic space after the precursor is proteolytically processed and the mature STIp translocates across the outer membrane through the secretory system including TolC, an outer membrane protein of E . coli . However, it remains unknown how the mature STIp is recognized by this secretory system . In this study, we investigated the amino acid residues of STIp involved in its translocation across the outer membrane . We prepared mutant STIp genes by site-directed mutagenesis and analyzed translocation of the mutant STIps across the outer membrane . Deletion of the Phe or Tyr residue at position 3 or 18, respectively, decreased the efficiency of translocation of STIp across the outer membrane . To confirm the involvement of these amino acid residues, we further mutated the codons for these amino acid residues to that for Gly . These mutations also decreased the efficiency of extracellular secretion of STIp . In contrast, substitution of Phe-3 and Syr-18 with Tyr and Phe, respectively, did not affect the efficiency of translocation of the toxin . These results indicated that the aromatic amino acid residues at positions 3 and 18 in the mature region are important for the ability of STIp to cross the outer membrane.

Cancer Immunol Immunother, 2000 Jul, 49(4-5), 186 - 92
Immunological properties of a single-chain fragment of the anti-idiotypic antibody ACA125; Reinartz S et al.; Vaccination with anti-idiotypic antibodies has been described as a promising concept for treatment of several malignant diseases . The murine monoclonal anti-idiotypic antibody ACA125 imitates a specific epitope of the tumor-associated antigen CA125 expressed by 80% of ovarian carcinomas . In the first clinical trial it could be shown that mAb ACA125 is able to elicit anti-anti-idiotypic antibodies (Ab3) with anti-CA125 specificity in patients with advanced ovarian cancer . In order to improve the capabilities of anti-idiotype vaccines we generated a genetically engineered single-chain fragment (scFv) ACA125 composed of heavy- and light-chain variable regions connected by a flexible linker . The antigenicity of scFv ACA125 was demonstrated by immunizing rats i.p . with scFv or complete mAb in complete/incomplete Freund's adjuvants (CFA/IFA) or precipitated by aluminium hydroxide . Negative control groups included applications of irrelevant mouse IgG or adjuvants alone . Anti-anti-idiotypic antibodies (Ab3) directed against the mAb ACA125 as well as specific anti-CA125 antibodies (Ab1') could be detected in all animals treated with scFv in CFA/IFA . Nevertheless, antibody titers were lower than when the complete mAb ACA 125 was used . Suprisingly, an increase of specificity could not be observed in scFv-immunized animals, which had been expected because of the lack of heavy- and light-chain constant regions that could raise rather unspecific anti-isotypic and anti-allotypic rat anti-(mouse Ig) antibodies (RAMA) . In contrast, the RAMA responses detected in these rats were even stronger than those following immunization with complete mAb ACA125 . In conclusion, the anti-idiotypic scFv ACA125 alone cannot improve the immunogenic features of the corresponding mAb, but provides a useful tool for the further development of genetic vaccines.

Lipids, 2000 Jul, 35(7), 709 - 20
Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava); Tijet N et al.; Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity . The enzyme proved stable to chromatographic procedures and was purified to homogeneity . Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD . Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3' and 5' rapid amplification of cDNA ends . The sequence shows approximately 60-70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5 . The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-beta-D-thiogalactopyranoside and delta-aminolevulinic acid . The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids . 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry . The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was approximately 2000/s, comparable to values reported for the related allene oxide synthases . Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 149 - 56
RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro; Karthikeyan G et al.; To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro . Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes . Escherichia coli SSB, causes no such improvement in Klenow fidelity . RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP . Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime . These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.

Can J Microbiol, 2000 Aug, 46(8), 764 - 9
Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2); Loke P et al.; With the rapid generation of genetic information from the Streptomyces coelicolor genome project, deciphering the relevant gene products is critical for understanding the genetics of this model streptomycete . A putative malate synthase gene (aceB) from S . coelicolor A3(2) was identified by homology-based analysis, cloned by polymerase chain reaction, and fully sequenced on both strands . The putative malate synthase from S . coelicolor has an amino acid identity of 77% with the malate synthase of S . clavuligerus, and possesses an open reading frame which codes for a protein of 540 amino acids . In order to establish the identity of this gene, the putative aceB clones were subcloned into the expression vector pET24a, and heterologously expressed in Escherichia coli BL21(DE3) . Soluble cell-free extracts containing the recombinant putative malate synthase exhibited a specific activity of 1623 (nmol.mg-1.min-1), which is an increment of 92-fold compared to the non-recombinant control . Thus, the gene product was confirmed to be a malate synthase . Interestingly, the specific activity of S . coelicolor malate synthase was found to be almost 8-fold higher than the specific activity of S . clavuligerus malate synthase under similar expression conditions . Furthermore, the genomic organisation of the three Streptomyces aceB genes cloned thus far is different from that of other bacterial malate synthases, and warrants further investigation.

Eur J Immunol, 2000 Jul, 30(7), 1939 - 47
Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity; Tighe H et al.; Immunostimulatory DNA sequences (ISS) are a potent Th1 adjuvant . We hypothesized that conjugation of ISS to protein antigens would strongly enhance their immunogenicity because both antigen and adjuvant (ISS) would be delivered to the same locale/antigen-presenting cell . To test this hypothesis, we conjugated a 22-mer immunostimulatory oligodeoxynucleotide (ISS-ODN) to two test antigens of differing intrinsic immunogenicity, namely Escherichia coli beta-galactosidase and the HIV-1 envelope glycoprotein gp120 . We show that the antigen-ISS conjugates rapidly induce Th1 cells secreting high levels of IFN-gamma, strong CTL activity, and high titer IgG2a and HIV-neutralizing antibodies, exceeding gene and protein vaccination alone or immunization with mixtures of antigen and ISS-ODN . The data suggest that this procedure generates a novel and unique vaccine that rapidly triggers strong humoral and cell-mediated immunity.

Exp Nephrol, 2000 Jul-Oct, 8(4-5), 275 - 82
Endotoxin-induced renal failure . II . A role for tubular hypoxic damage; Heyman SN et al.; Endotoxin-induced hypotension and altered renal microcirculation could lead to tubular injury, particularly at the physiologically hypoxic outer medulla . We explored this hypothesis in isolated perfused kidneys and in vivo in rats subjected to endotoxemia . Rat kidneys were removed 15 min after endotoxin injection in vivo (from Escherichia coli 0127:B8, 1 mg/kg i.p.) and perfused with oxygenated medium supplemented with 20 amino acids and endotoxin . Glomerular filtration rate and filtration fraction markedly declined (0.4 +/- 0 . 1 ml/min and 1.1 +/- 0.1, respectively) as compared with control kidneys (0.7 +/- 0.1 ml/min and 1.8 +/- 0.1, n = 8-12 per group; p < 0.05) . Hypoxic injury to medullary thick ascending limbs in the innermost outer medulla increased (47 +/- 9% of tubules vs . 16 +/- 8% in controls, p < 0.05) . When rats were preconditioned with an additional endotoxin injection 16 h earlier (a manipulation that markedly reduces cortical and medullary blood flow), glomerular filtration rate and filtration fraction further declined to 0.1 +/- 0.0 ml/min and 0.4 +/- 0.1, respectively (p < 0.01), and tubular sodium reabsorption fell to 81 +/- 12 vs 98 +/- 0% in controls (p < 0.05) . Tubular damage, however, did not increase (20 +/- 7%), probably reflecting a decline in reabsorptive workload and oxygen requirement . In rats subjected to a single or two repeated daily doses of endotoxin (1 mg/kg i.p.) plasma creatinine comparably rose 41% on the average over 24 h, creatinine clearance fell by 27% (p < 0.0001), but tubular damage was absent . By contrast, in rats preconditioned with indomethacin and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10 mg/kg), the addition of endotoxin markedly augmented outer medullary hypoxic tubular damage both in S(3) segments (27 +/- 10 vs 1 +/- 1%) and in medullary thick ascending limbs (38 +/- 11 vs . 10 +/- 5%, n = 7-8; p < 0.05) . It is concluded that under special conditions, such as altered medullary oxygen balance or defective nitric oxide or prostaglandin synthesis, endotoxin may predispose to hypoxic outer medullary tubular damage .

Exp Nephrol, 2000 Jul-Oct, 8(4-5), 266 - 74
Endotoxin-induced renal failure . I . A role for altered renal microcirculation; Heyman SN et al.; The pathogenesis of sepsis-induced renal failure is multifactorial and only partially understood . In these studies we evaluated intrarenal microcirculatory changes during endotoxemia and the potential role of nitric oxide (NO) and endothelin in these changes . In anesthetized rats endotoxin infusion {lipopolysaccharide (LPS), Escherichia coli serotype 0127:B8; 10 mg/kg/h} resulted in hypotension and a transient enhancement of renal blood flow, with cortical vasodilation and a loss of outer medullary vasodilatory response to hypotension . The initial cortical vasodilation was abolished by the NO synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin . Direct NO measurements disclosed a gradual rise in cortical NO, despite the waning vasodilatory effect, suggesting antagonizing vasoconstrictive stimuli . In rats pretreated by LPS (1 mg/kg i.p . 1 day earlier) the renal blood flow was reduced to 55% of that of controls . Moreover, the vasodilatory response to LPS infusion was converted into profound cortical and medullary vasoconstriction . In these preconditioned rats the endothelin receptor antagonist bosentan evoked a vasodilatory response and attenuated the vasoconstrictive reaction to LPS infusion . The infusion of another LPS (E . coli serotype 0111:B4) exerted predominant and protracted renal vasodilation without hypotension . In conclusion, different LPS exert diverse systemic and renal hemodynamic responses . The 0127:B8 serotype attenuates renal medullary vasodilation during hypotension, exerts transient cortical vasodilation, and following repeated exposure induces profound renal vasoconstriction . NO and endothelin participate in LPS-induced vascular responses that may predispose to hypoxic tubular damage .

Gene, 2000 Aug 8, 253(2), 237 - 47
Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana; Jost R et al.; The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria . Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana . Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated . The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications . OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities . However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism . In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B . Multiple database accessions for each of the A . thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.

Gene, 2000 Aug 8, 253(2), 221 - 9
Genetic engineering of Escherichia coli to produce a 1:1 complex of the anabaena sp . PCC 7120 nuclease NucA and its inhibitor NuiA; Korn C et al.; A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp . PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA . With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression . Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA . In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio . The complex can be solubilized with 6M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity . 2mg of the complex are obtained from 1l Escherichia coli culture . As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies . Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation.

Mol Cell Endocrinol, 2000 Jul 25, 165(1-2), 107 - 13
DNA immunization in vivo down-regulates nuclear all-trans retinoic acid receptors in mouse spleen cells; Brtko J et al.; Nuclear retinoid receptors - retinoic acid inducible transcription factors - participate in pathways influencing many components of the immune system . In the present study in vivo effects of DNA-based immunization of mice on binding parameters of all-trans retinoic acid receptors (RARs) in spleen cell nuclei was investigated . A eucaryotic expression vector encoding the gene for the model enzyme beta-galactosidase of Escherichia coli (pCMV-beta) was used for intradermal injection . Furthermore, immunostimulatory CpG motifs, which stimulate the expression of various cytokines and may serve as a 'danger signal' for the mammalian immune system, were coinjected as oligodeoxynucleotides . The results demonstrate that the concentration of RARs was significantly reduced in the late phase of the primary immune response (21 days after injection of plasmid DNA-indicated by high affinity IgG antibodies and IFN-gamma expression) . Coinjection of CpG motifs did not change the course of the humoral response but enhanced and accelerated the proliferative response and expression of IFN-gamma, which correlated with the reduced RARs concentration.

FEBS Lett, 2000 Aug 11, 479(1-2), 72 - 7
Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp . PCC 6803; Mann NH et al.; The Synechocystis sp . PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study . Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes . Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.

Eur J Pharmacol, 2000 Aug 18, 402(1-2), 11 - 8
Lipopolysaccharide decreases bradykinin receptor-induced acidification responses in cultured bovine aortic endothelial cells; Bentley KR et al.; The effects of bacterial lipopolysaccharide (Escherichia coli 0127-B8) on bradykinin receptor function in bovine aortic endothelial cells were investigated using a microphysiometer . Bradykinin and Lys(0)-desArg(10)-bradykinin produced concentration-dependent acidification responses with pEC(50) values of 8.87+/-0.20 and 9.78+/-0.08, respectively . These responses were competitively and selectively antagonised by the bradykinin B(2) receptor antagonist, icatibant and the bradykinin B(1) receptor antagonist, desArg(9)-Leu(8)-bradykinin, respectively . The non-peptide bradykinin B(2) receptor antagonist, FR173657 (0.3 and 3 nM), selectively antagonised bradykinin-induced acidification responses, causing rightward shifts of the concentration-response curves to bradykinin, but at the same time, significantly decreasing the maximum response . A preincubation with lipopolysaccharide (0.01 and 0.1 microg/ml) for 24 h caused a significant concentration-dependent decrease in maximal response to bradykinin (27.2+/-1.9 and 9.7+/-0.4% of control) and the bradykinin B(1) receptor agonist, Lys(0)-desArg(10)-bradykinin (59.0+/-7.14 and 25.3+/-7.8% of control), without affecting the EC(50) . These results suggest that bradykinin B(1) receptors are constitutively expressed in cultured bovine aortic endothelial cells and that the microphysiometer provides a rapid, sensitive technique to characterise bradykinin receptors and investigate their regulation by cytokines . Interactions between bradykinin receptors and lipopolysaccharide may play a part in the cascade of deleterious effects that occur during septic shock.

Annu Rev Nutr, 2000, 20, 153 - 67
Biosynthesis of vitamin b2 (riboflavin); Bacher A et al.; The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates . The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation . Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine . Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway . The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

J Biol Chem, 2000 Aug 18, 275(33), 25540 - 6
A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc; Kvaratskhelia M et al.; Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA . They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species . Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified . We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange . Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes . The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis . This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.

Plant Sci, 2000 Aug 8, 157(1), 77 - 88
Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L . during berry development; Tesniere C et al.; Three full-length cDNAs (VvAdh1, VvAdh2, and VvAdh3) encoding alcohol dehydrogenases (EC 1.1.1.1) were obtained from grape berries (Vitis vinifera L.) by means of PCR and RACE . Pairwise comparisons at the nucleotide level showed that the three cDNAs displayed strong homology in the coding region, but were highly divergent in the 5' and 3' untranslated regions . VvAdh1 and VvAdh2 corresponded to the two previously characterised Adh genes from grapevine, but VvAdh3 was unrelated to known grapevine Adh sequences . The two first cDNAs presented a single ORF of 380 amino acids, whereas the last one has two additional residues . Moreover, the three encoded polypeptides possessed the 22 residues strictly conserved between Adh from different kingdoms . Expression pattern of the individual isogenes was investigated during fruit development . Specific primers were designed, and quantitative RT-PCR experiments were performed to increase the sensitivity of detecting isogenes with a low expression level . Results presented here revealed different developmental regulation of the three Adh isogenes during fruit ripening . VvAdh1 and VvAdh3 transcripts were temporarily accumulated in young, developing berry, whereas VvAdh2 was overexpressed later in fruit development, from the onset of ripening (veraison) . Expression analysis also indicated that VvAdh2 accounted for most of the Adh mRNAs present in berries during development . The increased ADH activity detected in berries correlated with the expression pattern of VvAdh2 transcripts . The VvAdh2 and VvAdh3 encoded enzymes were purified from overexpressing E . coli cells . Comparison of kinetic properties of the two ADH enzymes showed a difference in affinity with either ethanol or acetaldehyde as substrates . Significance of multiple Adh expressed in berries is discussed.

J Bacteriol, 2000 Sep, 182(17), 5025 - 8
Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12; Zhao J et al.; Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA . Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase . In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant . These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.

J Bacteriol, 2000 Sep, 182(17), 5013 - 6
Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea; Howell DM et al.; Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate . Neither enzyme was found to use any of the isomers of isocitrate as a substrate . The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate) . These intermediates included (-)-threo-isohomocitrate {(-)-threo-1-hydroxy-1,2, 4-butanetricarboxylic acid}, (-)-threo-iso(homo)(2)citrate {(-)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid}, and (-)-threo-iso(homo)(3)citrate {(-)-threo-1-hydroxy-1,2, 6-hexanetricarboxylic acid} . The protein product of MJ0720 was found to be alpha-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine . The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100 degrees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80 degrees C for 10 min.

J Bacteriol, 2000 Sep, 182(17), 4959 - 69
Genetic evidence that transcription activation by RhaS involves specific amino acid contacts with sigma 70; Bhende PM et al.; RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators . We wished to determine whether sigma(70) might be an activation target for RhaS . We found that sigma(70) K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP . To determine whether these positively charged sigma(70) residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS . Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters . Finally, we assayed combinations of the RhaS and sigma(70) substitutions and found that RhaS D241 and sigma(70) R599 met the criteria for interacting residues at both promoters . Molecular modeling suggests that sigma(70) R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact sigma(70) . More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with sigma(70) may be conserved.

J Bacteriol, 2000 Sep, 182(17), 4882 - 8
Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background; Misra R et al.; Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers . In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype . Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability . These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly . The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP(S210A) protein was overproduced . Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane . Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP(S210A) was overproduced . Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP(S210A) also fractionated . These results suggested that the DegP(S210A)-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.

J Bacteriol, 2000 Sep, 182(17), 4875 - 81
Identification of the mob genes of plasmid pSC101 and characterization of a hybrid pSC101-R1162 system for conjugal mobilization; Meyer R; Similarities in DNA base sequence indicate that pSC101 and R1162 encode related systems for conjugal mobilization, although these plasmids are otherwise very different . The mob region of pSC101 was cloned, and two genes that are required for transfer were identified . One gene, mobA, encodes a protein similar in amino acid sequence to the DNA processing domain of the R1162 MobA protein . The other gene, mobX, is within the same transcriptional unit as the pSC101 mobA and is located just downstream, at the same position occupied by mobB in R1162 . Despite this, the MobB and MobX proteins do not appear to be closely related based on a comparison of their amino acid sequences . Complementation analysis indicated that neither of the pSC101 Mob proteins could substitute for, or be replaced by, their R1162 counterparts, nor were they active together at the R1162 origin of transfer (oriT) . However, the full set of R1162 Mob proteins did recognize the pSC101 oriT . A hybrid system for mobilization, active at the R1162 oriT site, was constructed . This system consists of MobX and a chimeric protein made up of the DNA cleaving-ligating domain of the R1162 MobA protein joined to a fragment of pSC101 MobA . Previous results suggested that MobB and a region of MobA distinct from the DNA processing domain together formed a functional unit in transfer . The present results support this model because the chimeric MobA, although active on R1162 oriT, requires the pSC101 protein MobX for efficient plasmid mobilization.

J Bacteriol, 2000 Sep, 182(17), 4862 - 7
Identification of the gene encoding sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coenzyme M; Graupner M et al.; The products of two adjacent genes in the chromosome of Methanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in Streptomyces wedmorensis . These two M . jannaschii genes were recombinantly expressed in Escherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of alpha-ketoacids . Both subunits are required to form an alpha(6)beta(6) dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde . This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism . The M . jannaschii sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite . The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.

J Bacteriol, 2000 Sep, 182(17), 4856 - 61
Effects of organic antagonists of Ca(2+), Na(+), and K(+) on chemotaxis and motility of escherichia coli; Tisa LS et al.; Various Ca(2+) antagonists used in animal research, many of them known to be Ca(2+) channel blockers, inhibited Escherichia coli chemotaxis (measured as entry of cells into a capillary containing attractant) . The most effective of these, acting in the nanomolar range, was omega-conotoxin GVIA . The next most effective were gallopamil and verapamil . At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant) . Various other Ca(2+) antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility . Cells treated with each of these Ca(2+) antagonists swam with a running bias, i.e., tumbling was inhibited . Similarly, some Na(+) antagonists used in animal research inhibited bacterial chemotaxis . E . coli chemotaxis was inhibited by saxitoxin at concentrations above 10(-7) M, while more than 10(-4) M was needed to inhibit motility . Cells treated with saxitoxin swam with a tumbling bias . In the case of other Na(+) antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration . In the case of K(+) antagonists used in animal research, 4-aminopyridine blocked E . coli chemotaxis between 10(-3) M and, totally, 10(-2) M, while motility was not affected at 10(-2) M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10(-2) M.

J Bacteriol, 2000 Sep, 182(17), 4829 - 35
Inactivation of the ampDE operon increases transcription of algD and affects morphology and encystment of Azotobacter vinelandii; Nunez C et al.; Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii . We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of an algD::lacZ fusion . AC28 cells were morphologically abnormal and unable to encyst . The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDE operon . Tn5 was located within the ampD gene, encoding a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments . The ampE gene encodes a transmembrane protein, but the function of the protein is not known . We constructed strains carrying ampD or ampE mutations and one with an ampDE deletion . The strain with a deletion of the ampDE operon showed a phenotype similar to that of mutant AC28 . The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

J Bacteriol, 2000 Sep, 182(17), 4803 - 10
Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump; Tsukagoshi N et al.; Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases . The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance . We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent . Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5) . The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant . The solvent entered the E . coli cells rapidly . Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels . The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6 . 0) more abundantly than the parent or the delta acrAB mutant . The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction . The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.

Anal Chem, 2000 Jul 15, 72(14), 3138 - 41
MutS-mediated detection of DNA mismatches using atomic force microscopy; Sun HB et al.; We have developed an atomic force microscopy-based method for detecting DNA base-pair mismatches using MutS protein isolated from E . coli . MutS is a biological sensor and a locator of DNA base-pair mismatches . It binds specifically to a mismatched DNA base pair and initiates a process of DNA repair . To test the possibility of visually detecting mismatched base pairs by atomic force microscopy, we prepared DNA templates approximately 500 bp in length consisting of a single or multiple base-pair mismatches . We demonstrate that MutS binding sites on individual DNA molecules were readily detectable by atomic force microscopy and that the observed positions were in good agreement with the predicted sites of base-pair mismatches at a few-nanometer resolution . The technique described here is rapid and sensitive and is expected to be useful in screening mutations and DNA polymorphisms.

J Exp Bot, 2000 Jan, 51(342), 81 - 8
Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance; Sakamoto A et al.; Metabolic acclimation via the accumulation of compatible solutes is regarded as a basic strategy for the protection and survival of plants in extreme environments . Certain plants accumulate significant amounts of glycinebetaine (betaine), a compatible quaternary amine, in response to high salinity, cold and drought . It is likely that betaine is involved in the protection of macrocomponents of plant cells, such as protein complexes and membranes, under stress conditions . Genetic engineering of the biosynthesis of betaine from choline has been the focus of considerable attention as a potential strategy for increasing stress tolerance in stress-sensitive plants that are incapable of synthesizing this compatible/protective solute . Three distinct pathways for the synthesis of betaine have been identified in spinach, Escherichia coli and Arthrobacter globiformis, and various genes and cDNAs for the proteins involved are available . Moreover, each of the pathways has been exploited to a greater or lesser extent in efforts to convert betaine-deficient plants to betaine accumulators . In this review, the potential of several recent examples of transgenic approaches to the enhancement of stress tolerance in plants is summarized and discussed.

Bull Math Biol, 2000 Jul, 62(4), 775 - 91
Modelling run-and-tumble chemotaxis in a shear flow; Bearon RN et al.; The biased random walk undergone by chemotactic bacteria such as Escherichia coli will be influenced at the microscopic level by flow in the ambient medium . In this paper, we model swimming bacteria being advected and rotated by a simple shear flow . Under certain scaling assumptions, we obtain an advection-diffusion equation for cell density, when the chemotactic response is small, which shows a coupling between the rotation and chemotaxis . We also present an alternative method for calculating the chemotactic flux in an unbounded region which is valid for more general chemotactic responses.

Int J Oncol, 2000 Sep, 17(3), 467 - 71
Mutation analysis of the hMTH1 gene in sporadic human ovarian cancer; Takama F et al.; 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) is a major oxidation product in the nucleotide pool of the cell and is a potent mutagen, because it can be incorporated into DNA with equal frequency opposite either template C or A . The human MTH1 gene (hMTH1) is a homologue of the E . coli mutator gene mutT, which encodes 8-oxo-dGTPase . hMTH1 protein reduces spontaneous mutations by removing 8-oxo-dGTP from the triphosphate pool . To determine whether this gene is associated with carcinogenesis of human ovarian cancer, the present study examined, for the first time, the hMTH1 sequence in 49 ovarian cancers and 9 ovarian cancer cell lines by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analyses . A Gright curved arrow A transition at codon 83 was detected in one patient and one cell line (3.4%), followed by an amino acid change (valineright curved arrow methionine) which was known to cause the protein to be less active in vitro . This one base substitution was found in normal and corresponding tumor DNA, and its allele type was heterozygous . The same change has been detected in HNPCC (hereditary non-polyposis colorectal cancer) and gastric cancer patients, and thus it may not represent a mutation specific for ovarian cancer . A silent Tright curved arrow C transition at codon 119 was detected in 12 patients and 2 cell lines (24.1%) . No specific mutations in hMTH1 were found in either ovarian cancer patients or cell lines . Thus, it appears that hMTH1 is not directly associated with ovarian cancer.

Plant Physiol, 2000 Aug, 123(4), 1561 - 70
AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein; Mueller LA et al.; AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole . For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump . Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates . Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration . These results suggest that no conjugate is formed in vitro . However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching . We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.

Plant Physiol, 2000 Aug, 123(4), 1427 - 36
A plastid terminal oxidase associated with carotenoid desaturation during chromoplast differentiation; Josse EM et al.; The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation . Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes . In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase . This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase . This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits . Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and zeta-carotene desaturase gene expression during fruit ripening . Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits . These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.

Plant Physiol, 2000 Aug, 123(4), 1235 - 46
A germination-specific endo-beta-mannanase gene is expressed in the micropylar endosperm cap of tomato seeds; Nonogaki H et al.; Endo-beta-mannanase (EC 3.2.1.78) is involved in hydrolysis of the mannan-rich cell walls of the tomato (Lycopersicon esculentum Mill.) endosperm during germination and post-germinative seedling growth . Different electrophoretic isoforms of endo-beta-mannanase are expressed sequentially in different parts of the endosperm, initially in the micropylar endosperm cap covering the radicle tip and subsequently in the remaining lateral endosperm surrounding the rest of the embryo . We have isolated a cDNA from imbibed tomato seeds (LeMAN2) that shares 77% deduced amino acid sequence similarity with a post-germinative tomato mannanase (LeMAN1) . When expressed in Escherichia coli, the protein encoded by LeMAN2 cDNA was recognized by anti-mannanase antibody and exhibited endo-beta-mannanase activity, confirming the identity of the gene . LeMAN2 was expressed exclusively in the endosperm cap tissue of tomato seeds prior to radicle emergence, whereas LeMAN1 was expressed only in the lateral endosperm after radicle emergence . LeMAN2 mRNA accumulation and mannanase activity were induced by gibberellin in gibberellin-deficient gib-1 mutant seeds but were not inhibited by abscisic acid in wild-type seeds . Distinct mannanases are involved in germination and post-germinative growth, with LeMAN2 being associated with endosperm cap weakening prior to radicle emergence, whereas LeMAN1 mobilizes galactomannan reserves in the lateral endosperm.

J Biol Chem, 2000 Oct 27, 275(43), 33449 - 56
Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis; Hardeland U et al.; Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G.T and G.U mismatches . Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions . Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG . Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal . Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity . These results establish that the structure function model postulated for the E . coli enzyme is largely applicable to the human TDG . We further provide evidence for G.U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.

Mol Cell Biol, 2000 Sep, 20(17), 6550 - 67
In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions; Melnick A et al.; The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia . PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression . X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface . In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches . To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed . We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein . Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes . The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain . In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription . Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription . In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions . However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize . These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Science, 2000 Aug 11, 289(5481), 920 - 30
The structural basis of ribosome activity in peptide bond synthesis; Nissen P et al.; Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site . Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized . The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin . The unusual pK(a) (where K(a) is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E . coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases . The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.

Metab Eng, 1999 Jul, 1(3), 270 - 4
Flow cytometric analysis of metabolic stress effects due to recombinant plasmids and proteins in Escherichia coli production strains; Soriano E et al.; Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells . Using flow cytometry, we analyzed the temporal development of the cellular content of DNA, total protein, and the recombinant product (human superoxide dismutase) in different strains . In cells carrying plasmids utilizing the phage T7 promoter 10 (pET vectors), induction with IPTG leads to an increase in protein content and size, an increase and a wide spreading of DNA content distribution, and a termination of cell division . These effects occurred with pET plasmids with or without an insert, but not with another plasmid which utilizes the tac promoter.

Metab Eng, 1999 Oct, 1(4), 320 - 33
Dynamics of glucose uptake by single Escherichia coli cells; Natarajan A et al.; The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells . When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate . The dependence upon substrate concentration could be described according to Michaelis-Menten kinetics with apparent saturation constant KM = 1.75 microM, and maximum 2-NBDG uptake rate= 197 molecules/cell-second . Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by D-glucose, 3-o-methyl glucose, and D-glucosamine, and lack of inhibition by L-glucose . Inhibition of 2-NBDG uptake by D-glucose was competitive in nature . The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of D-glucose in the culture medium (approximately 0.2 microM) is detectable . The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity . The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics . A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates . The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation.

Invest Ophthalmol Vis Sci, 2000 Aug, 41(9), 2445 - 55
Cloning and functional characterization of salamander rod and cone arrestins; Smith WC et al.; PURPOSE: To clone, localize, and determine functional binding characteristics of rod and cone arrestins from the retina of the tiger salamander (Ambystoma tigrinum) . METHODS: Two arrestins from salamander retina were cloned on the basis of their homology to known arrestins from other species . The expression pattern of these arrestins (SalArr1 and SalArr2) in the retina was determined by immunocytochemistry and in situ hybridization . SalArr1 and SalArr2 were expressed and functionally characterized . RESULTS: Both immunocytochemistry and in situ hybridization show that SalArr1 and SalArr2 localized specifically to rod and cone photoreceptors, respectively . SalArr1 demonstrated a characteristic high selectivity for light-activated phosphorylated rhodopsin (P-Rh*) and significant species selectivity, binding preferentially to amphibian rhodopsin over bovine rhodopsin . Mutant constitutively active forms of SalArr1 demonstrated a 2- to 4-fold increase in P-Rh* binding (compared with wild-type protein) and an even more dramatic (up to 25-fold) increase in binding to unphosphorylated Rh* and dark P-Rh . Constitutively active SalArr1 mutants also showed a reduced specificity for amphibian rhodopsin . The ability of Escherichia coli-expressed SalArr1, SalArr2, and an SalArr1-3A (L369A,V370A,F371A) mutant to bind to frog Rh* and P-Rh* and to compete with tritiated SalArr1 for amphibian P-Rh* was compared . SalArr1 and its mutant form bound to amphibian P-Rh* with high affinity (Ki = 179 and 74 nM, respectively), whereas the affinity of SalArr2 for P-Rh* was substantially lower (Ki = 9.1 microM) . CONCLUSIONS: SalArr1 and SalArr2 are salamander rod and cone arrestins, respectively . Crucial regulatory elements in SalArr1 are conserved and play functional roles similar to those of their counterparts in bovine rod arrestin . Rod and cone arrestins are relatively specific for their respective receptors.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 311 - 20
Phosphoenolpyruvate is a signal metabolite in transcriptional control of the cbb CO2 fixation operons in Ralstonia eutropha; Grzeszik C et al.; The two highly homologous cbb operons of the facultative chemoautotroph Ralstonia eutropha H16 encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle . Their transcriptional regulation was investigated both in vitro and in vivo to identify a metabolic signal involved in this process . For this purpose an in vitro transcription system employing the DNA-dependent RNA polymerase purified from R . eutropha was established . The enzyme from Escherichia coli was also used in verifying comparative studies . Plasmid DNA carrying the control region of the chromosomal cbb operon served as template . In the homologous as well as the heterologous system specific transcripts synthesized under the control of the operon promoter PcbbL were observed, depending on the structure of the tested promoter variant as well as the presence or absence of the activator protein CbbR . Unlike mutationally improved PcbbL variants, the wild-type promoter remained inactive, even in the presence of CbbR together with various potential signal metabolites . CbbR stimulated PcbbL mutants with intermediate basal activity . Phosphoenolpyruvate (PEP) was identified as a negative effector of CbbR that inhibited PcbbL-directed transcription and increased the operator-binding affinity of the protein . This CbbR-mediated inhibition was confirmed by assaying wild-type PcbbL operon fusions in glucose- or succinate-grown cells of E . coli, which contain greatly different concentrations of PEP . It is concluded that at least one additional protein must participate in the overall control of the cbb operons in R . eutropha.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 301 - 10
Role of DNA methylation at GATC sites in the dnaA promoter, dnaAp2; Kedar GC et al.; DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids . The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle . Methylation of GATC sites in the dnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating dnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication . Mutations that eliminate these two GATC sites but do not substantially change the activity of the promoter were introduced into a reporter gene fusion and into the chromosome . The chromosomal mutants are able to initiate DNA replication synchronously at both moderately slow and fast growth rates, demonstrating that GATC methylation at these two sites is not directly involved in providing the necessary amount of DnaA for precise timing of initiation during the cell cycle . Either sequestration does not involve these GATC sites, or cell cycle control of DnaA expression is not required to supply the concentration necessary for correct timing of initiation.

Rev Soc Bras Med Trop, 2000 Jul-Aug, 33(4), 401 - 2
South American rattlesnake bite and soft-tissue infection: report of a case; Nishioka Sde A et al.; The case of a man bitten by a South American rattlesnake (Crotalus durissus) and who developed an abscess at the site of the bite is reported . Abcesses are a rare complication of this type of envenoming, possibly due to the lack of a strong cytotoxic action of Crotalus durissus venom.

J Biotechnol, 2000 Jul 28, 81(1), 15 - 25
A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli; Ohiso I et al.; A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli . Six oligonucleotide probes labeled with FITC were designed and evaluated . One of these gave a detection limit of 10(3) colony forming units per assay, and assay results could be obtained within 5 min after PCR . It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product . The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences.

Vet Parasitol, 2000 Sep 10, 92(1), 37 - 49
Inhibition of Eimeria tenella replication after recombinant IFN-gamma activation in chicken macrophages, fibroblasts and epithelial cells; Heriveau C et al.; We have previously shown that activation of primary cultures of chicken bone-marrow macrophages and embryo fibroblasts with supernatants of concanavaline A-stimulated or reticuloendotheliosis virus (REV)-transformed chicken spleen cells as source of IFN-gamma significantly decreases Eimeria tenella growth in vitro . In the present study, we used various chicken cell lines, HD11 macrophages and DU24 fibroblasts, both virally transformed, CHCC-OU2 fibroblasts and LMH hepatic epithelial cells, both chemically transformed, to replicate E . tenella in vitro . We confirmed the previous results by showing that HD11 macrophages pre-treated for 24h with recombinant chicken IFN-gamma (either produced in E . coli or by transfected COS cells), at doses ranging from 1000 to 10U/ml, drastically inhibited E . tenella replication as measured by {3H} uracil uptake after a further 70h of culture, as when treated with REV supernatant . Likewise the fibroblast and epithelial cell lines exhibited significant inhibitory activity on E . tenella replication after pre-treatment with recombinant chicken IFN-gamma, but were less sensitive (1000-100U/ml) than when treated with REV supernatant . Recombinant chicken IFN-alpha pre-treatment of all cell lines had no inhibitory effect on parasite development.

J Neurochem, 2000 Sep, 75(3), 1190 - 9
Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-Galpha fusion proteins in Escherichia coli; Stanasila L et al.; Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg) . Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine . Affinities of the micro-selective agonists morphine, {D-Ala(2),N-Me-Phe(4),Gly(5)-ol}enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-{(35)S}thiotriphosphate) ({(35)S}GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits . Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated {(35)S}GTPgammaS binding . In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras . Potencies of the four agonists in stimulating {(35)S}GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2 . The intrinsic activities of the four agonists at the two fusion constructs were similar . The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.

Genomics, 2000 Aug 1, 67(3), 291 - 300
The RS447 human megasatellite tandem repetitive sequence encodes a novel deubiquitinating enzyme with a functional promoter; Saitoh Y et al.; We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite . In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit . Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells . We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat . When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo . An antibody against USP17 detected USP17 protein in human cells . Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme . Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain .

Science, 2000 Aug 11, 289(5481), 947 - 50
A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center; Muth GW et al.; Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation . To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification . A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction . In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function . These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

Metab Eng, 1999 Apr, 1(2), 180 - 7
Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase; Richardson MT et al.; BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction . MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts . These developments have set the stage for detailed mechanistic studies of this model system . RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E . coli . As reported earlier for the enzyme isolated from P . patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH . Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo . The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA . Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate . CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks . The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E . coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides . Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.

Metab Eng, 2000 Apr, 2(2), 149 - 54
Effect of glucose analog supplementation on metabolic flux distribution in anaerobic chemostat cultures of Escherichia coli; Berrios-Rivera SJ et al.; Previous work in our laboratories investigated the use of methyl alpha-glucoside (alpha-MG), a glucose analog that shares a phosphotransferase system with glucose, to modulate glucose uptake and therefore reduce acetate accumulation . The results of that study showed a significant improvement in batch culture performance and a reduction in acetate excretion without any significant effect on the growth rate in complex medium . The current study investigates the effect of supplementing the culture medium with the glucose analog alpha-MG on the metabolic fluxes of Escherichia coli under anaerobic chemostat conditions at two different dilution rates . Anaerobic chemostat studies utilizing complex media supplemented with glucose or glucose and alpha-MG at dilution rates of 0.1 and 0.4 h(-1), were performed, and the metabolic fluxes were analyzed . It was found that the addition of the glucose analog alpha-MG has an effect on the specific production rate of various extracellular metabolites . This effect is slightly greater at the higher dilution rate of 0.4 h(-1) . However, the glucose analog does not cause any major shift in the central metabolic patterns . It was further observed that alpha-MG supplementation does not result in the reduction in specific acetate synthesis rate in anaerobic chemostat cultures . These results emphasize the importance of testing different strategies for metabolic manipulation under the actual operating conditions.

Metab Eng, 2000 Apr, 2(2), 104 - 14
In vitro determined kinetic properties of mutant phosphoglucomutases and their effects on sugar catabolism in Escherichia coli; Brautaset T et al.; Based on primary amino acid sequence comparisons with other phosphoglucomutases, 12 conserved residues in the Acetobacter xylinum phosphoglucomutase (CelB) were substituted by site-directed mutagenesis, resulting in mutant enzymes with Kcat values {glucose-1-phosphate (G-1-P) to glucose-6-phosphate} ranging from 0 to 46% relative to that of the wild-type enzyme . In combination with a versatile set of plasmid expression vectors these proteins were used in a metabolic engineering study on sugar catabolism in Escherichia coli . Mutants of E . coli deficient in phosphoglucomutase synthesize intracellular amylose when grown on galactose, due to accumulation of G-1-P . Wild-type celB can complement this lesion, and we show here that the ability of the mutant enzymes to complement is sensitive to variations in their respective in vitro determined Kcat and Km G-1-P values . Reduced catalytic efficiencies could be compensated by increasing the CelB expression level, and in this way a mutant protein (substitution of Thr-45 to Ala) displaying a 7600-fold reduced catalytic efficiency could be used to eliminate the amylose accumulation . Complementation experiments with the homologous phosphoglucomutase indicated that a Km G-1-P value significantly below that of CelB is not critical for the in vivo conversion of the substrate.

Neoplasia, 1999 Oct, 1(4), 315 - 20
A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase; Tjuvajev JG et al.; Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients . We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene . We show that the expression and imaging of HSV1-tk (a marker gene) can be used to monitor the expression of the LacZ gene (a second gene) under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site . In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo . We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.

Chin J Biotechnol, 1999, 15(3), 153 - 8
Expression, purification, and bone-inducing activity of recombinant human bone morphogenetic protein-3 mature peptide; Zhu B et al.; It was inferred that the mature peptide of human bone morphogenetic protein-3 (hBMP-3m) consists of the carboxyl terminal 127 amino acid residues of hBMP-3 . A plasmid, pDH-B3m, was constructed by inserting the cDNA sequences encoding hBMP-3m into pDH, a PL-containing expression vector . pDH-B3m was transformed into Escherichia coli DH5 alpha . The highest expression level of recombinant hBMP-3m (rhBMP-3m) could be reached after 6 hours of induction at 42 degrees C, accounting for 28% of the total bacterial proteins . The rhBMP-3m was found in the inclusion bodies . After being washed and partially purified, the inclusion bodies were solubilized in urea and purified efficiently through ion-exchange chromatography . The purity of the rhBMP-3m was at least 95% . The rhBMP-3m was refolded by dilution method and then 1 mg was implanted into mouse thigh muscle to assay its activity . A classic pattern of cartilaginous osteogenesis was observed . The results showed that the purified and refolded rhBMP-3m had ectopic bone-inducing activity.

Am J Respir Crit Care Med, 2000 Aug, 162(2 Pt 1), 465 - 70
Effect of aminoguanidine on lung fluid filtration after endotoxin in awake sheep; Evgenov OV et al.; It has been suggested that enhanced generation of nitric oxide by inducible nitric oxide synthase (iNOS) may contribute to acute lung injury . We hypothesized that aminoguanidine (AG), a proposed selective inhibitor of iNOS, would alter pulmonary hemodynamics, fluid filtration, and gas exchange after endotoxin in chronically instrumented awake sheep . Eighteen sheep were randomly assigned to receive either AG (10 mg/kg + 1 mg/kg/h), or NaCl 0.9% intravenously for 4 h, beginning 2 h after injection of Escherichia coli endotoxin (1 microgram/kg) . After endotoxin, pulmonary artery pressure (Ppa), capillary pressure (Pc), and vascular resistance index (PVRI) rose concomitantly with six-fold increments in lung lymph flow (Q L) and protein clearance (CL) . Extravascular lung water (EVLW) doubled, as assessed with the thermal dye dilution technique; Pa(O(2)) decreased, AaPO(2) and venous admixture (Q S/Q T) increased . After AG, Q L and CL increased further by approximately 30%, whereas EVLW remained unchanged, despite an additional increase in Pc . Ppa, PVRI, and systemic vascular resistance index rose, whereas cardiac index and pulmonary blood volume index declined . In addition, Pa(O(2)) rose, and AaPO(2) and Q S/Q T decreased . We conclude that in endotoxemic sheep, AG improves gas exchange and increases Q L and CL, whereas EVLW remains unchanged in spite of enhanced Pc . Apparently, increased lymphatic drainage prevents EVLW from rising after AG.

J Cell Sci, 2000 Sep, 113 ( Pt 17), 3093 - 101
The protease-activated receptor-2 upregulates keratinocyte phagocytosis; Sharlow ER et al.; The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini . Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage . PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes . Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis . PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E . coli K-12 bioparticles . This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity . Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes . PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

Arch Biochem Biophys, 2000 Aug 15, 380(2), 373 - 9
2,4-Dienoyl-CoA reductase from Escherichia coli is a novel iron-sulfur flavoprotein that functions in fatty acid beta-oxidation; Liang X et al.; 2,4-Dienoyl-CoA reductase is an enzyme that is required for the beta-oxidation of unsaturated fatty acids with even-numbered double bonds . The 2,4-dienoyl-CoA reductase from Escherichia coli was studied to explore the catalytic and structural properties that distinguish this enzyme from the corresponding eukaryotic reductases . The E . coli reductase was found to contain 1 mol of flavin mononucleotide and 4 mol each of acid-labile iron and sulfur in addition to 1 mol of flavin adenine dinucleotide per mole of protein . Redox titrations revealed a requirement for 5 mol of electrons to completely reduce 1 mol of enzyme and provided evidence for the formation of a red semiquinone intermediate . The reductase caused a significant polarization of the substrate carbonyl group as indicated by an enzyme-induced red shift of 38 nm in the spectrum of 5-phenyl-2,4-pentadienoyl-CoA . However, suspected cis --> trans isomerase and Delta(3),Delta(2)-enoyl-CoA isomerase activities were not detected in this enzyme . It is concluded that the 2, 4-dienoyl-CoA reductases from E . coli and eukaryotic organisms are structurally and mechanistically unrelated enzymes that catalyze the same type of reaction with similar efficiencies .

Arch Biochem Biophys, 2000 Aug 15, 380(2), 267 - 73
Copper-mediated toxicity of 2,4,5-trichlorophenol: biphasic effect of the copper(I)-specific chelator neocuproine; Zhu BZ et al.; The lipophilic copper(I)-specific chelator neocuproine has been frequently used as an inhibitor of copper-mediated damage in biological systems . In this communication we report that the copper-mediated toxicity of 2,4,5-trichlorophenol is markedly potentiated by neocuproine at levels which are near-stoichiometric with respect to the copper concentration but is inhibited at higher concentrations . However, no potentiation was observed when neocuproine was substituted by bathocuproinedisulfonic acid, a negative charged ligand with essentially the same copper-binding characteristics as neocuproine . We found that the potentiation by neocuproine was due to the formation of a lipophilic copper complex, while the inhibition by bathocuproinedisulfonic acid was due to the formation of a hydrophilic one . Caution in the use of neocuproine to study copper-mediated toxicity is advised .

Biotechnol Prog, 2000 Jul-Aug, 16(4), 642 - 9
GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing; Dauner M et al.; Gas chromatography-mass spectrometry (GC-MS) is a rapid method that provides rich information on isotopomer distributions for metabolic flux analysis . First, we established a fast and reliable experimental protocol for GC-MS analysis of amino acids from total biomass hydrolyzates, and common experimental pitfalls are discussed . Second, a suitable interface for the use of mass isotopomer data is presented . For this purpose, a general, matrix-based correction procedure that accounts for naturally occurring isotopes is introduced . Simulated and experimentally determined mass distributions of unlabeled amino acids showed a deviation of less than 3% for 89% of the analyzed fragments . Third, to investigate general properties of GC-MS-based isotopomer balancing, altered flux ratios through glycolysis and pentose phosphate pathway and/or exchange fluxes were simulated . Different fluxes were found to exert specific and significant influence on the mass isotopomer distributions, thus indicating that GC-MS data contain valuable information for metabolic flux analysis . Fourth, comparison of different methods revealed that GC-MS analysis provides the largest number of independent constraints on amino acid isotopomer abundance, followed by correlation spectroscopy and fractional enrichment analysis by nuclear magnetic resonance.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 595 - 9
Production of alpha-galactosyl epitopes via combined use of two recombinant whole cells harboring UDP-galactose 4-epimerase and alpha-1,3-galactosyltransferase; Chen X et al.; alpha-Galactosyl epitopes (or alpha-Gal, oligosaccharides with a terminal Galalpha1,3Gal sequence) are a class of biologically important oligosaccharides in great demand in bulk quantities for basic and clinical studies on preventing hyperacute rejection in pig-to-primate organ xenotransplantation . A truncated bovine alpha-1, 3-galactosyltransferase, the key enzyme responsible for the biosynthesis of the terminal structure of alpha-Gal, was cloned and overexpressed previously . The acceptor specificity was further studied in the present paper, and lactose and galactose derivatives were found to be good acceptors . To develop a more proficient reaction process, we report herein an example of an efficient enzymatic synthesis of alpha-Gal oligosaccharides catalyzed by the combination of two recombinant Escherichia coli whole cells harboring the genes of a UDP-galactose 4-epimerase and the alpha-1, 3-galactosyltransferase, respectively . Using lactosyl azide (LacN(3)) as the acceptor for the glycosyltransferase, the combined use of the two recombinant cells efficiently produced alpha-Gal epitope Gal alpha1,3LacN(3) in 60-68% yield.

Biochemistry, 2000 Aug 15, 39(32), 9804 - 10
Asp338 controls hydride transfer in Escherichia coli IMP dehydrogenase; Kerr KM et al.; IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) . This reaction involves the formation of a covalent adduct with an active site Cys . This intermediate, E-XMP, hydrolyzes to produce XMP . The mutation of Asp338 to Ala severely impairs the activity of Escherichia coli IMPDH, decreasing the value of k(cat) by 650-fold . No (D)V(m) or (D)V/K(m) isotope effects are observed when 2-(2)H-IMP is the substrate for wild-type IMPDH . Values of (D)V(m) = 2.6 and (D)V/K(m) (IMP) = 3.4 are observed for Asp338Ala . Moreover, while a burst of NADH production is observed for wild-type IMPDH, no burst is observed for Asp338Ala . These observations indicate that the mutation has decreased the rate of hydride transfer by at least 5 x 10(3)-fold . In contrast, k(cat) for the hydrolysis of 2-chloroinosine-5'-monophosphate is decreased by only 8-fold . In addition, the rate constant for inactivation by 6-chloropurine riboside 5'-monophosphate is increased by 3-fold . These observations suggest that the mutation has little effect on the nucleophilicity of the active site Cys residue . These results are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2'-OH of IMP, and the amide group of NAD(+) {Colby, T . D., Vanderveen, K., Strickler, M . D., Markham, G . D., and Goldstein, B . M . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 3531-3536}.

Biochemistry, 2000 Aug 15, 39(32), 9746 - 53
Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"; Snider MJ et al.; To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis . Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited . Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release . Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy . As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation . As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature . The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C) . This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex . Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature . When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4) . This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding . This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex . In an Appendix, some objections to the conventional scheme for transition state binding are discussed.

Biochemistry, 2000 Aug 15, 39(32), 9709 - 17
Crystal structure at 2.5 A resolution of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli; Chen Z et al.; Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, utilizing molecular oxygen as a terminal electron acceptor . To accomplish this transformation, CAOs utilize two cofactors: a mononuclear copper, and a unique redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ or TOPA quinone) . TPQ is derived via posttranslational modification of a specific tyrosine residue within the protein itself . In this study, the structure of an amine oxidase from Hansenula polymorpha has been solved to 2.5 A resolution, in which the precursor tyrosine is unprocessed to TPQ, and the copper site is occupied by zinc . Significantly, the precursor tyrosine directly ligands the metal, thus providing the closest analogue to date of an intermediate in TPQ production . Besides this result, the rearrangement of other active site residues (relative to the mature enzyme) proposed to be involved in the binding of molecular oxygen may shed light on how CAOs efficiently use their active site to carry out both cofactor formation and catalysis.

Biochemistry, 2000 Aug 15, 39(32), 9687 - 97
Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein; Chelius D et al.; (R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function . A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH) . A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)) . HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function . The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH . A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242 . With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters) . Six cysteine mutants each have an increased K(m)(NADH) (2-6-fold) but an unchanged K(m)(NAD)+ . The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site . Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.

J Virol, 2000 Sep, 74(17), 8011 - 7
Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies; Riddell MA et al.; Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited . A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein . Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F . Li, J . Torresi, S . A . Locarnini, H . Zhuang, W . Zhu, X . Guo, and D . A . Anderson, J . Med . Virol . 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457 . The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera . Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2 . 1 and/or VLP ELISAs, suggesting a complex antigenic structure . MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity . These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.

J Virol, 2000 Sep, 74(17), 7834 - 41
Involvement of the zinc-binding capacity of Sendai virus V protein in viral pathogenesis; Huang C et al.; The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice . The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity . In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins . When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type . We then recovered two mutant SeVs from cDNA, which have V-C(341)S and V-C(365)R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively . The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells . However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V(DeltaC), which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice . We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

J Virol, 2000 Sep, 74(17), 7781 - 6
A new mutant class, made by targeted mutagenesis, of phage PRD1 reveals that protein P5 connects the receptor binding protein to the vertex; Bamford JK et al.; Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions . We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations . The role of protein P5 in the vertex structure was examined by this method . Mutation in gene V revealed that protein P5 is essential . The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing . P5(-) particles also lost their DNA upon purification . Based on this and previously published information we propose a spatial model for the spike structure at the vertices . This resembles to the corresponding structure in adenovirus.

J Virol, 2000 Sep, 74(17), 7720 - 9
Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes; Hobom U et al.; We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli . Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette . We report on a fast and efficient screening procedure for a Tn insertion library . Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates . A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established . Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone . We applied this method to retrieve mutants of HCMV envelope glycoprotein genes . To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts . In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit . Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth . We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

Protein Sci, 2000 Jul, 9(7), 1334 - 9
The dihydrolipoyl acyltransferase (BCE2) subunit of the plant branched-chain alpha-ketoacid dehydrogenase complex forms a 24-mer core with octagonal symmetry; Mooney BP et al.; Little is known of the plant branched-chain alpha-ketoacid dehydrogenase complex . We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach . Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli . The soluble recombinant protein was purified using a Superose 6 size-exclusion column to >90% homogeneity and was catalytically active . The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24-mer . Negative-staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry . Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.

Bone Marrow Transplant, 2000 May, 25 Suppl 2, S80 - 2
Development of a cytomegalovirus vector for somatic gene therapy; Borst E et al.; The development of new and improved vector systems is central for realization of new concepts for gene therapy . The tropism of human cytomegalovirus (CMV) for hematopoietic progenitor cells and the large genome size (230 kbp) that offers a unique cloning capacity make this virus a promising vector candidate for gene transfer into hematopoietic cells and for therapy of congenital and acquired diseases of the hematopoietic system . Recently, we cloned the CMV genome as a bacterial artificial chromosome (BAC) in Escherichia coli and established efficient mutagenesis procedures for CMV - a prerequisite for vector construction . Here, we report on the construction of a recombinant GFP virus that will be used to re-evaluate the tropism of CMV and to monitor gene transfer into target cells . Further goals of CMV vector development are the evaluation of the cloning capacity and the construction of replication-deficient vectors.

Rev Latinoam Microbiol, 1999 Oct-Dec, 41(4), 263 - 5
{Colibacillosis in swine: proof of vaccine efficacy}; Cicuta ME et al.; The damage caused in the economy and animal sanity by the porcine colibacilosis are significant and they deserve the investigation of preventive measures that give answers to the producers . Existing at the present time approximately 21,000 pigs in Corrientes and 110,000 in Chaco provinces of Argentine, the losses for diarrhea that exterminate whole litters, acquire relevance, specially if they can be prevented or cured . For that reason, having 21 strains of enteropathogenic and verocitotoxigenic E . coli (ETEC/VTEC) isolated from pigs of the North East of Argentine, that were recognized by PCR, two were selected, containing the genes for STIa, STIb, LTI, VT2e (SLT-IIv) and F4 (K88) . They were spread on nutrient agar and Minca medium, to obtain the suspension in PBS, which was inactivated with formol . After the sterility and innocuity controls, it was diluted to a 12 x 10(8) concentration to make the mouse protection test in 20 mice, of 18-20 g, inoculating the vaccine the days 1, 4, 7 and 10, by the intraperitoneal route, doses of 0.25 ml each one . The day 21 after beginning the test the animals were challenged with 50 LD50, and a protection of 85% was obtained . To determine the LD50, we prepared a suspension in physiologic solution, corresponding to Mac Farland's tube No 10, making dilutions from 10(-1) to 10(-5) and applying the statistical method of Reed and Muench . These first results encourage us to continue working after a prophylactic measure that were effective, potent and elaborated with strains of this area.

Rev Latinoam Microbiol, 1997 Jul-Dec, 39(3-4), 145 - 51
Frequency of adhesive factors and enterotoxins in strains of Escherichia coli isolated from piglets with diarrhea; Flores-Abuxapqui JJ et al.; Neonatal colibacillosis is one of the most prevalent illnesses in pig farms . In order to examine the frequency of adherence factors and the production of enterotoxins in strains of E . coli, we collected stool specimens from 500 piglets between 1 and 10 days of age with diarrhea, including piglets from several different farms on the periphery of Merida, Yucatan . One thousand and eighty (1080) strains of E . coli were isolated, of which 127 (11.76%) produced STa, and 62 (5.74%) produced adherence factors . Of these, 30 (48.39%) produced factor K88, 18 (29.03%) produced factor 987P, 12 (19.35%) produced K99, and 2 (3.23%) produced F41 . Of the 62 strains which produced adherence factors, 42 (67.74%) also produced STa, and of these, 17 (40.84%) produced factor K88, 13 (30.95%) produced 987P, 10 (23.81%) produced K99, and 2 (4.76%) produced F41 . In summary, of the 1080 strains isolated, 42 (3.89%) produced both STa toxin and adherence factors, 85 (7.87%) produced STa but did not produce adherence factors, and 933 (86.39%) produced neither STa or adherence factors . No LT-producing E . coli was detected in this study.

Rev Latinoam Microbiol, 1997 Jan-Jun, 39(1-2), 47 - 56
Electron microscopy and biological properties of pBR322 DNA condensed with the trivalent cations spermidine and hexammine cobalt (III); Baeza I et al.; Electron microscopy and the biological properties of susceptibility to DNase I, genetic transcription, and transformation of pBR322 DNA compacted with spermidine or hexammine cobalt (III), were analyzed in order to characterize the association of DNA in its compacted form with these two different trivalent cations . Spermidine and hexammine cobalt (III) produced an average 4-fold reduction of the DNA perimeter in compact DNA forms, which were doughnut-shaped toroids and cylinders . Both compacted DNAs were resistant to the hydrolytic activity of DNase I . However, spermidine-condensed pBR322 DNA was 10-fold and 4 to 6-fold more active in transcription and transformation, respectively, than naked pBR322 . I . Hexammine cobalt (III)-condensed pBR322 was inactive in both biological properties . An inhibitory effect of hexammine cobalt (III) on RNA polymerase and genetic transformation activities was discarded because at higher ionic strength, in which DNA is not compacted by hexammine cobalt (III), transcription and transformation were similar to those observed with naked DNA . This information showed that the interaction of hexammine cobalt (III) with the DNA converted the pBR322 DNA into an inert molecule . In contrast, pBR322 did not loose its biological properties after its interaction with the polyamine spermidine; i.e., experimental condensation of pBR322 DNA by spermidine produced compacted DNA that is more similar to compact native genomes than relaxed DNA . These experiments led us to conclude that spermidine-condensed DNA can be used to study the roll of the native supercoiling of DNA in the regulation of genetic replication and transcription, as well as to study the mechanisms that allow the accessibility of the supercoiled or condensed DNA substrate for enzymes.

Nat Struct Biol, 2000 Aug, 7(8), 644 - 7
Probing protein-protein interactions in real time; Viani MB et al.; We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules . In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support . This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.

Nat Biotechnol, 2000 Aug, 18(8), 843 - 6
Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli; Albrecht M et al.; We have used combinatorial biosynthesis to synthesize novel lipophilic carotenoids that are powerful cellular antioxidants . By co-expressing three different carotenoid desaturases in combination with a carotenoid hydratase, a cyclase, and a hydroxylase on compatible plasmids in Escherichia coli, we synthesized four novel carotenoids not previously detected in biological material or chemically synthesized . Their identification was based on their relative retention times on HPLC, spectroscopic properties, molecular weights, number of hydroxy groups, and 1H-NMR spectra . The carotenoids were designated as 1-HO-3', 4'-didehydrolycopene, 3, 1'-(HO)2-gamma-carotene, 1,1'-(HO)2-3, 4, 3', 4'-tetradehydrolycopene, and 1, 1'-(HO)2-3, 4-didehydrolycopene . These novel acyclic derivatives differ from structurally related compounds by extension of the conjugated polyene chain as well as additional hydroxy groups at position C-1' . We determined their antioxidative activity in a liposome-membrane model system, which showed that their ability to protect against photooxidation and radical-mediated peroxidation reactions was linked to the length of the conjugated double-bond system and the presence of a single hydroxy group . The protection of membrane degradation was superior to the related 1-HO and 1, 1'-(HO)2 lycopene derivatives, making them interesting pharmaceutical candidates.

Microbiology, 2000 Aug, 146 ( Pt 8), 1815 - 28
Phylogeny of related functions: the case of polyamine biosynthetic enzymes; Sekowska A et al.; Genome annotation requires explicit identification of gene function . This task frequently uses protein sequence alignments with examples having a known function . Genetic drift, co-evolution of subunits in protein complexes and a variety of other constraints interfere with the relevance of alignments . Using a specific class of proteins, it is shown that a simple data analysis approach can help solve some of the problems posed . The origin of ureohydrolases has been explored by comparing sequence similarity trees, maximizing amino acid alignment conservation . The trees separate agmatinases from arginases but suggest the presence of unknown biases responsible for unexpected positions of some enzymes . Using factorial correspondence analysis, a distance tree between sequences was established, comparing regions with gaps in the alignments . The gap tree gives a consistent picture of functional kinship, perhaps reflecting some aspects of phylogeny, with a clear domain of enzymes encoding two types of ureohydrolases (agmatinases and arginases) and activities related to, but different from ureohydrolases . Several annotated genes appeared to correspond to a wrong assignment if the trees were significant . They were cloned and their products expressed and identified biochemically . This substantiated the validity of the gap tree . Its organization suggests a very ancient origin of ureohydrolases . Some enzymes of eukaryotic origin are spread throughout the arginase part of the trees: they might have been derived from the genes found in the early symbiotic bacteria that became the organelles . They were transferred to the nucleus when symbiotic genes had to escape Muller's ratchet . This work also shows that arginases and agmatinases share the same two manganese-ion-binding sites and exhibit only subtle differences that can be accounted for knowing the three-dimensional structure of arginases . In the absence of explicit biochemical data, extreme caution is needed when annotating genes having similarities to ureohydrolases.

J Cell Biol, 2000 Aug 7, 150(3), 689 - 94
Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon; Koch HG et al.; Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane . We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins . Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level . These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon . In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

J Cell Biol, 2000 Aug 7, 150(3), 447 - 60
Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle; Agrawal RK et al.; Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution . The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined . Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy . Now, the positions of the A- and E-site tRNAs are determined with similar accuracy . The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation . The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.

J Biol Chem, 2000 Oct 20, 275(42), 32974 - 82
PRMT3 is a distinct member of the protein arginine N-methyltransferase family . Conferral of substrate specificity by a zinc-finger domain; Frankel A et al.; S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins . At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5 . To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity . Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus . Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts . The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl(2) as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups . We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected . We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine . Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein . These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.

J Biol Chem, 2000 Nov 10, 275(45), 35176 - 84
Mechanism of pH-dependent N-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor; Feinberg H et al.; Efficient release of ligands from the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of the hepatic asialoglycoprotein receptor at endosomal pH requires a small set of conserved amino acids that includes a critical histidine residue . When these residues are incorporated at corresponding positions in an homologous galactose-binding derivative of serum mannose-binding protein, the pH dependence of ligand binding becomes more like that of the receptor . The modified CRD displays 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic of the asialoglycoprotein receptor . In the crystal structure of the modified CRD bound to N-acetylgalactosamine, the histidine (His(202)) contacts the 2-acetamido methyl group and also participates in a network of interactions involving Asp(212), Arg(216), and Tyr(218) that positions a water molecule in a hydrogen bond with the sugar amide group . These interactions appear to produce the preference for N-acetylgalactosamine over galactose and are also likely to influence the pK(a) of His(202) . Protonation of His(202) would disrupt its interaction with an asparagine that serves as a ligand for Ca(2+) and sugar . The structure of the modified CRD without sugar displays several different conformations that may represent structures of intermediates in the release of Ca(2+) and sugar ligands caused by protonation of His(202).

J Biol Chem, 2000 Nov 10, 275(45), 34853 - 7
The role of alpha,beta -dicarbonyl compounds in the toxicity of short chain sugars; Okado-Matsumoto A et al.; The extent to which sugars serve as targets for superoxide was examined using glycolaldehyde as the simplest sugar and using superoxide dismutase (SOD)-replete and SOD-null strains growing under aerobic and anaerobic conditions . Glycolaldehyde was more toxic to the SOD-null strain than to its SOD-replete parent, and this differential effect was oxygen-dependent . The product, glyoxal, could be trapped in the medium by 1,2-diaminobenzene and assayed as quinoxaline . The SOD-null strain produced more glyoxal and eliminated it more slowly than the SOD-replete parent strain . Glyoxal was approximately 10 times more toxic than glycolaldehyde and was more toxic to the SOD-null strain than to the parental strain . 1,2-Diaminobenzene protected against the toxicity of glycolaldehyde . These Escherichia coli strains contained the glutathione-dependent glyoxalases I and II, as well as the glutathione-independent glyoxalase III . Of these enzymes, glyoxalase III was most abundant, and it was inactivated within the aerobic SOD-null strain and also in extracts when exposed to the flux of superoxide and hydrogen peroxide imposed by the xanthine oxidase reaction . Thus, it appears that short chain sugars are oxidized by superoxide yielding toxic dicarbonyls . Moreover, the defensive glyoxalase III is also inactivated by the oxidative stress imposed by the lack of SOD, thereby exacerbating the deleterious effect of sugar oxidation.

Radiat Res, 2000 Aug, 154(2), 208 - 16
Effect of ELF magnetic fields on protein synthesis in Escherichia coli K12; Nakasono S et al.; Escherichia coli K12 was used as a model system to determine whether ELF magnetic fields (MFs) are a general stress factor . The cells were exposed to ELF MFs (5-100 Hz) at a maximum intensity of 14 mT r . m.s . for circularly polarized MFs and 10 mT r.m.s . for vertically polarized MFs . The response of the cells to the MFs was estimated from the change in protein synthesis by using 2D PAGE . Approximately 1,000 proteins were separated on the 2D gels . The stress-responsive proteins such as CH10, DNAK, CH60, RECA, USPA, K6P1 and SODM were identified from the SWISS-2DPAGE database on the 2D gels . These proteins respond to most stress factors, including temperature change, chemical compounds, heavy metals, and nutrients . When the bacterial cells were exposed to each MF at 5-100 Hz under aerobic conditions (6.5 h) or at 50 Hz under anaerobic conditions (16 h) at the maximum intensity (7.8 to 14 mT r.m.s.), no reproducible changes were observed in the 2D gels . Changes in protein synthesis were detected by 2D PAGE with exposure to heat shock (50 degrees C for 30 min) or under anaerobic conditions (no bubbling for 16 h) . Increases in the levels of synthesis of the stress proteins were observed in heat-shocked cells (CH60, CH10, HTPG, DNAK, HSLV, IBPA and some unidentified proteins) and in cells grown under anaerobic conditions (DNAK, PFLB, RECA, USPA and many unidentified proteins) . These results suggest that 2D PAGE is sufficient to detect cell responses to environmental stress . The high-intensity ELF MFs (14 mT at power frequency) did not act as a general stress factor.

Mol Microbiol, 2000 Aug, 37(3), 680 - 6
Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N(4)-ethenocytosine via an SOS-induced mechanism; Dunman PM et al.; The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4-ethenocytosine (epsilon C) lesions borne on transfected single-stranded M13 vector DNA . All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E . coli . To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response . Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at epsilon C residues . In contrast to normal cells that show approximately 6% mutagenesis at epsilon C lesions, but approximately 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at epsilon C lesion sites in untreated cells . However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA . These results imply that recN cells are constitutively active for SOS mutagenesis functions . The observation that epsilonC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at epsilonC can also be independently elevated by the SOS pathway.

Mol Microbiol, 2000 Aug, 37(3), 629 - 38
The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein; Torheim NK et al.; In wild-type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth . In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two- to threefold relative to wild type . Here, we show that the DnaA protein concentration was two- to threefold lower in the dnaA204 mutant compared with the wild-type strain . The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein . Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells . During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild-type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable . Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half-life of the protein . Our results suggest that the DnaA204 protein has essentially wild-type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions . The basis for the stabilization in the absence of SeqA is not known . We suggest that the formation of stable DnaA-DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.

Mol Microbiol, 2000 Aug, 37(3), 555 - 60
The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins; Seitz EM et al.; The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities . Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues . The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences . These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.

Mol Microbiol, 2000 Jul, 37(2), 410 - 23
Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery; Justice SS et al.; SulA and MinCD are specific inhibitors of cell division in Escherichia coli . In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites . SulA prevented the formation of high-molecular-weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization . In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo . However, MinCD did prevent the formation of FtsA rings . Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition . These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.

Mol Microbiol, 2000 Jul, 37(2), 371 - 81
Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two-component regulator; Mukhopadhyay S et al.; The stationary phase expression of many conserved, adaptive bacterial proteins is dependent on RpoS, a second vegetative sigma factor . The regulation of RpoS itself, however, is complex and not fully understood, particularly at the level of transcription . In this report, we show that the observed hydrogen peroxide sensitivity of a mutant defective in expression of barA, a bacterial virulence factor, can be explained by a reduction in catalase activity, an RpoS-controlled function . Levels of katE mRNA, encoding the major catalase of Escherichia coli, were much lower in the barA mutant, suggesting that BarA is required for the expression of this RpoS-regulated gene . Expression of another RpoS-regulated gene, osmY, was also found to be severely reduced in the barA mutant . Employing Western analyses with anti-RpoS antisera and Northern analyses using probes specific for rpoS, we found that BarA is required for the exponential phase induction of RpoS itself . Operon lacZ fusion expression studies and Northern analyses indicate that BarA itself is maximally expressed in early exponential phase cultures immediately preceding the transcriptional induction of RpoS . Results of primer extension studies indicate that exponential phase expression from the rpoSp1 promoter is reduced by more than 85% in a barA mutant but could be efficiently complemented by a plasmid-borne copy of barA in trans . These results suggest that regulatory signals that are operant in exponentially growing cultures play an important role in effecting stationary phase gene expression.

Mol Microbiol, 2000 Jul, 37(2), 331 - 44
Membrane topology of the Mep/Amt family of ammonium transporters; Thomas GH et al.; The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature . Members from bacteria, yeast and plants have been identified experimentally as high-affinity ammonium transporters . We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family . This was established using a minimal set of AmtB-PhoA fusion proteins with a complementary set of AmtB-LacZ fusions . These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning helices, with the N-terminus on the exterior face of the membrane and the C-terminus on the interior . A small subset, including E . coli AmtB, probably have an additional twelfth membrane-spanning region at the N-terminus . Addition of PhoA or LacZ alpha-peptide to the C-terminus of E . coli AmtB resulted in complete loss of transport activity, as judged by measurements of {14C}-methylammonium uptake . This C-terminal region, along with four membrane-spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family . Structural modelling of the E . coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp-182 . The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.

Mol Microbiol, 2000 Jul, 37(2), 226 - 38
Escherichia coli translocase: the unravelling of a molecular machine; Manting EH et al.; Protein translocation across the bacterial cytoplasmic membrane has been studied extensively in Escherichia coli . The identification of the components involved and subsequent reconstitution of the purified translocation reaction have defined the minimal constituents that allowed extensive biochemical characterization of the so-called translocase . This functional enzyme complex consists of the SecYEG integral membrane protein complex and a peripherally bound ATPase, SecA . Under translocation conditions, four SecYEG heterotrimers assemble into one large protein complex, forming a putative protein-conducting channel . This tetrameric arrangement of SecYEG complexes and the highly dynamic SecA dimer together form a proton-motive force- and ATP-driven molecular machine that drives the stepwise translocation of targeted polypeptides across the cytoplasmic membrane . Recent findings concerning the translocase structure and mechanism of protein translocation are discussed and shine new light on controversies in the field.

Mol Microbiol, 2000 Jul, 37(1), 180 - 91
Resolution of holliday junctions by RuvABC prevents dimer formation in rep mutants and UV-irradiated cells; Michel B et al.; In this work, we present evidence that indicates that RuvABC proteins resolve Holliday junctions in a way that prevents dimer formation in vivo . First, although arrested replication forks are rescued by recombinational repair in cells deficient for the Rep helicase, rep mutants do not require the XerCD proteins or the dif site for viability . This shows that the recombination events at arrested replication forks are generally not accompanied by the formation of chromosome dimers . Secondly, resolution of dimers into monomers is essential in the rep ruv strain because of an increased frequency of RecFOR recombination events in the chromosome of this mutant . This suggests that, in the absence of the Ruv proteins, chromosomal recombination leads to frequent dimerization . Thirdly, dif or xerC mutations increase the UV sensitivity of ruv-deficient cells 100-fold, whereas they do not confer UV sensitivity to ruv+ cells . This shows that recombinational repair of UV lesions is not accompanied by dimer formation provided that the RuvABC proteins are active . The requirement for dimer resolution in ruv strains is suppressed by the expression of the RusA Holliday junction resolvase; therefore, RusA also prevents dimer formation . We conclude that the inviability arising from a high frequency of dimer formation in rep or UV-irradiated cells is only observed in the absence of known enzymes that resolve Holliday junctions.

Mol Microbiol, 2000 Jul, 37(1), 145 - 55
Site-specific DNA binding and bending by the Borrelia burgdorferi Hbb protein; Kobryn K et al.; The Borrelia burgdorferi Hbb protein shows sequence similarity to members of the Escherichia coli HU/integration host factor (IHF) family of DNA accessory factors . We have overexpressed the hbb gene product in E . coli and purified the protein to near homogeneity . Biochemical analyses have revealed that Hbb has unique properties and is neither a strict HU nor IHF analogue . Hbb was found to bind specifically to a site in the putative origin of DNA replication between dnaA and dnaN . DNA footprinting studies have shown that this site is unrelated to the consensus sequence recognized by IHF proteins . Hbb induces a dramatic bend (> 126 degrees ) at this site and was also shown to restrain negative supercoils efficiently upon DNA binding . These features of the protein suggest that Hbb may act as a DNA accessory factor that facilitates the assembly of higher order protein-DNA complexes, such as those involved in DNA replication, transcription, recombination, packaging and perhaps other DNA metabolic processes unique to Borrelia.

Mol Microbiol, 2000 Jun, 36(6), 1494 - 503
The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway; Diez A et al.; The link between cell division defects and the induction of the universal stress response is demonstrated to operate via the RecA regulator of the SOS response . An insertion in the cell division gene ftsK upregulates uspA in a recA-dependent manner . Unlike true SOS response genes, this upregulation only occurs in growth-arrested cells and is LexA independent . Thus, besides ppGpp-dependent starvation signals, DNA aberrations transduce RecA-dependent signals to the uspA promoter, which only affect the promoter during stasis . Further, we show that ftsK itself, like uspA, is induced in stationary phase and that this induction requires the stringent control modulon rather than activated RecA . Thus, ftsK, like uspA, is regulated by at least two global regulators: ppGpp of the stringent control network and RecA of the SOS modulon . We suggest that UspA is a new bona fide member of the RecA-dependent DNA protection and repair system, as mutants lacking functional UspA were found to be sensitive to UV irradiation and mitomycin C exposure . Moreover, the UV sensitivity of uspA mutants is enhanced in an additive manner by the ftsK1 mutation.

Mol Microbiol, 2000 Jun, 36(6), 1470 - 80
Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli; Zangrossi S et al.; Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock-induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures . We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10-fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift-like pattern in a PNPase-independent manner . The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs . The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase . In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho-dependent termination site within pnp . In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.

Mol Microbiol, 2000 Jun, 36(6), 1360 - 70
ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells; Thomas JG et al.; DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli . A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear . Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions . We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES . However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation . Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates . Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants . By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells . These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E . coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.

Mol Microbiol, 2000 Jun, 36(6), 1349 - 59
FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli; Gullbrand B et al.; In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli . In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication . This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication . Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions . Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length . These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid . These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide . Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation . The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring . A narrow cell length interval was found, under and over which primarily central and non-central FtsZ rings, respectively, were observed . This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.

Mol Microbiol, 2000 Jun, 36(6), 1319 - 26
The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC; Skarstad K et al.; The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro . The mechanism of inhibition is, however, not known . SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein . We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1 . Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies . The affinity of SeqA for hemi-methylated oriC was higher than for fully methylated oriC . The binding was in both cases strongly cooperative . We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein-protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.

Eur J Biochem, 2000 Aug, 267(16), 5247 - 56
Functional characterization of green fluorescent protein-profilin fusion proteins; Wittenmayer N et al.; To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful . As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function . We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP . These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose . Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2 . The affinity for actin was quantitatively determined in polymerization assays . Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding . In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP . Binding to PtdIns(4,5)P2 was not significantly altered . Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin . In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor . However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites . Therefore, other constructs should be preferred for further in vivo studies.

Eur J Biochem, 2000 Aug, 267(16), 5156 - 67
Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin; Fleischmann G et al.; A new procedure was devised for reversibly removing the flavin from flavocytochrome b2 . It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P . From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution . The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states . Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction . On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states . From coupling constant measurements it is shown that the N(3) proton is not solvent accessible . Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins . With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure . In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane . In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins . 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

Eur J Biochem, 2000 Aug, 267(16), 5142 - 8
Quantitative analysis of Na+-Ca2+ exchanger expression in guinea-pig heart; McDonald RL et al.; In previous studies, regional variations in the expression of the Na+-Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody {Wang, J., Schwinger, R.H., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E . & McDonough, A.A . (1996) J . Clin . Invest . 98, 1650-1658} . Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue . NCX has been located in the general sarcolemma and t-tubules of ventricular muscle and as t-tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles {Wang et al . (1996)} . To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea-pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and right ventricle . NCX protein expression was 17.5 +/- 3.9 pmol.mg-1 of protein in the left atrium and 29.2 +/- 6.1 pmol.mg-1 of protein in the right atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 +/- 15.2, 76.8 +/- 19.5 and 69.4 +/- 14.1 pmol.mg-1 of protein, respectively, n = 7) . These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions . To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t-tubular location) . We found that the general membrane staining was 79.0 +/- 1.2% in cells from the atria which was significantly higher (P < 0 . 001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 +/- 1.1%, 48.2 +/- 1.8% and 45.6 +/- 1.3%, respectively (n = 20) . These results illustrate a similar pattern of NCX expression in guinea-pig and human, with expression in atrial tissue significantly lower than in ventricular tissue . However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t-tubules).

Clin Exp Immunol, 2000 Aug, 121(2), 364 - 74
Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients withtype-1 autoimmune hepatitis; Costa M et al.; We previously described autoantibodies against a UGA serine tRNA-protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis {1} and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein . The presence of anti-tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47.5% of patients were positive compared with none of the control subjects . To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera . Two clones (19 and 13) were isolated . Clone 19 encodes a protein with a predicted molecular mass of 48.8 kD . Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35.9-kD protein . Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins . Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex . Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec.

Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 920 - 1
Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli CyaY, a structural homologue of human frataxin; Lee MG et al.; CyaY is a 106-residue protein from Escherichia coli . It shows amino-acid sequence similarity to human frataxin and a frataxin homologue in Saccharomyces cerevisiae, Yfh1p . The former is associated with the disease Friedreich ataxia and the latter plays a key role in iron homeostasis in mitochondria . CyaY has been overexpressed in soluble form in E . coli . The recombinant protein with a His(6) tag at its C-terminus has been crystallized at 296 K using polyethylene glycol (PEG) 4000 as a precipitant . Native diffraction data have been collected to 1.8 A using Cu Kalpha X-rays . The crystals belong to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 44.66, c = 99.87 A, alpha = beta = 90.0, gamma = 120.0 degrees . The asymmetric unit contains one molecule of recombinant CyaY, with a corresponding V(m) of 2.13 A(3) Da(-1) and solvent content of 42.3%.

Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 795 - 804
Structure of m-carboxyphenyl-alpha-D-galactopyranoside complexed to heat-labile enterotoxin at 1.3 A resolution: surprising variations in ligand-binding modes; Minke WE et al.; In the quest to develop drugs against traveller's diarrhoea and cholera, the structure of the B pentamer of heat-labile enterotoxin (LT) complexed with a new receptor-binding antagonist, m-carboxyphenyl-alpha-D-galactopyranoside, has been determined . The high resolution obtained for this structure allowed anisotropic refinement of the model . It was also now possible to confirm at a near-atomic resolution the structural similarity between the B subunits of LT and the closely related cholera toxin (CT), including the similarity in deviations of planarity of the same peptide unit in LT and CT . The structure of the LT complex clearly revealed different conformations for the m--carboxyphenyl moiety of the ligand in the five B subunits of LT, while the binding modes of the well defined galactopyranoside moieties were identical . In two binding sites the m-carboxyphenyl moiety displayed no significant electron density, demonstrating significant flexibility of this moiety . In a third binding site the m-carboxyphenyl moiety could be modelled unambiguously into the density . The two remaining binding sites were involved in crystal packing contacts and the density for the ligands in these two binding sites clearly revealed different binding modes, of which one conformation was identical to and one completely different from the conformation of m-carboxyphenyl-galactopyranoside in the third subunit . The multiple binding modes observed in the crystal may represent the ensemble of conformations of m-carboxyphenyl-alpha-D-galactopyranoside complexed to LT in solution.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 239 - 46
Characterisation of a Rrhodobacter sphaeroides gene that encodes a product resembling Eescherichia coli cytochrome b(561) and R . sphaeroides cytochrome b(562); Duggan PS et al.; Analysis of the photoactive yellow protein (pyp) gene region of Rhodobacter sphaeroides has revealed the presence of an additional open reading frame, orfD, that had not previously been identified . Here we report the location of this new gene and the predicted amino acid sequence of the encoded protein . The translation product resembles a group of small cytochrome b-like proteins, including Escherichia coli cytochrome b(561), R . sphaeroides cytochrome b(562), and two new cytochrome b(561)-like proteins identified using the E . coli genome sequence, for which functions have not yet been established . To determine OrfD function in R . sphaeroides, an orfD mutant was constructed . The OrfD mutant exhibited growth rates and yields very similar to those of the wild-type strain when grown under a variety of growth conditions . Respiration rates, reduced-minus-oxidised spectra and levels of photosynthetic complexes were also very similar in the two strains . Although the role of OrfD was therefore not determined here, we demonstrate that the orfD gene is expressed in R . sphaeroides under aerobic, semi-aerobic and photosynthetic growth conditions.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 195 - 200
Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue; Guo K et al.; The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis . Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate . Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase . After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal . Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene . Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 171 - 5
An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003; Du L et al.; We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003 . We report here the identification and characterization of an oxidation domain in BlmIII . The oxidation domain shows local homology to a family of oxidoreductases and is present in all thiazole-forming non-ribosomal peptide synthetase modules known to date . Both the blmIII-Ox domain and blmIII gene were expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity . The oxidation domain contains one molar equivalent of non-covalently bound FMN as a prosthetic group . These results provide experimental evidence for an oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in bleomycin biosynthesis.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 159 - 64
Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli; Toida J et al.; Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively) . We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes . Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp . The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi . Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved . The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.

FEBS Lett, 2000 Aug 4, 478(3), 299 - 303
A putative binding protein for lipophilic substances related to butterfly oviposition; Tsuchihara K et al.; A unique protein of 23 kDa (Jf23) was found in the tarsus of the female swallowtail butterfly, Atrophaneura alcinous . Jf23 has 38% identity with a bilin-binding protein, which was found in the cabbage butterfly, Pieris brassicae, and which has two consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands . Western blot analysis showed that Jf23 was expressed only in the female, and not in the male . Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchman's pipe (Aristolochia debilis) . The stimulated response was depressed by the presence of Jf23 antiserum . These results suggest that Jf23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition.

FEBS Lett, 2000 Aug 4, 478(3), 271 - 5
Evidence for an active role of the DnaK chaperone system in the degradation of sigma(32); Tatsuta T et al.; Under non-stressed conditions in Escherichia coli, the heat shock transcription factor sigma(32) is rapidly degraded by the AAA protease FtsH . The DnaK chaperone system is also required for the rapid turnover of sigma(32) in the cell . It has been hypothesized that the DnaK chaperone system facilitates the degradation of sigma(32) by sequestering it from RNA polymerase core . This hypothesis predicts that mutant sigma(32) proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system . We examined the in vivo stability of such mutant sigma(32) proteins . Results indicated that the mutant sigma(32) proteins as similar as authentic sigma(32) were stabilized in DeltadnaK and DeltadnaJ/DeltacbpA cells . The interaction between sigma(32) and DnaK/DnaJ/GrpE was not affected by these mutations . These results strongly suggest that the degradation of sigma(32) requires an unidentified active role of the DnaK chaperone system.

Mol Biol Cell, 2000 Aug, 11(8), 2733 - 41
Nuclear dynamics in Arabidopsis thaliana; Chytilova E et al.; The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments . Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments . To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions . This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to beta-glucuronidase (GUS) from E . coli . This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm . This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement . Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes . The GFP-based assay is simple and of general applicability . It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

J Biol Chem, 2000 Jul 28, 275(30), 22743 - 9
The N-terminal sequence (residues 1-65) is essential for dimerization, activities, and peptide binding of Escherichia coli DsbC; Sun XX et al.; Limited proteolysis of DsbC with trypsin resulted in a compact and stable C-terminal fragment (residues 66-216), fDsbC, which retains the active site sequence, -Cys(98)-Gly-Tyr-Cys(101)-, and shows only minor differences in conformation compared with that of the intact molecule . The pK(a) of active site thiol and the K(SS) with glutathione are very close to that of DsbC, respectively; however, fDsbC is inactive as an isomerase in catalyzing the formation of correct disulfide bonds in scrambled RNase A and denatured and reduced bovine pancreatic trypsin inhibitor and shows only 13% thiol-protein oxidoreductase activity (TPOR) of DsbC . In contrast to the dimeric DsbC, fDsbC exists as a monomer and has no chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase . The heterodimer of DsbC with the inactive DsbC carboxymethylated at both active site thiols shows about 50% TPOR activity of DsbC but no isomerase activity, indicating that the DsbC subunit in the heterodimer displays full TPOR activity but little, if any, isomerase activity . It is concluded that the N-terminal sequence (residues 1-65) is essential for dimer formation and chaperone activity of DsbC . The active sites in both subunits of the dimeric DsbC appear to be essential for its isomerase activity.

J Biol Chem, 2000 Oct 27, 275(43), 33346 - 52
Inhibition of six serine proteinases of the human coagulation system by mutants of bovine pancreatic trypsin inhibitor; Grzesiak A et al.; A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P(4) and P(3) positions of the canonical binding loop containing additional K15R and M52L mutations were used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a), thrombin, and protein C . The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding . The mutants inhibited the blood-clotting proteinases with association constants in the range of 10(3)-10(10) m(-)(1) . Inhibition of plasma kallikrein, factors X(a) and XII(a), thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution . The highest increase in the association constant for P(3) mutant was measured for factor XII(a); P13S substitution increased the K(a) value 58-fold . Several other substitutions at P(3) resulted in about 10-fold increase for factor X(a), thrombin, and protein C . The cumulative P(3) and P(1) effects on K(a) values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2.2- (plasmin) to 4,000-fold (factors XII(a) and X(a)) . The substitutions at the P(4) site always caused negative effects (a decrease in the range from over 1,000- to 1.3-fold) on binding to all studied enzymes, including trypsin . Thermal stability studies showed a very large decrease of the denaturation temperature (about 22 degrees C) for all P(4) mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.

Am J Hum Genet, 2000 Sep, 67(3), 549 - 62 Epub 2000 Aug 04.
Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia; Adamec J et al.; Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease . Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes . Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress . Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA . These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals . The functional mechanism of frataxin, however, is still unknown . We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro . Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate . Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1 . 1 MDa (megadaltons) and a diameter of 13+/-2 nm . Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron . Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly . Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form . In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000 . After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p . We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

Cytokine, 2000 Aug, 12(8), 1211 - 7
The production and biological assessment of cervine interferon gamma; Slobbe L et al.; Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Escherichia coli expression system pET-32 . The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to the IFN-gamma molecule . The ability of IFN-gamma to inhibit the killing of Madin-Darby bovine kidney cells by Semliki forest virus was used as a measure of the bioactivity of the recombinant cervine IFN-gamma (rIFN-gamma) . It was shown that the presence of the thioredoxin fusion partner 5' to the IFN-gamma molecule did not affect its biological activity . As in the mouse model, it was shown that cervine rIFN-gamma was able to down-regulate the transcription of interleukin 10 mRNA while up-regulating the transcription of interleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood mononuclear cells . A prototype ELISA was tested for its ability to detect both recombinant and native IFN-gamma . The ELISA was able to detect rIFN-gamma at concentrations greater than 100 pg/ml . It was also used to detect native IFN-gamma produced by peripheral blood lymphocytes from Mycobacterium bovis infected or vaccinated deer after in vitro restimulation with antigen . The rIFN-gamma and the cervine IFN-gamma specific ELISA provide valuable tools with which to study important zoonotic infections in farmed and wild deer .

Can J Anaesth, 2000 Jul, 47(7), 673 - 9
Milrinone improves intestinal villus blood flow during endotoxemia; Schmidt W et al.; PURPOSE: To determine whether the compromised intestinal villus blood flow in a rat model of endotoxemia could be improved by continuous infusion of the phosphodiesterase (PDE) inhibitor milrinone . METHODS: Twenty-four anesthetized and ventilated rats were laparotomized and an ileal portion was exteriorized and opened by an antimesenteric incision . The ileal segment was fixed with the mucosal surface upward . Microcirculatory parameters were assessed by intravital videomicroscopy . The animals were randomly assigned to receive one of three treatments: infusion of Escherichia coli lipopolysaccharides without phosphodiesterase inhibitor pretreatment (=LPS group); or infusion of LPS with milrinone pretreatment (= milrinone group), or without infusion of LPS or milrinone (=control group) . Macrohemodynamic parameters (MAP, HR) and microhemodynamic parameters of ileal mucosa (mean diameter of central arterioles = D(A) and mean erythrocyte velocity within the arterioles= V(E)) were measured 30 min before and at 0, 60, and 120 min after induction of endotoxemia . Mucosal villus blood flow was calculated from D(A) and V(E) . RESULTS: In the milrinone group MAP decreased 60 min after induction of endotoxemia whereas it remained stable in the control and the LPS group . In both groups given endotoxin V(E) decreased after start of LPS infusion . In contrast, D(A) decreased in the LPS group, but increased in the milrinone group after 120 min of endotoxemia . Thus, the endotoxin-induced decrease of intestinal villus blood flow was diminished but not fully restored by milrinone infusion . CONCLUSION: Our results indicate that milrinone has some beneficial microcirculatory effects during endotoxemia . Although it contributed to systemic hypotension, it attenuated intestinal mucosal hypoperfusion.

J Hypertens, 2000 Jul, 18(7), 901 - 9
Does hypertension confer a hypercoagulable state in stroke-prone spontaneously hypertensive rats?
Abumiya T, Sakata T, Enjyoji K, Kato H, Kawai J, Suzuki T, Masuda J, Sasaguri T, Ogata J.
OBJECTIVE: To verify whether hypertension confers a hypercoagulable state in a hypertensive animal model . DESIGN: The parameters of blood coagulation were compared between stroke-prone spontaneously hypertensive rats (SHR-SP) and Wistar-Kyoto (WKY) rats . Each rat group consisted of a younger subgroup at 8-12 weeks old (n = 12) and an older subgroup at 16-20 weeks old (n = 12) . METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fluorogenic PT, fibrinogen, fibrin/fibrinogen degradation products (FDP), thrombin-anti-thrombin III complex (TAT), factor Xa activity, anti-thrombin III (AT-III), tissue factor pathway inhibitor (TFPI), protein C and C1 inhibitor were measured in both rat groups . RESULTS: There was no significant difference in FDP and TAT levels between SHR-SP and WKY rats even at 16-20 weeks when SHR-SP developed severe hypertensive vascular lesions . Contrary to expectations, fluorogenic PT and factor Xa activity were significantly lower in SHR-SP than in WKY rats . While there was no significant difference in AT-III, TFPI and protein C activities between SHR-SP and WKY rats, C1 inhibitor activity was significantly higher in SHR-SP than in WKY rats . The elevated C1 inhibitor activity was inversely correlated with the reduced factor Xa activity . Gel-filtered fractionated plasma with C1 inhibitor activity had an inhibitory effect on the purified rat factor Xa, and immunodepletion of C1 inhibitor from the fractionated plasma attenuated the inhibitory effect CONCLUSION: These results suggest that SHR-SP get into a hypocoagulable state rather than a hypercoagulable state, and that the reduction of factor Xa activity in SHR-SP may be related to the elevation of C1 inhibitor activity.

Toxicol Pathol, 2000 Jul-Aug, 28(4), 580 - 7
Effects of antioxidants apocynin and the natural water-soluble antioxidant from spinach on cellular damage induced by lipopolysaccaride in the rat; Lomnitski L et al.; Oxidative damage plays a key role in septic shock induced by the endotoxin lipopolysaccaride (LPS) by enhancing the formation of reactive oxygen species such as superoxide anion radicals, peroxides, and their secondary product, malondialdehyde, especially in the liver . In this study, histopathologic changes in several organs were compared among groups of male Wistar rats that had been injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group that had been injected with LPS without pretreatment with antioxidants, an untreated control group, and groups that had been injected with either of the 2 antioxidants only . The antioxidants used were a water-soluble natural antioxidant from spinach (NAO) and the NADPH oxidase inhibitor apocynin . Hematoxylin-and-eosin-stained slides were prepared, and lesions were semiquantitatively scored . Exposure to LPS alone was associated with multifocal hepatocellular necrosis and acute inflammation, thymic and splenic lymphoid necrosis, ocular retinal hemorrhage and acute endophthalmitis, adrenal medullary vacuolation and necrosis and acute inflammation, and decreased adrenal cortical cytoplasmic vacuolation (consistent with depletion of steroidal hormone contents) . Results indicated that pretreatment with both antioxidants for 8 days reduced, in some organs, the necrotic and inflammatory changes associated with the LPS challenge . These findings suggest a potential therapeutic application for these antioxidants in clinical sepsis.

Plant Cell Physiol, 2000 May, 41(5), 644 - 8
Action spectra of DNA photolyases for photorepair of cyclobutane pyrimidine dimers in sorghum and cucumber; Hada M et al.; DNA photolyases that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs) were extracted and partially purified from sorghum and cucumber . The action spectra of CPD photolyases in these plant species had a maximum at 400 nm, which differ from those in Drosophila, Escherichia coli and Anacystis.

Plant Cell Physiol, 2000 May, 41(5), 627 - 38
Vascular tissue-specific gene expression of xylem sap glycine-rich proteins in root and their localization in the walls of metaxylem vessels in cucumber; Sakuta C et al.; Root-specific cDNAs of glycine-rich protein (cucumber root glycine rich protein-1 and -2; CRGRP-1 and CRGRP-2) were cloned previously by use of an antiserum raised against whole xylem sap of Cucumis sativus . The accumulation of the corresponding mRNA at high levels was detected in the root-hair zone of cucumber tap root {Sakuta et al . (1998) Plant Cell Physiol . 39: 1330} . The RNA gel blot analysis with the CRGRP-1- and -2-specific probes revealed that the CRGRP genes expressed only in root but not at all in aboveground organs . When the localization of these mRNAs were examined by in situ hybridization, CRGRP mRNAs were found only in the parenchyma cells in the central cylinder of young lateral roots and it was most abundant in the cells that surrounded xylem vessels in the root-hair zone of the tap root . In immunoblotting of xylem sap collected from cucumber stem with an antiserum raised against CRGRP-1 that had been produced in an E . coli expression system, the antibodies, which did not cross-react with GRP1.8 of kidney bean, reacted with two proteins, whose mobilities corresponded to those of proteins deduced from the CRGRP-1 and -2 cDNAs . Immunohistochemical staining revealed that the CRGRPs accumulated specifically in the lignified walls of metaxylem vessels in the root, stem and leaf and in the lignified cell walls of perivascular fibers in cucumber stems . Immunostaining was also detected in the walls of metaxylem vessels and in the cell walls of adjacent sclerenchyma in the hypocotyl of kidney bean . These data clearly indicate that the novel glycine-rich proteins were produced in the vascular tissue of the root, transported systemically over a long distance via the xylem sap and immobilized in the walls of metaxylem vessels and sclerechyma cells in aboveground organs.

Plant Cell Physiol, 2000 May, 41(5), 617 - 26
Characterization of the arabidopsis formin-like protein AFH1 and its interacting protein; Banno H et al.; A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA . The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins . In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmembrane domain suggesting its association with membrane . Cell fractionation by differential centrifugation demonstrated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction . An Arabidopsis cDNA library was screened to identify proteins that interact with the C-terminal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2 . Experiments using recombinant proteins expressed in E . coli demonstrated that FIP2 interacted directly with AFH1 . The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases . AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton.

Nucleic Acids Res . 2000 Aug 15;28(16):E76.
Efficient purification of DNA fragments using a protein binding membrane; Ihle O et al.; A novel and efficient method has been developed for isolation of correctly digested DNA fragments without the use of classic size-dependent electrophoretic separation methods . To achieve this, DNA fragments are end-labelled by haptens . After specific endonuclease digestion of the hapten-labelled DNA, the DNA is incubated with a protein that specifically binds to the hapten . The incubation mixture is then passed through a cartridge containing a protein-binding membrane that does not bind DNA . Undigested and partly digested DNA are retained on the membrane, while correctly digested DNA is selectively recovered for use in further downstream applications.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3178 - 84
The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli; Pan X et al.; A 24 triplet TGG.CCA repeat array shows length- and orientation-dependent propagation when present in the plasmid pUC18 . When TGG(24) is present as template for leading-strand synthesis, plasmid recovery is normal in all strains tested . However, when it acts as template for lagging-strand synthesis, plasmid propagation is seriously compromised . Plasmids carrying deletions in the 5' side of this sequence can be isolated and products carrying 15 TGG triplets do not significantly interfere with plasmid propagation . Mutations in sbcCD, mutS and recA significantly improve the recovery of plasmids with TGG(24) on the lagging-strand template . These findings suggest that TGG(24) can fold into a structure that can interfere with DNA replication in vivo but that TGG(15) cannot . Furthermore, since the presence of the MutS and SbcCD proteins are required for propagation interference, it is likely that stabilisation of mismatched base pairs and secondary structure cleavage are implicated . In contrast, there is no correlation of triplet repeat expansion and deletion instability with predicted DNA folding . These results argue for a dissociation of the factors affecting DNA fragility from those affecting trinucleotide repeat expansion-contraction instability.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3117 - 24
In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase; Nozawa K et al.; Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis . A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat . In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions . Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer . It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis . Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA . The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length . Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion . On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli . The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3105 - 16
Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate; Hsu AW et al.; RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein) . M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells . In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping . In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3' immediately to the cleavage site were similar to those found in the absence of C5 . Meanwhile, some of the ribozyme regions (e.g . P12 and J11/12) that were crosslinked to the leader sequence 5' immediately to the cleavage site in the presence of C5 were different from those regions (e.g . P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA . Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3083 - 91
DNA bending induced by DNA (cytosine-5) methyltransferases; Rasko T et al.; DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay . The following bend angles were obtained: M.BSP:RI (GG(m5)CC), 46-50 degrees; M.HAE:III (GG(m5)CC), 40-43 degrees; M.SIN:I (GGW(m5)CC), 34-37 degrees; M.SAU:96I (GGN(m5)CC), 52-57 degrees; M.HPA:II (C(m5)CGG), 30 degrees; and M.HHA:I (G(m5)CGC), 13 degrees . M . HAE:III was also tested with fragments carrying a methylated binding site, and it was found to induce a 32 degrees bend . A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HAE:III and M . BSP:RI bend DNA toward the minor groove . The DNA curvature induced by M.HAE:III contrasts with the lack of DNA bend observed for a covalent M.HAE:III-DNA complex in an earlier X-ray study . Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3' to the target cytosine tend to induce greater bends than enzymes with guanine in this position . We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.

Br J Pharmacol, 2000 Aug, 130(7), 1531 - 8
Cytotoxicity associated with induction of nitric oxide synthase in rat duodenal epithelial cells in vivo by lipopolysaccharide of Helicobacter pylori: inhibition by superoxide dismutase; Lamarque D et al.; The products released by Helicobacter pylori (H . pylori) in the gastric antral and duodenal mucosa may be involved in mucosal ulceration by stimulating the local formation of cytotoxic factors such as nitric oxide (NO), superoxide or peroxynitrite . The present study investigates the ability of purified H . pylori lipopolysaccharide (LPS) to induce nitric oxide synthase (iNOS) in rat duodenal epithelial cells following in vivo challenge and its interaction with superoxide in promoting cellular damage and apoptosis . H . pylori LPS (0.75-3 mg kg(-1) i.v . or 3-12 mg kg(-1) p.o.) induced a dose - dependent expression of iNOS activity after 5 h in the duodenal epithelial cells, determined by {(14)C} arginine conversion to citrulline . The epithelial cell viability, as assessed by Trypan Blue exclusion and MTT conversion, was reduced 5 h after challenge with H . pylori LPS, while the incidence of apoptosis was increased . The iNOS activity and reduction in cell viability following H . pylori LPS challenge i.v . was inhibited by the selective iNOS inhibitor, 1400 W (0.2-5 mg kg(-1) i.v.) . Concurrent administration of superoxide dismutase conjugated with polyethylene glycol (250 - 500 i.u . kg(-1), i.v.), which did not modify the cellular iNOS activity, reduced the epithelial cell damage provoked by i.v . H . pylori LPS, and abolished the increased incidence of apoptosis . These results suggest that expression of iNOS following challenge with H . pylori LPS provokes duodenal epithelial cell injury and apoptosis by a process involving superoxide, implicating peroxynitrite involvement . These events may contribute to the pathogenic mechanisms of H . pylori in promoting peptic ulcer disease.

Z Naturforsch {C}, 2000 May-Jun, 55(5-6), 410 - 2
Instability of three-dimensional structures in ribosomal cores evidenced by microcalorimetric studies; Bonincontro A et al.; In this paper we show a microcalorimetric investigation carried out on the so-called cores, i.e . ribosomes deprived of select proteins by LiCl treatment . Thermal degradation of native ribosomes gives rise to two thermal transitions occurring at different temperatures . In the cores the high temperature peak persists even after treatment at very high ion strength (2 M LiCl) . This strongly suggests the existence of a very stable structure that was previously observed also in particles treated with agents that hydrolyze the RNA moiety . The low temperature peak gradually but dramatically decreases even though it never disappears completely . This indicates that the treatment to obtain ribosomal cores does not cause complete unfolding of the particle but only the destabilization of a structural three-dimensional domain present in native ribosomes . These data are discussed in the light of previous results obtained by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.

Thromb Haemost, 2000 Jul, 84(1), 71 - 7
Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha; Arza B et al.; The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis . MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E . coli and purified to apparent homogeneity . Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha . Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha . Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1) . In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen . In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin . Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.

Circ Res, 2000 Aug 4, 87(3), 254 - 60
beta(2)-Integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice; Xu N et al.; Interaction of CD11/CD18 beta(2) integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier . Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (Ancylostoma caninum), binds to the I domain of CD11a and CD11b and inhibits beta(2) integrin-dependent PMN adhesion . We describe a novel strategy using the endothelial cell-specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells . A construct containing NIF cDNA driven by the inducible endothelial cell-specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs) . Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs . NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression . To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct . Analysis of lung tissue showed that intraperitoneal challenge of Escherichia coli resulted in NIF expression . Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by E coli challenge . These studies suggest the feasibility of conditionally blocking beta(2) integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.

Biochem J, 2000 Aug 15, 350 Pt 1, 69 - 73
Mutagenesis studies on the sensitivity of Escherichia coli acetohydroxyacid synthase II to herbicides and valine; Lee YT et al.; Acetohydroxyacid synthase (EC 4.1.3.18, also known as acetolactate synthase) isoenzyme II from Escherichia coli is inhibited by sulphonylurea and imidazolinone herbicides, although it is much less sensitive than the plant enzyme . This isoenzyme is also unusual in that it is not inhibited by valine . Mutating S100 (Ser(100) in one-letter amino acid notation) of the catalytic subunit to proline increases its sensitivity to sulphonylureas, but not to imidazolinones . Mutating P536 to serine, as found in the plant enzyme, had little effect on the properties of the enzyme . Mutating E14 of the regulatory subunit to glycine, either alone or in combination with the H29N (His(29)-->Asn) change, did not affect valine-sensitivity.

J Membr Biol, 2000 Jul 15, 176(2), 159 - 68
The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli . Analysis of suppressor mutations; Cain SM et al.; The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9 . In three previous studies (Jessen-Marshall, A.E., & Brooker, R.J . 1996 . J . Biol . Chem . 271:1400-1404; Jessen-Marshall, A.E., Parker, N., & Brooker, R.J . 1997 . J . Bacteriol . 179:2616-2622; and Pazdernik, N., Cain, S.M., & Brooker, R.J . 1997 . J . Biol . Chem . 272:26110-26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif . In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif . The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both . The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4% lactose transport activity . Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation . Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9 . In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease . From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V(max) values for lactose transport compared with the single G64S and P280L strains . These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface.

J Mol Biol, 2000 Aug 11, 301(2), 389 - 99
Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis; Wagner T et al.; The crystal structure of the phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex with Mn(2+) and the substrate analog, 2-phosphoglycolate (PGL), was determined by molecular replacement using X-ray diffraction data to 2.0 A resolution . DAHPS*Mn*PGL crystallizes in space group C2 (a=210.4 A, b=53.2 A, c=149.4 A, beta=116.1 degrees ) with its four (beta/alpha)(8) barrel subunits related by non-crystallographic 222 symmetry . The refinement was carried out without non-crystallographic symmetry restraints and yielded agreement factors of R=20.9 % and R(free)=23.9 % . Mn(2+), the most efficient metal activator, is coordinated by the same four side-chains (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb(2+) . A fifth ligand is a well-defined water molecule, which is within hydrogen bonding distance to an essential lysine residue (Lys97) . The distorted octahedral coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site . However, unlike PEP in the Pb*PEP complex, PGL binds the Mn(2+) via one of its carboxylate oxygen atoms . A model of the active site is discussed in which PEP binds in the same orientation as does PGL in the DAHPS*Mn*PGL structure and the phosphate of E4P is tethered at the site of a bound sulfate anion . The re face of E4P can be positioned to interact with the si face of PEP with only small movement of the protein .

J Mol Biol, 2000 Aug 11, 301(2), 265 - 83
Function of tyrosyl-tRNA synthetase in splicing group I introns: an induced-fit model for binding to the P4-P6 domain based on analysis of mutations at the junction of the P4-P6 stacked helices; Chen X et al.; We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core . We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants . Most mutations at either position inhibit self-splicing, but can be suppressed by CYT-18 . CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking, based on the analysis of DNA four-way junctions . However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking . Thermal denaturation and Fe(II)-EDTA analysis showed that mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain . CYT-18-suppressible mutants bind the protein with K(d) values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k(off) value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K(d) value reflects primarily an increased k(on) value for the binding of CYT-18 to the misfolded intron . Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core .

J Mol Biol, 2000 Aug 11, 301(2), 233 - 8
3-Deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli . Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate; Wagner T et al.; The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli was determined by molecular replacement using coordinates given to us by Radaev and co-workers prior to publication . The KDOPS crystals reported by Radaev et al . were grown in the presence of 1.4 M (NH(4))(2)SO(4) and 0.4 M (K/H)(3)PO(4) . They are in the cubic space group I23 (a=228.6 A) with a tetramer in the asymmetric unit; the structure has been refined with data to 2.4 A . Our crystals of E . coli KDOPS, grown in 24 % (w/v) polyethylene glycol (PEG) 1500 in the presence of the substrates, 2-phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P), are also in space group I23 (a=118.2 A), with one subunit in the asymmetric unit.The medium of crystallization, 1.8 M SO(4)/PO(4) versus 24 % PEG, does not significantly affect the conformation of KDOPS . The inter-monomer contacts in both structures are the same . The beta(8)/alpha(8) loop (residues 246 to 251) situated near the entrance to the active site is not seen in the 229 A structure but can be traced in the 118 A structure.Most significantly, Radaev et al . interpreted two SO(4)/PO(4) sites in the 229 A structure as marking the phosphate positions of the substrates, PEP and A5P, after the precedent of DAHPS . In the 118 A structure the inner of these two SO(4)/PO(4) peaks is present at the same position as in the 229 A structure of KDOPS . The outer phosphate peak in the 118 A KDOPS is 3.7 A from the outer SO(4)/PO(4) peak in the 229 A structure and is within hydrogen bonding distance of Arg63 of the same subunit and Arg120 of another subunit . Based on the precedent of the d-erythrose-4-phosphate (E4P) modeled in the active site of DAHPS, we have modeled PEP and A5P in KDOPS and compared the coordination of PEP and A5P in KDOPS with that of PEP and E4P in DAHPS .

J Mol Biol, 2000 Aug 4, 301(1), 219 - 27
On the role of the N-terminal group in the allosteric function of glucosamine-6-phosphate deaminase from Escherichia coli; Lara-Gonzalez S et al.; Glucosamine-6-phosphate deaminase (EC 3.5.99.6) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosphate . It is a homohexamer and has six allosteric sites located in clefts between the subunits . The amino acid side-chains in the allosteric site involved in phosphate binding are Arg158, Lys160 and Ser151 from one subunit and the N-terminal amino group from the facing polypeptide chain . To study the functional role of the terminal amino group, we utilized a specific non-enzymic transamination reaction, and we further reduced the product with borohydride, to obtain the corresponding enzyme with a terminal hydroxy group . Several experimental controls were performed to assess the procedure, including reconditioning of the enzyme samples by refolding chromatography . Allosteric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein . Its kinetic study suggests that the allosteric equilibrium for this modified enzyme is displaced to the R state, with the consequent loss of co-operativity . The deaminase with a terminal hydroxy acid, obtained by reducing the transaminated enzyme, showed significant recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme . It had lost, however, the pH-dependence of homotropic co-operativity shown by the unmodified deaminase in the pH range 6-8 . These results show that the terminal amino group plays a part in the co-operativity of the enzyme and, more importantly, indicate that the loss of this co- operativity at low pH is due to the hydronation of this amino group .

J Mol Biol, 2000 Aug 4, 301(1), 61 - 73
Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus; Pisani FM et al.; Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC) . SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively . The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio . The small subunit was individually expressed in E . coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized . The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S . solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S . solfataricus (039p and 048p) . Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT) . As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity . The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed .

Exp Mol Med, 2000 Jun 30, 32(2), 72 - 8
The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes; Park EJ et al.; Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens . Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes . Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT . LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization . LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization . In addition, quantitative H . pylori culture of stomach tissue following challenge with H . pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant . These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H . pylori urease may differ between the IN and IG mucosal immunization routes.

Zh Mikrobiol Epidemiol Immunobiol, 2000 May-Jun, (3), 92 - 9
{The ecological and medical aspects of the symbiosis between Escherichia coli and man}; Gritsenko VA et al.; In this work different variants of the symbiosis of E . coli with a human body are analyzed, and the symbiotic relationships between them are shown to follow the type mutualism, commensalism, parasitism and habitation . The authors emphasize that the multiplicity of variants of bacteria-host relationships is based on the phenotypic polymorphism of E . coli clones (clone lines) . Taking into account their ecological (symbiotic) features and biomedical importance, all E . coli clones are divided into 4 groups (clusters): mutualists as nonpathogenic organisms; commensals as potential pathogens (causing extraintestinal E . coli infections); parasites as real pathogens (causing acute intestinal infections); "occasional" symbionts of man . The proposition on the cluster structure of E . coli as a species is formulated.

Zh Mikrobiol Epidemiol Immunobiol, 2000 May-Jun, (3), 71 - 3
{Changes in the cell size of Escherichia coli M-17 during induced destruction}; Akaizin ES; The autolysis of E . coli, induced by their deprivation of nutrition in combination with the action of oleic acid and a temperature of 45 degrees C, was studied . The study revealed that an increase in the number of cells during 4 hours after the induction of the process was accompanied by a decrease in their size and an increase in the surface/volume ratio . Changes in the size of bacteria in the course of induced destruction resulting from their deprivation of nutrition in combination the action of oleic acid and a temperature of 45 degrees C were found to occur in two phases: (1) a decrease in size with an increase in the surface/volume ratio; (2) an increase in size with a decrease in the surface/volume ratio.

Res Rep Health Eff Inst, 2000 Mar, (92), 49 - 87; discussion 141-9
1,3-butadiene: cancer, mutations, and adducts . Part II: Roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity; Recio L et al.; 1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being more susceptible than rats to its carcinogenic effects . 1,3-Butadiene is mutagenic in the bone marrow and spleen cells of B6C3F1 lacI transgenic mice . The goal of this research was to assess the roles of two BD metabolites, 1,2-epoxy-3-butene (BDO) and 1,2,3,4-diepoxybutane (BDO2), in the mutagenicity and mutational spectrum of the parent compound BD by determining the mutagenicity and mutational spectra of BDO and BDO2 in human and rodent cells in vitro and in vivo . In human TK6 lymphoblastoid cells (TK6 cells), BDO exposure increased the frequency of G.C-->A.T transitions and A.T-->T.A transversions (Fisher exact test; p < 0.05) . The most striking difference in the type of base-substitution mutations between BDO-exposed and BDO-unexposed TK6 cells was the 19-fold increase in A.T-->T.A transversions . 1,2,3,4-Diepoxybutane increased the frequency of A.T-->T.A transversions (Fisher exact test; p < 0.05) and the frequency of deletions in exposed TK6 cells compared with unexposed controls . Exposure of Rat2 lacI transgenic fibroblasts (Rat2 cells) to BDO increased the frequency of three types of base-substitution mutations: G.C-->A.T transitions, G.C-->T.A transversions, and A.T-->T.A transversions . Exposure of Rat2 cells to BDO2-induced dose-dependent increases in micronuclei at exposure levels that apparently did not induce mutagenicity at the lacI transgene . The lack of detectable mutagenicity at the lacI transgene in Rat2 cells exposed to BDO2 probably reflects the poor recovery of large deletions by this lambda phage-based mutagenicity assay . Inhalation exposure of B6C3F1 lacI transgenic mice (lacI mice) and F344 lacI transgenic rats (lacI rats) to BDO (29.9 parts per million {ppm}; 6 hours/day; 5 days/week for 2 weeks) did not increase the lacI mutant frequency (MF) in bone marrow or spleen cells of mice and rats, but in the cells of mouse lung (a tumor target organ for BD), significant mutagenicity was observed . An increased lacI MF was also observed in the bone marrow cells of rats exposed to BDO . Inhalation exposure of lacI mice and lacI rats to BDO2 (3.8 ppm; 6 hours/day; 5 days/week for 2 weeks) did not increase the lacI MF in bone marrow or spleen cells of mice or in the spleen cells of rats . An increased lacI MF was observed in the bone marrow cells of rats exposed to BDO2 . In the present study, BDO specifically induced G.C-->A.T and A.T-->T.A transversions in vitro at both the endogenous hypoxanthine phosphoribosyltransferase (hprt) gene and the lacI transgene in Rat2 cells . It also induced an increased frequency of G.C-->T.A transversions in Rat2 cells . These types of mutations also occur at an increased frequency in mice exposed to the parent compound, BD . This finding demonstrates the induction of consistent mutational types across biological systems by BDO and indicates that BDO, but not BDO2, probably has a role in mediating the mutations recovered at the lacI transgene in animals exposed to the parent compound, BD . Therefore, it is apparent that in mice exposed to BD at carcinogenic levels, BDO and BDO2 act in concert to mediate the range of genotoxic responses . These data demonstrate that certain DNA adducts (guanine or adenine) may be useful biomarkers for BD genetic effects . However, other DNA lesions that can account for BDO2-induced deletions and chromosomal alterations also need to be considered as biomarkers for BD-induced genotoxicity.

Somat Cell Mol Genet, 1999 Jan, 25(1), 49 - 57
Introduction of plasmid DNA and oligonucleotides into lung epithelial cells by the hemagglutinating virus of Japan (HVJ)-liposome method; Yoshida M et al.; The hemagglutinating virus of Japan (HVJ) fused with liposomes provides a unique transfection vehicle with characteristics of both virus vector and liposome . Here we investigate the efficiency and safety of the HVJ-liposome technique in delivering foreign genes and oligonucleotides into the lung of the Wistar rat . A plasmid vector containing the Escherichia coli beta-galactosidase (beta-gal) gene and the chicken beta-actin promoter was transfected via the trachea using the HVJ-liposome method . Cytochemical staining showed expression of exogenous beta-gal activity in airway epithelial cells, alveolar macrophages, and alveolar type II cells . This activity persisted at least 28 days after administration of the genes . FITC-labeled oligonucleotides also were introduced into the same types of lung cells as those expressing beta-gal . After instillation of HVJ-liposome, anti-HVJ antibodies were detected in the sera of the rats, but even after repeated administration of HVJ-liposome, no marked histopathologic change was observed while exogenous beta-gal expression was detected in pulmonary cells.

Gene, 2000 Jul 25, 253(1), 67 - 75
Construction and application of mycobacterial reporter transposons; Machowski EE et al.; The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions . A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions . Mutants containing lacZ produced varying intensities of blue colour on indicator media . This reporter activity could be used as a quantitative measure of promoter strength . Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions . Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin . This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria.

Vet Microbiol, 2000 Sep 15, 76(1), 51 - 9
Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil; Martins MF et al.; Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes . These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41 . Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones . The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones . Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources . Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.

Vet Microbiol, 2000 Sep 15, 76(1), 41 - 9
Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC); Amorim CR et al.; The adhesin F18ac purified on Sepharose CL 4B column chromatography and SDS-PAGE stained with Coomassie Blue and Western blotting using specific anti-F18ac serum presented one band of approximately 17kDa . Gold immunolabeling revealed that the adhesin F18ac has a fimbrial structure on the bacterial surface . The first 27 amino acid residues of the N-terminal portion of the adhesin F18ac, showed 92.5% homology (25 amino acids) with the F107 (F18ab) fimbriae.

Biochim Biophys Acta, 2000 Jul 20, 1459(1), 49 - 60
Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis; Schnick C et al.; When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop . In the plasma membrane of Escherichia coli, however, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F1 domain . The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations . Residues 12-24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain . Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays . The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric complex . Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulations . Different stoichiometries and monomer orientations were applied in these calculations . A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data.

J Biol Chem, 2000 Nov 3, 275(44), 34609 - 18
The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli; Leu FP et al.; In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA . When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta . DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments . The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand . Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)) . Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading . Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E . coli cells reveals an excess of delta, free from gamma complex and pol III* . Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.

J Biol Chem, 2000 Oct 13, 275(41), 32187 - 92
Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1) . Impact on oligomerization, stability, and kinetic properties; Berenstein D et al.; Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization . Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106 . The TK1 sequence in NCBI GenBank(TM) has methionine at this position . The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively) . Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure . Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively) . Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.

J Biol Chem, 2000 Nov 10, 275(45), 35185 - 91
The metabotropic GABAB receptor directly interacts with the activating transcription factor 4; Nehring RB et al.; G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences . In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element . Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family . As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes . Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo . In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays . Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.

Biochemistry, 2000 Aug 8, 39(31), 9604 - 11
Recombinant antitoxic and antiinflammatory factor from the nonvenomous snake Python reticulatus: phospholipase A2 inhibition and venom neutralizing potential; Thwin MM et al.; From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics . A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) . It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein . PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure . PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance . The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics . Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa . PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin . It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice . Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

Biochemistry, 2000 Aug 8, 39(31), 9583 - 90
New probes of the F1-ATPase catalytic transition state reveal that two of the three catalytic sites can assume a transition state conformation simultaneously; Nadanaciva S et al.; MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F(1)-ATPase . The F(1).MgADP.ScFx complex mimics a catalytic transition state . Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3 . These results indicate that sites 1 and 2 may form a transition state conformation . A new direct optical probe of F(1)-ATPase catalytic transition state conformation is also reported, namely, substantial enhancement of fluorescence emission of residue beta-Trp-148 observed upon binding of MgADP.ScFx or MgIDP . ScFx . Using this fluorescence signal, titrations were performed with MgIDP.ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot . Supporting data were obtained using MgIDP-fluoroaluminate . Current models of the MgATP hydrolysis mechanism uniformly make the assumption that only one catalytic site hydrolyzes MgATP at any one time . The fluorometal analogues demonstrate that two sites have the capability to form the transition state simultaneously.

Biochemistry, 2000 Aug 8, 39(31), 9540 - 50
Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles; Ionescu RM et al.; The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents . Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures . The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model . Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes . The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1) . The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state . The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C . The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions . The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain . The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact . Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.

Biochemistry, 2000 Aug 8, 39(31), 9508 - 13
Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis; Kamiya H et al.; The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I . In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E . coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template . Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G . On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G . These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E . coli.

Biochemistry, 2000 Aug 8, 39(31), 9459 - 65
Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues; Spedaliere CJ et al.; On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered {Koonin, E . V . (1996) Nucleic Acids . Res . 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P . J., and Santi, D . V . (1996) Nucleic Acids Res . 24, 3756-3762} . We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families . Contrary to our expectations, the altered enzymes display only very mild kinetic impairment . Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB {Zerbarjadian, Y., King, T., Fournier, M . J., Clarke, L., and Carbon, J . (1999) Mol . Cell . Biol . 19, 7461-7472} . Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated . By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.

Biochemistry, 2000 Aug 8, 39(31), 9451 - 8
Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer . Implications for the interpretation of thio effects in reactions of phosphatases; Holtz KM et al.; It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters . This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters . The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states . Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate . In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate . Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0 . 06) are the same . The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes . The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction . This result has important implications for the interpretation of thio effects in enzymatic reactions.

Biochemistry, 2000 Aug 8, 39(31), 9373 - 83
Characterization of interactions among the heme center, tetrahydrobiopterin, and L-arginine binding sites of ferric eNOS using imidazole, cyanide, and nitric oxide as probes; Berka V et al.; Endothelial nitric oxide synthase (eNOS) is a self-sufficient P450-like enzyme . A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahydrobiopterin (BH(4)) . This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis . To probe the relationships among these three components, we examined the binding kinetics of three different small heme ligands in the presence and absence of either L-arginine, BH(4), or both . Imidazole binding was strictly competitive with L-arginine, indicating a domain overlap . BH(4) had no obvious effect on imidazole binding but slightly increased the k(on) for L-arginine . L-Arginine decreased the k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruction" mechanism . BH(4) slightly enhanced cyanide binding . Nitric oxide (NO) binding kinetics were more complex . Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium . In both BH(4)-abundant and BH(4)-deficient eNOS, half of the NO binding sites showed a sizable decrease of the binding rate by L-arginine, with the rate of NO binding at the other half of the sites remaining essentially unaltered by L-arginine, implying that the two heme centers in the eNOS dimer are functionally distinct.

Biochemistry, 2000 Aug 8, 39(31), 9164 - 73
Interruption of the internal water chain of cytochrome f impairs photosynthetic function; Sainz G et al.; The structure of cytochrome f includes an internal chain of five water molecules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range of photosynthetic organisms from higher plants, algae, and cyanobacteria . The in vivo electron transfer capability of Chlamydomonas reinhardtii cytochrome f was impaired in site-directed mutants of the conserved Asn and Gln residues that form hydrogen bonds with water molecules of the internal chain {Ponamarev, M . V., and Cramer, W . A . (1998) Biochemistry 37, 17199-17208} . The 251-residue extrinsic functional domain of C . reinhardtii cytochrome f was expressed in Escherichia coli without the 35 C-terminal residues of the intact cytochrome that contain the membrane anchor . Crystal structures were determined for the wild type and three "water chain" mutants (N168F, Q158L, and N153Q) having impaired photosynthetic and electron transfer function . The mutant cytochromes were produced, folded, and assembled heme at levels identical to that of the wild type in the E . coli expression system . N168F, which had a non-photosynthetic phenotype and was thus most affected by mutational substitution, also had the greatest structural perturbation with two water molecules (W4 and W5) displaced from the internal chain . Q158L, the photosynthetic mutant with the largest impairment of in vivo electron transfer, had a more weakly bound water at one position (W1) . N153Q, a less impaired photosynthetic mutant, had an internal water chain with positions and hydrogen bonds identical to those of the wild type . The structure data imply that the waters of the internal chain, in addition to the surrounding protein, have a significant role in cytochrome f function.

Biochemistry, 2000 Aug 8, 39(31), 9146 - 56
Solution structure of ZipA, a crucial component of Escherichia coli cell division; Moy FJ et al.; ZipA, an essential component of cell division in Escherichia coli, interacts with the FtsZ protein at the midcell in one of the initial steps of septum formation . The high-resolution solution structure of the 144-residue C-terminal domain of E . coli ZipA (ZipA(185)(-)(328)) has been determined by multidimensional heteronuclear NMR . A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2758 experimental NMR restraints . The atomic root means square distribution about the mean coordinate positions for residues 6-142 for the 30 structures is 0.37 +/- 0.04 A for the backbone atoms, 0 . 78 +/- 0.05 A for all atoms, and 0.45 +/- 0.04 A for all atoms excluding disordered side chains . The NMR solution structure of ZipA(185)(-)(328) is composed of three alpha-helices and a beta-sheet consisting of six antiparallel beta-strands where the alpha-helices and the beta-sheet form surfaces directly opposite each other . A C-terminal peptide from FtsZ has been shown to bind ZipA(185)(-)(328) in a hydrophobic channel formed by the beta-sheet providing insight into the ZipA-FtsZ interaction . An unexpected similarity between the ZipA(185)(-)(328) fold and the split beta-alpha-beta fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.

Biochemistry, 2000 Aug 8, 39(31), 9139 - 45
DNA phase transition promoted by replication initiator; Yoshimura SH et al.; DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction . DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination . It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions . Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures . A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2) . When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state . This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand . Furthermore, RepE54 is not wrapped by DNA repeatedly . These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.

Appl Environ Microbiol, 2000 Aug, 66(8), 3415 - 20
Quantitative determination of the biodegradable polymer Poly(beta-hydroxybutyrate) in a recombinant Escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics; Kansiz M et al.; Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(beta-hydroxybutyrate) (PHB) contents of bacterial cells . A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents . Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988 . With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.

Cancer Res, 2000 Jul 15, 60(14), 3732 - 7
Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer; Chakrabarti S et al.; Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations . Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples . We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence . To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations . Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY . Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch . For PCR amplification, synthetic linkers are then ligated to the DNA fragments . Biotinylated DNA is then isolated and PCR amplified . Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation . This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene . In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified . The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude . Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated . This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

Transplantation, 2000 Jul 15, 70(1), 191 - 8
Immune response after adenoviral gene transfer in syngeneic heart transplants: effects of anti-CD4 monoclonal antibody therapy; Schroder G et al.; BACKGROUND: E1-deleted adenoviral vectors are frequently used for in vivo gene therapy . However, gene expression after adenovirus-(ad) mediated gene transfer is known to be transient due to the generation of an immune response against virus-infected cells . In this study, we asked whether an anti-CD4 mAb (RIB 5/2) treatment may improve the gene transfer into rat cardiac grafts . METHODS: We injected recombinant ad-constructs encoding for Escherichia coli beta-gal into syngeneic rat heart transplants via the proximal aorta . One-half of the recipients of genetically modified grafts received the anti-CD4 mAb RIB 5/2, whereas the other half received no monoclonal antibody treatment . Genetically unmodified isografts without any treatment of the recipients were used as additional controls . At different time points hearts were harvested and analyzed for reporter gene expression, intragraft cellular infiltration, and cytokine gene expression (quantitative "real time" reverse transcriptase polymerase chain reaction) . Serum samples were analyzed for anti-ad-Ig using enzyme-linked-immunosorbent-assay . RESULTS: In control animals the beta-gal reporter gene expression slowly increased until day 7 and then declined . The immunohistological and reverse transcriptase polymerase chain reaction intragraft analyses revealed a strong inflammatory response (cellular infiltration, cytokine expression) in ad-transfected grafts that may explain the delayed expression and fast down-regulation of the transgene . Treatment with RIB 5/2 mAb resulted in a faster and prolonged reporter gene expression, reduced graft infiltration, reduced anti-ad-Ig titers and less interferon-gamma up-regulation . CONCLUSIONS: Our results indicate that modulation of the anti-ad immune response using a nondepleting anti-CD4 mAb may increase the efficiency of ad-vectors for gene therapy in the transplant setting.

Res Microbiol, 2000 Jun, 151(5), 333 - 41
Interest of partial 16S rDNA gene sequences to resolve heterogeneities between Leptospira collections: application to L . meyeri; Postic D et al.; This paper describes the advantage of using the first 330 bp (positions 46 to 375, Escherichia coli numbering) of the 16S rDNA gene for comparison of Leptospira isolates . Phylogenetic analysis conducted from the whole 16S rDNA sequences available in databanks as well as that conducted from the partial sequences yielded quite similar results, in accordance with data inferred from previous DNA-DNA relatedness studies . This tool was used for the comparison of Leptospira strains from different reference collections . Consistent results were obtained from the analysis of the polymorphism generated by pulsed-field gel electrophoresis . The study focused on different serovars of L . meyeri species, the classification of which has been controversial . The results revealed large collection heterogeneities, and suggest that the classification of the L . meyeri species should be revised.

Biol Pharm Bull, 2000 Jul, 23(7), 834 - 7
Immunostimulatory effects of cycloartane-type triterpene glycosides from astragalus species; Bedir E et al.; In the course of our research on the oligoglycosidic constituents of Turkish Astragalus species, we have isolated a number of cycloartane-type triterpene glycosides . The current study examines the immunostimulatory effects of nineteen of these cycloartane-type compounds using a transcription-based bioassay for Nuclear Factor kappa B (NF-kappaB) activation in a human macrophage/monocyte cell line, THP-1 . All compounds were inactive at 100 microg/ml except astragaloside I which increased NF-kappaB directed luciferase expression to levels about 65% as compared with maximal stimulation by E . coli lipopolysaccharide (LPS) at 10 microg/ml . None of the compounds were active at low dosage levels (0.1 microg/ml) in combination with 50 ng/ml LPS . Astragaloside I also increased mRNA expression of the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as measured using reverse transcriptase-polymerase chain reaction (RT)-PCR . Based on these results it is clear that certain structural features are required for immunostimulation of cycloartane-type triterpene glycosides.

Environ Mol Mutagen, 2000, 36(1), 1 - 4
Tris(2,3-dibromopropyl)phosphate causes a gradient of mutations in the cortex and outer and inner medullas of the kidney of lacI transgenic rats; de Boer JG et al.; Tris(2,3-dibromopropyl)phosphate (TDBP) is a kidney carcinogen in rats in which exposure results in tumors specifically in the outer medulla . We have previously shown that TDBP induces mutation in the rat kidney . Here we demonstrate that TDBP induces mutation in the kidney of the F344 Big Blue lacI transgenic rat in a gradient with the highest induction (6.4-fold) in the cortex and lowest induction (2.2-fold) in the inner medulla, when given at 2000 ppm in the feed for 45 days . Similar results were obtained at 100 ppm, although the gradient effect was less pronounced . Because exposure to TDBP results in increased cell proliferation in the outer medulla, our results suggest that tissue-specific targeting of TDBP-induced kidney tumors reflects the combination of cell proliferation and mutation induction . This is also the first known case when transgenic animals have been used to study mutation at the suborgan level .

Bioessays, 2000 Aug, 22(8), 738 - 46
DNA topology and the thermal stress response, a tale from mesophiles and hyperthermophiles; Lopez-Garcia P et al.; During heat shock and cold shock, plasmid DNA supercoiling changes transiently both in mesophilic bacteria and in hyperthermophilic archaea, despite a different overall topology (negative supercoiling versus relaxation to positive supercoiling) . Transient changes in DNA supercoiling might be essential to generate the stress response, but they could also be a consequence of the physical effects of temperature on cellular components . Indeed, both appear intertwined . Comparison of the mechanisms acting in the two biological systems suggests that the dependence on temperature of the activity of different DNA topoisomerases, as well as of protein binding, are key factors for the control of DNA topology during stress, which may in turn be relevant for the expression of stress-induced genes .

Biotechnol Bioeng, 2000 Sep 20, 69(6), 664 - 78
Large-scale prediction of phenotype: concept; Varner JD; The capability to gather organism wide data has far outstripped the ability to understand it . Transforming large-scale data into a "better" cell requires tools that integrate physiology with its environment . One such tool is large-scale mathematical models that marry stoichiometry and kinetics with metabolic regulation and control . It is straightforward to determine stoichiometry (at least for central pathways), and kinetics can be roughly approximated where need be . However, the molecular details of the "metabolic wiring" managing the cell are often missing . Presented here is a surrogate for these missing details based on a simple premise; over evolutionary time, biological systems have developed objective-based programs that frugally manage gene expression and enzyme activity . Mathematically, this notion can be represented as sets of nonlinear control or "management" problems which, when solved in parallel with the model balances, offer a prediction of how gene expression and enzyme activity are modulated, in the absence of specific mechanistic details . We present a model of Escherichia coli central carbon metabolism, describing batch aerobic growth on glucose, in which transcription, translation, and activity of the gene products of 45 genes is "managed" using this approach . The model consists of 122 species (metabolites, enzymes, mRNA pools, and biomass) and describes 46 reactions (17 reversible) . The model is identified (kinetic parameters as well as management structure) from metabolic flux ratio (METAFoR) analysis and physiological measurements . Simulations of a pyruvate kinase knockout strain are compared with experiments and it is shown the model is capable of accurately capturing the metabolic reprogramming resulting from the deletion . Analysis of the mRNA expression pattern, translational pattern and enzyme activity pattern of the wild-type versus mutant indicates a combination of expression and specific activity shifts are responsible for observed differences . While being only a first step toward large-scale physiological modeling, this work is important in two ways . First, it strengthens the hypothesis that unknown mechanism can be reasonably approximated using objective-based management criteria . Second, it provides a dynamic means to couple large-scale analysis technologies with physiology at the single-gene, single-protein level .

Biotechnol Bioeng, 2000 Sep 20, 69(6), 627 - 32
Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic beta-glycosidase; Garcia-Garibay M et al.; Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems . Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively . When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme . When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature . The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction . As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside . Both reactions followed Michaelis-Menten kinetics behavior . Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol . However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield .

J Biol Chem, 2000 Nov 3, 275(44), 34375 - 81
Promoter trapping of a novel medium-chain acyl-CoA oxidase, which is induced transcriptionally during Arabidopsis seed germination; Eastmond PJ et al.; The first step of peroxisomal fatty acid beta-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities . Here we identify a new acyl-CoA oxidase gene from Arabidopsis (AtACX3) following the isolation of a promoter-trapped mutant in which beta-glucuronidase expression was initially detected in the root meristem . In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged . Despite this reduction in activity lipid catabolism and seedling development were not perturbed . AtACX3 was cloned and expressed in Escherichia coli . The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity . Analysis of beta-glucuronidase activity in acx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings . This suggests that beta-oxidation is regulated predominantly at the level of transcription in germinating oilseeds . After the discovery of AtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo.

Biotechnol Appl Biochem, 2000 Aug, 32 ( Pt 1), 73 - 9
Gene delivery via polyomavirus major capsid protein VP(1), isolated from recombinant Escherichia coli; Yang YW et al.; In this study we attempted to investigate the feasibility of transferring exogenous DNA into the recipient host via polyomavirus major capsid protein VP(1) pseudocapsids generated from recombinant Escherichia coli, designated PyVP(1,E), both in vitro and in vivo . NIH-3T3 and FR3T3 cells were transfected with pCMV beta and pPyMT-1 plasmid DNA, respectively . In vitro DNA transfection was carried out via Pseudofect- and PyVP(1,E)-mediated methods, or by co-precipitation with calcium phosphate . Expression of beta-galactosidase and PyMT reporter genes was examined by Western-blot analysis . Parallel experiments were performed in vivo by direct injection of pCMV beta and pPyMT-1 plasmid DNA, complexed with PyVP(1,E), into the livers of Wistar rats, followed by Western-blot analysis and histochemical staining . The results obtained from in vitro transfection experiments showed that expression of the reporter genes can be detected in the recipient cells at 48 h post-transfection . PyVP(1,E) was shown to exhibit similarly efficient in vitro DNA transfection properties to Pseudofect, which was obtained from recombinant baculovirus.

Biotechnol Appl Biochem, 2000 Aug, 32 ( Pt 1), 53 - 60
Effect of amino acid substitution at the P(3) and P(4) subsites of fusion proteins on kex2 protease activity; Suzuki Y et al.; The effect of amino acid substitution at the P(3) and P(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins . They were constructed from a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 {hPTH(1-34)} with a junction designed to allow cleavage with Kex2-660 . Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P(3)-P(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys . As for Asp and Glu at P(4), they precluded the cleavage almost completely . Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction . The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the introduction of Asp at P(4) of the peptide substrate resulted in a large increase in K(m) and a change in the optimum pH . The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P(4)-P(3) . The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution . Thus Kex2 exhibited extended substrate specificity beyond P(2)-P(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.

RNA, 2000 Jul, 6(7), 1031 - 43
Unusual synthesis by the Escherichia coli CCA-adding enzyme; Hou YM; The tRNA 3' end contains the conserved CCA sequence at the 74-76 positions . The CCA sequence is synthesized and maintained by the CCA-adding enzymes . The specificity of the Escherichia coli enzyme at each of the 74-76 positions was investigated using synthetic minihelix substrates that contain permuted 3' ends . Results here indicate that the enzyme has the ability to synthesize unusual 3' ends . When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs . Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not . In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme . However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA . The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs . Thus, the E . coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP . These features led to a model of CCA synthesis that is independent of a nucleic acid template . The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.

RNA, 2000 Jul, 6(7), 952 - 61
Nonsense-mediated decay mutants do not affect programmed -1 frameshifting; Bidou L et al.; Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough . These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules . Cis and trans-acting factors sensitively modulate the efficiency of recoding events . In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency . We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites . Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting . This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression . Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Nature, 2000 Jul 20, 406(6793), 318 - 22
A ratchet-like inter-subunit reorganization of the ribosome during translocation; Frank J et al.; The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms . It is composed of two unequal subunits: the smaller subunit binds messenger RNA and the anticodon end of transfer RNAs, and helps to decode the mRNA; and the larger subunit interacts with the amino-acid-carrying end of tRNAs and catalyses the formation of the peptide bonds . After peptide-bond formation, elongation factor G (EF-G) binds to the ribosome, triggering the translocation of peptidyl-tRNA from its aminoacyl site to the peptidyl site, and movement of mRNA by one codon . Here we analyse three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states, and show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S subunit relative to the large 50S subunit . Furthermore, our finding indicates a two-step mechanism of translocation: first, relative rotation of the subunits and opening of the mRNA channel following binding of GTP to EF-G; and second, advance of the mRNA/(tRNA)2 complex in the direction of the rotation of the 30S subunit, following GTP hydrolysis.

Cancer Gene Ther, 2000 Jul, 7(7), 1015 - 22
Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine; Koyama F et al.; The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells . The E . coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway . To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E . coli CD and E . coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system) . The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system . Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo . These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.

Chem Pharm Bull (Tokyo), 2000 Jul, 48(7), 1101 - 3
Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus . cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase; Kojima N et al.; cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method . Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs . The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate . To investigate the factor determining the product chain length of plant GGPPSs, S . dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed . Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate . These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.

Chem Pharm Bull (Tokyo), 2000 Jul, 48(7), 1051 - 4
Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro; Suh DY et al.; Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively . We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS . When expressed in eucaryotic cells, or in E . coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction . STS refolded from inclusion bodies also showed reduced cross-reaction . While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation . These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T . et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1247 - 54
Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression; Nakagawa R et al.; Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus . cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum . The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314) . When introduced into E . coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity . The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins . When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner . The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus . In view of these results, HTA I is considered to be a jasmonate-induced protein.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1189 - 96
Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli; Matsui K et al.; Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids . A cDNA encoding tomato fruit HPL (LeHPL) was obtained . An active LeHPL was expressed in E . coli and purified . It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid . 9-Hydroperoxides were poor substrates . The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained . LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it . LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides . The inactivation is prevented by inhibitors of LeHPL . Thus, HPL catalytic activity is thought to be essential to its inactivation . During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.

Planta, 2000 Jun, 211(1), 62 - 71
Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding, and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae; Tamayo MC et al.; The mpi gene encodes a maize proteinase inhibitor (MPI) protein whose mRNA accumulates in response to mechanical wounding . In this study, mpi gene expression in response to different types of damage was investigated . In mechanically damaged leaves of maize (Zea mays L.), mpi mRNA accumulation was affected by the degree of damage inflicted on the leaf . Consecutive wounds resulted in higher levels of mpi transcripts . The MPI protein was expressed in Escherichia coli and purified . Polyclonal antibodies were then produced and used to study MPI accumulation in insect-wounded and mechanically wounded maize leaves . When larvae of the lepidopteran insect Spodoptera littoralis were fed on maize leaves, MPI accumulated in tissues adjacent to the wound site . The level of inhibitor accumulation was higher in leaves chewed by larvae than in leaves that had been damaged mechanically . Longer feeding periods also resulted in higher levels of MPI accumulation . Additionally, the inhibitory properties of MPI toward mammalian and insect digestive serine proteinases were determined . Contrary to the majority of the plant proteinase inhibitors described, MPI is an inhibitor of mammalian elastase that only weakly inhibits mammalian chymotrypsin . However, both elastase and chymotrypsin-like activities from the larval midgut of S . littoralis were effectively inhibited by MPI . We discuss these results with regard to the function and evolution of plant proteinase inhibitors . The availability of a plant proteinase inhibitor which is able to inhibit the two types of insect digestive proteinase, elastase and chymotrypsin, might be useful for engineering protection against lepidopteran insect pests in transgenic plants.

Zhonghua Yi Xue Za Zhi, 1998 Apr, 78(4), 272 - 4
{Engineered glutamic acid decarboxylase fusion protein in diagnosis of type I diabetes mellitus}; Wu R et al.; OBJECTIVE: To get recombinant GAD 65 proteins which have immunogenicity by the method of gene engineering so that the recombinant protein can be used for early diagnosis in type I diabetes mellitus . METHOD: We amplified the complete fragment encoding GAD65 gene by PCR, and expressed its protein in E . coli . DH5 alpha after inserting it in the vector and determining its nucleotide sequence . Consequentially, we attempted to prove the immunogenicity of expresion products and construct a method for detecting antibodies against GAD65 in Diabetic serum . RESULT: The sequence analysis showed that the amplified fragments contained 1758 bp, encoded 585 amino acid, and had been correctly inserted into pGEX-3X vector . The recombinant proteins expressed in E . Coli . DH5 alpha had immunogenicity and could be used to detect agtibodies against GAD65 in diabetic serum . CONCLUSION: We have got recombinant GAD65 proteins which have immunogenicity and have used them to detect preliminarily antibodies against GAD65 in diabetic serum.

Zhonghua Yi Xue Za Zhi, 1998 Apr, 78(4), 257 - 60
{Cloning and expression of subunit genes of pertussis toxin and its immunological evaluation}; Han F et al.; OBJECTIVE: To clone the gene encoding pertussis toxin (PT) from Bordetella Pertussis CS strain and five genes encoding subunits of PT, to investigate the possibility of expressing the genes encoding for mature methionyl subunits of PT in insect cell and to evaluate the immunological characteristics of recombinant subunits . METHODS: The genes encoding for PT and its subunits were cloned by PCR and confirmed by restriction enzyme digestion and Southern blot . Using recombinant baculovirus technology, the recombinant subunits were expressed in Sf9 insect cells and analyzed by immunofluorescence assay . The immunological properties of recombinant subunits were analyzed by ELISA . RESULTS: The genes encoding for PT and its subunits were cloned and expressed in Sf9 cells . The level of specific antibodies against nature PT induced by five recombinant subunits polymerized was higher than that unpolymerized and polymerized by four recombinant subunits (without S1) in mice . The results indicate that PT subunits had weak ability to induce specific antibodies against PT, and also anti-nature PT sera had poor recognition to PT subunits . CONCLUSION: The ability of PT to induce specific antibody is conformation dependent of intact PT, and antibody induction domains are located dominantly in S1 subunit of intact PT.

Zhonghua Yi Xue Za Zhi, 1998 Jan, 78(1), 23 - 6
{Effects of specific removal of circulating tumor necrosis factor-alpha by immunoadsorption on nitric oxide in endotoxin shock}; Long H et al.; OBJECTIVES: To evaluate the effects of specific removal of circulating TNF-alpha by immunoadsorption on nitric oxide (NO) in endotoxin shock . METHODS: Immunoadsorbent against TNF-alpha was produced by the attachment of anti-TNF-alpha monoclonal antibody (McAb) to agarose beads . Blank columns were made of agarose beads without the attachment of anti-TNF-alpha McAb . New Zealand white rabbits were injected intravenously with Lipopolysaccharide (LPS, Escherichia coli O111: B4, 8.0 x 10(9) cfu/kg . B.W), and then were randomly divided into three groups: (1) control group(n = 25); without other treatment . (2) pseudoperfusion group(n = 25): rabbits underwent hemoperfusion through the blank columns . (3) perfusion group (n = 15): rabbits underwent hemoperfusion through the immunoadsorbent columns . Hemoperfusion was started at 1 h after the injection of LPS, and was sustained for two hours with blood flow rate of 5 ml/min . RESULTS: Mean arterial pressure in the perfusion group was significantly increased at 30 min after hemoperfusion (P < 0.05) . It maintained a level higher than that before hemoperfusion (P < 0.05), and was higher (P < 0.05) than that in the control and pseudoperfusion groups at 3 h (end of the monitoring period) . The plasma TNF-alpha level in the perfusion group was significantly lower than that in the other two groups at 2, 3 and 6 hour after LPS injection (P < 0.05) . Although the concentration of plasma nitrite (NO2-, one of the stable end products of NO) in the perfusion group was significantly lower (P < 0.05) than that in the other two groups from 3 h after LPS infusion, it was significantly higher (P < 0.05) than the baseline value from 30 min to 12 h . The activities of NO synthase (NOS) in the heart and lung were significantly lower (P < 0.05) in the perfusion group than in the other two groups at 24 h . The serum levels of alanine transaminase, aspartate transaminase, urea nitrogen, creatinine, lactate dehydrogenase, alpha-hydrobulyric dehydrogenase and alkaline phosphatase in the perfusion group were significantly lower (P < 0.05) than those in the other two groups at 24 h . Moreover, the survival rate of rabbits in the perfusion group was higher (P < 0.05) than that of the other two groups at 24 h . CONCLUSION: Specific removal of circulating TNF-alpha by immunoadsorption actually acts as the selective inhibition of the inducible NOS(iNOS) and may be a new and effective therapy for endotoxin shock.

Bioorg Khim, 2000 Jun, 26(6), 442 - 7
{A rapid method for testing the activity of the repair enzyme uracil-DNA-glycosylase}; Sud'ina AE et al.; A rapid and effective method of testing of a repair enzyme, uracil-DNA-glycosylase, was proposed . As a substrate, a deoxyuridine-containing 5'-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used . The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.

Bioorg Khim, 2000 Jun, 26(6), 423 - 32
{Design of the human tumor-associated VNTR(Muc1) antigen gene, fused with streptavidin, its expression in Escherichia coli . Study of properties of the hybrid protein}; Gul'ko LB et al.; A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed . It was shown that the streptavidin leader peptide ensures an effective secretion of the hybrid protein into the periplasmic space of Escherichia coli cells . The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.

Surgery, 2000 Aug, 128(2), 198 - 205
Hypertonic saline solution induces prostacyclin production by increasing cyclooxygenase-2 expression; Arbabi S et al.; BACKGROUND: Previously, we demonstrated that hypertonic saline solution (HTS) and endotoxin (lipopolysaccharide {LPS}) induce prostacyclin (PGI(2)) production in human endothelial cells . Here, we hypothesized that HTS and LPS may induce PGI(2) production by increasing cyclooxygenase (COX) expression . We further examined the activation of p38 and extracellular signal-regulated kinases (ERK) and questioned whether these transduction cascades might mediate COX expression . METHODS: Human umbilical vein endothelial cells were stimulated with varying concentrations of NaCl or LPS . RESULTS: HTS and LPS induced prompt activation of both p38 and ERKs that peaked at 30 minutes . HTS and LPS also induced a dose-related increase in COX-2 with maximal expression within 4 to 6 hours; there was no change in COX-1 . This correlated with an increase in supernatant PGI(2) levels, which became statistically significant for NaCl of more than 40 mmol/L and for all LPS doses . The inhibition of p38 with SB202190 abrogated the osmotic and LPS-induced COX-2 expression and PGI(2) production . Inhibition of ERK activation had no effect on COX-2 expression . CONCLUSIONS: Hyperosmolarity and LPS induce, in chronologic order, p38 and ERK activation, COX-2 expression, and PGI(2) production . Because COX is the rate-limiting enzyme in prostaglandin synthesis, it is likely that the increase in PGI(2) production is due to, at least in part, the increased COX-2 expression . The data also suggest that p38 mitogen-activated protein kinase is involved in the signaling cascade for COX-2 expression.

FEBS Lett, 2000 Jul 28, 478(1-2), 133 - 6
Magnetic field exposure enhances DNA repair through the induction of DnaK/J synthesis; Chow K et al.; In contrast to the common impression that exposure to a magnetic field of low frequency causes mutations to organisms, we have demonstrated that a magnetic field can actually enhance the efficiency of DNA repair . Using Escherichia coli strain XL-1 Blue as the host and plasmid pUC8 that had been mutagenized by hydroxylamine as the vector for assessment, we found that bacterial transformants that had been exposed to a magnetic field of 50 Hz gave lower percentages of white colonies as compared to transformants that had not been exposed to the magnetic field . This result was indicative that the efficiency of DNA repair had been improved . The improvement was found to be mediated by the induced overproduction of heat shock proteins DnaK/J (Hsp70/40).

FEBS Lett, 2000 Jul 28, 478(1-2), 127 - 32
Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles; Preikschat P et al.; The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease . To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed . Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

FEBS Lett, 2000 Jul 28, 478(1-2), 13 - 8
Membrane topology of the N-terminus of the Escherichia coli FtsK division protein; Dorazi R et al.; The Escherichia coli FtsK protein targets the septum, is essential for cell division and may play a role in DNA partitioning . Computer modelling suggests that the first 180 amino acids of the protein are embedded in the cytoplasmic membrane by up to six transmembrane domains . We demonstrate, using gene fusions, that the N-terminus contains four transmembrane helices that link two periplasmic domains . The first periplasmic domain contains an HEXXH amino acid sequence characteristic of zinc metalloproteases . We show by mutation analysis that the conserved glutamic acid of the HEXXH sequence is essential for FtsK function during septation.

J Biol Chem, 2000 Oct 20, 275(42), 32775 - 82
Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis; Tanioka T et al.; Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway . GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge . Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex . Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol . A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity . The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment . cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response . Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.

J Biol Chem, 2000 Oct 27, 275(43), 33814 - 9
Polyphosphate binding and chain length recognition of Escherichia coli exopolyphosphatase; Bolesch DG et al.; Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths . The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme . Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme . In addition, two independently folded domains were identified . The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily . The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate . This domain was also found to confer a highly processive mode of action to PPX . Collectively, these results were used to describe the interaction of polyphosphate with PPX.

J Biol Chem, 2000 Oct 27, 275(43), 33712 - 7
Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid; Mikkelsen MD et al.; Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides . We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis . Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime . Recombinant CYP79B2 has a K(m) of 21 microm and a V(max) of 7.78 nmol/h/ml culture . Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Muller, J . , and Hilgenberg, W . (1990) Phytochemistry, 29, 1397-1400) . CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots . Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis . Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides . Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis . Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid . Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.

J Med Genet, 2000 Aug, 37(8), 579 - 80
Identification of four novel PMM2 mutations in congenital disorders of glycosylation (CDG) Ia French patients; Vuillaumier-Barrot S et al.; We screened 11 unrelated French patients with congenital disorders of glycosylation (CDG) Ia for PMM2 mutations . Twenty one missense mutations on the 22 chromosomes (95%) including four novel mutations were identified: C9Y (G26A) in exon 1, L32R (TA95GC) in exon 2, and T226S (C677G) and C241S (G722C) in exon 8 . We studied the PMM activity of these four novel mutant proteins and of the R141H mutant protein in an E coli expression system . The T226S, C9Y, L32R, and C241S mutant proteins have decreased specific activity (23 to 41% of normal), are all more or less thermolabile, and R141H has no detectable activity . Our results indicate that the new mutations identified here are less severe than the inactive R141H mutant protein, conferring residual PMM activity compatible with life.

Arch Surg, 2000 Aug, 135(8), 959 - 66
Decreased systemic polymorphonuclear neutrophil (PMN) rolling without increased PMN adhesion in peritonitis at remote sites; Swartz DE et al.; BACKGROUND: Previous in vitro studies have demonstrated that the host response to intra-abdominal infection produces increased generalized polymorphonuclear neutrophil (PMN) adherence to vascular endothelial cells (ECs), which may lead to subsequent endothelial damage, leaky capillaries, and organ dysfunction . There are scant data to demonstrate this enhanced systemic PMN adherence in vivo or the influence of PMN rolling on PMN endothelial adherence . HYPOTHESIS: Systemic PMN adherence in the animal with sepsis is increased . DESIGN: In vivo murine model of a 2-front infection using intravital microscopy of the cremasteric muscle to quantify PMN-EC adherence in a septic response . SETTING: Basic science laboratory and animal surgical facility . PATIENTS OR OTHER PARTICIPANTS: One hundred CD1 male mice . INTERVENTIONS: Animals underwent cecal ligation and puncture peritonitis, cremasteric muscle Escherichia coli infection, both infections, or neither (controls) . Eighteen hours later, the mice underwent exteriorization of the cremasteric muscle under an intravital microscope for measurement of PMN-EC interactions . Blood was then drawn for calculation of circulating PMN counts . MAIN OUTCOME MEASURES: Adherence of PMNs, PMN rolling flux, PMN rolling velocity, and circulating PMN counts . RESULTS: Circulatory mechanics did not differ between the groups . Unlike static in vitro systems, we could not detect an increase in PMN adherence after peritonitis with this dynamic in vivo model . A local (cremasteric) infection was associated with marked PMN adherence . Peritonitis was associated with reduced PMN adherence at a local infection site as well as reduced rolling adhesion and PMN rolling velocity . CONCLUSIONS: The data suggest that intra-abdominal infection does not increase remote PMN adherence, and may actually result in reduction of systemic adherence via modulation of PMN rolling.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8973 - 8
The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex; Gmachl M et al.; The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase that targets cyclin B, securin and other destruction box containing proteins for proteolysis . Nine APC subunits have been identified in vertebrates and eleven in yeast, but for none of them it is known how they contribute to the catalysis of ubiquitination reactions . Here we report the mass spectrometric identification of CDC26 and of the RING-H2 finger protein APC11 in the human APC . We have expressed these proteins and several other APC subunits in Escherichia coli and have tested their activities in vitro . We find that APC11 alone is sufficient to allow the synthesis of multiubiquitin chains in the presence of E1 and UBC4 . These multiubiquitin chains are partly unanchored and partly bound to APC11 itself . APC11 and UBC4 are also able to ubiquitinate securin and cyclin B, but these reactions show a decreased dependency on the destruction box . The integrity of the putative zinc binding RING-H2 finger is required for the ability of APC11 to support ubiquitination reactions . These results suggest that APC11 and UBC4 catalyze the formation of isopeptide bonds in APC-mediated ubiquitination reactions.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8916 - 20
Hydrolytic editing by a class II aminoacyl-tRNA synthetase; Beuning PJ et al.; Editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for accurate translation of the genetic code . To date, this activity, whereby misactivated amino acids are hydrolyzed either before or after transfer to noncognate tRNAs, has been characterized extensively only in the case of class I synthetases . Class II synthetases have an active-site architecture that is completely distinct from that of class I . Thus, findings on editing by class I synthetases may not be applicable generally to class II enzymes . Class II Escherichia coli proline-tRNA synthetase is shown here to misactivate alanine and to hydrolyze the noncognate amino acid before transfer to tRNA(Pro) . This enzyme also is capable of rapidly deacylating a mischarged Ala-tRNA(Pro) variant . A single cysteine residue (C443) that is located within the class II-specific motif 3 consensus sequence was shown previously to be dispensable for proline-tRNA synthetase aminoacylation activity . We show here that C443 is critical for the hydrolytic editing of Ala-tRNA(Pro) by this class II synthetase.

J Clin Microbiol, 2000 Aug, 38(8), 2989 - 93
Molecular analysis of H antigens reveals that human diarrheagenic Escherichia coli O26 strains that carry the eae gene belong to the H11 clonal complex; Zhang WL et al.; Fifty-seven Escherichia coli O26 strains isolated from patients in six countries were investigated by PCR restriction fragment length polymorphism (RFLP) analysis of the flagellin-encoding (fliC) gene (fliC RFLP analysis) . The strains were determined by serotyping to belong to five different H types or were nonmotile . The fliC RFLP analysis revealed only two different patterns among the 57 strains . One fliC RFLP pattern was displayed by 54 strains and was identical to that of E . coli H11 reference strain Su4321-41 . The other fliC RFLP pattern was observed for three strains and was identical to that for E . coli H32 reference strain K10 . The 54 strains with the H11 fliC RFLP pattern included 22 strains of serotype O26:H11, 23 nonmotile strains, and 9 strains that were initially serotyped as H2, H8, H21, and H32 but that were confirmed to express H11 by repeat serotyping . All 54 strains with the H11 fliC RFLP pattern contained the attaching-and-effacing (eae) gene . The three strains with the H32 fliC RFLP pattern belonged to serotype O26:H32, and all were eae negative . The fliC genes of 14 selected E . coli O26:H11 strains isolated between 1964 and 1999 had identical nucleotide sequences . Our results demonstrate that E . coli O26 strains that carry the eae gene belong exclusively to the H11 clonal complex . Since there were no H11 fliC allelic variations among the O26 strains tested, E . coli O26:H11 may have emerged recently . The fliC PCR-RFLP test is a reliable, easy-to-perform, and rapid method for determination of the H types of E . coli O26 isolates.

Genes Dev, 2000 Aug 1, 14(15), 1971 - 82
RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners; Muyrers JP et al.; The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5'-3' exonuclease/annealing protein pairs, RecE/RecT and Redalpha/Redbeta, were analyzed . Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates . The required orthologous function correlated with a specific protein-protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease . The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism . The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.

EMBO J, 2000 Aug 1, 19(15), 4101 - 10
Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation; Grill S et al.; Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met) . Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs . We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro . These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes . We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems . This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

EMBO J, 2000 Aug 1, 19(15), 3888 - 95
Roles of multimerization and membrane association in the proteolytic functions of FtsH (HflB); Akiyama Y et al.; FtsH (HflB) is an Escherichia coli ATP-dependent protease that degrades some integral membrane and cytoplasmic proteins . While anchored to the cytoplasmic membrane by the two transmembrane (TM) segments near the N-terminus, it has a large cytoplasmic domain . The N-terminal region also has a role in homo-oligomerization of this protein . To study the significance of the membrane integration and oligomer formation, we constructed FtsH derivatives in which the N-terminal region had been deleted or replaced with either the leucine zipper sequence from Saccharomyces cerevisiae GCN4 protein or TM regions from other membrane proteins . The cytoplasmic domain, which was monomeric and virtually inactive, was converted, by the attachment of the leucine zipper, to an oligomer with proteolytic function against a soluble, but not a membrane-bound substrate . In contrast, chimeric TM-FtsH proteins were active against both substrate classes . We suggest that the cytoplasmic domain has intrinsic but weak self-interaction ability, which becomes effective with the aid of the leucine zipper or membrane tethering, and that membrane association is essential for FtsH to degrade integral membrane proteins.

EMBO J, 2000 Aug 1, 19(15), 3849 - 56
Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism; Izard T et al.; Carbon-carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods . Here we describe the crystal structures of a novel class II enzyme, 2-dehydro-3-deoxy-galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate . The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues . The protomer displays a modified (alpha/beta)(8) barrel fold, in which the eighth alpha-helix points away from the beta-barrel instead of packing against it . Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

Crit Care Med, 2000 Jul, 28(7), 2515 - 21
Changes in circulating lymphocyte subpopulations and mitogen-stimulated response in a rat infusion model of intra-abdominal infection; Martineau L et al.; OBJECTIVE: To describe the alterations in circulating concentrations of immune cells as well as in in vitro mitogen-stimulated response in a recently developed rat model of intra-abdominal infection . DESIGN: Randomized, controlled study . SETTING: Government research facility . SUBJECTS: Male Sprague-Dawley rats . INTERVENTIONS: Infected animals received an intraperitoneal infusion of 6.0 x 10(8) colony forming units of Escherichia coli during 12 hrs, whereas control rats received a sterile inoculum . All experimental animals underwent laparotomy and peritoneal lavage at the end of the infusion period . Blood samples were obtained 12 hrs, 36 hrs, or 7 days after the onset of infusion . Splenocytes were concomitantly harvested and assayed for response to the mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and lipopolysaccharides, as well as for production of interleukin (IL)-2 . MEASUREMENTS AND MAIN RESULTS: Infected rats showed a marked leukopenia (-82% for 36 hrs), with leukocyte counts returning to normal at 7 days . They also developed a marked lymphocytopenia throughout the study; this was achieved through comparable reductions in circulating T and B cells . Con A responses in both groups were similar for 7 days . In contrast, splenocytes from infected animals showed reduced responses to lipopolysaccharides (-64%) and PHA (-30%) for 36 hrs compared with control splenocytes . IL-2 production from mitogen-stimulated splenocytes was suppressed in infected rats to 66% of that of control rats for 7 days . Suppressed PHA responses were not restored to control values in the presence of IL-2 . For all of the parameters assessed, control animals showed either no significant changes or relatively fewer changes than infected rats . CONCLUSIONS: This model of intra-abdominal infection is associated with changes in circulating concentrations of immune cells as well as with temporary functional defects in B and T cells, consistent with those often observed in patients with peritonitis . However, the role of IL-2 in limiting the adverse effects of infection in this experimental model seems to be limited . This model may be a useful tool in furthering our understanding of the pathophysiology of intra-abdominal infections and in assessing the efficacy of new therapeutic modalities.

Crit Care Med, 2000 Jul, 28(7), 2500 - 6
Effects of norepinephrine on the distribution of intestinal blood flow and tissue adenosine triphosphate content in endotoxic shock; Revelly JP et al.; OBJECTIVE: To investigate, during endotoxic shock, the effect of a treatment of norepinephrine (NE) administration on the distribution of blood flow and adenosine triphosphate (ATP) content in the intestinal wall . DESIGN: Randomized controlled trial . SETTING: Animal laboratory . SUBJECTS: Domestic pigs . INTERVENTION: A total of 18 pigs were anesthetized with ketamine and pentobarbital, mechanically ventilated, hemodynamically monitored, and then challenged with a continuous infusion of Escherichia coli endotoxin (ET) (15 microg/kg) for 2 hrs . Three groups of six animals were studied; one served as time control, one group received ET and fluid resuscitation, and a third group received ET, fluid resuscitation, and a perfusion of NE to maintain constant mean arterial pressure (MAP) . MEASUREMENTS AND MAIN RESULTS: Cardiac output, mesenteric arterial blood flow, MAP, pulmonary pressure, and portal pressure were measured . Intestinal mucosal intracellular pH (pHi) was determined with saline-filled balloon tonometers . Tissue blood flows to the intestinal mucosa and to the muscular layer were independently measured with fluorescent microspheres, using the arterial reference sample method . Measurements were performed before and 3 hrs after the start of the ET challenge . At the end of the experiments, muscularis and mucosal samples were quickly frozen for further enzymatic ATP measurements . ET administration with fluid resuscitation induced a distributive shock with increased mucosal blood flow and decreased muscularis blood flow, whereas pHi decreased and mucosal ATP content was significantly lower than in the control group . In the group receiving ET plus NE, MAP remained constant, mucosal blood flow did not increase, and mucosal ATP content was equal to the time control group . Meanwhile, mucosal acidosis was not prevented . CONCLUSIONS: Normodynamic endotoxic shock may induce an alteration in mucosal oxygenation, despite an increased tissue blood flow . A treatment of NE combined with fluid resuscitation has complex effects on tissue blood flow, ATP content, and pHi.

Crit Care Med, 2000 Jul, 28(7), 2415 - 9
End-tidal carbon dioxide as a noninvasive indicator of cardiac index during circulatory shock; Jin X et al.; OBJECTIVE: To document the relationships between cardiac index and end-tidal carbon dioxide tension (PetCO2 during diverse low-flow states of circulatory shock . DESIGN: Randomized, prospective, controlled studies on animal models of hemorrhagic, septic, and cardiogenic shock . SETTING: University-affiliated research laboratory . SUBJECTS: Sixteen anesthetized domestic pigs weighing 35-45 kg . INTERVENTIONS: Hemorrhagic shock was induced in five pigs by bleeding followed by reinfusion of shed blood . Septic shock was induced in five pigs by infusion of live Escherichia coli . Cardiogenic shock followed an interval of global myocardial ischemia after inducing and reversing ventricular fibrillation in six pigs . MEASUREMENTS AND MAIN RESULTS: PetCO2 was continuously measured . Cardiac index was measured intermittently by using conventional thermodilution techniques . Cardiac index was correlated with PetCO2 by polynomial regression and Bland-Altman analyses . PetCO2 was highly correlated with cardiac index during hemorrhagic shock (r2 = .69, p < .01), septic shock (r2 = .65, p < .01), and cardiogenic shock (r2 = .81, p < .01) . PetCO2 predicted thermodilution cardiac index with bias of -11+/-27 (+/-2 SD) mL/min/kg during hemorrhagic shock, 1.3+/-20.4 (+/- 2 SD) mL/min/kg during septic shock, and -1+/-12 (+/-2 SD) mL/min/kg during cardiogenic shock . CONCLUSIONS: Cardiac output and PetCO2 were highly related in diverse experimental models of circulatory shock in which cardiac output was reduced by >40% of baseline values . Therefore, measurement of PetCO2 is a noninvasive alternative for continuous assessment of cardiac output during low-flow circulatory shock states of diverse causes.

Zhonghua Zhong Liu Za Zhi, 1998 May, 20(3), 193 - 5
{Binding activity of retinoids with recombinant human retinoic acid receptor RAR alpha}; Cao X et al.; OBJECTIVE: To establish a model for the rapid screening of differentiation-inducing agents . METHODS: Human RAR alpha cDNA was cloned into the PT7-7 plasmids and the human recombinant RAR alpha protein was expressed in Escherichia coli . The ligand-binding activity of the receptor with all-trans retinoic acid was studied by competitive binding assay . For comparison of the binding activity, five retinoids, with different activities and chemical structures, were chosen . The binding of these retinoids to RAR alpha was investigated and compared with the NBT reduction and acid phosphatase activity of HL-60 cells induced by these compounds . RESULTS: The binding activity of these retinoids was well correlated to their ability to induce differentiation of HL-60 cell line . CONCLUSION: This model could be used to screen differentiation-inducing agents.

Zhonghua Zhong Liu Za Zhi, 1998 Jul, 20(4), 270 - 3
{Enhanced antitumor effect of suicide gene therapy by SCF, GM-CSF gene transfer in vivo and its immunological mechanism}; Huang X et al.; OBJECTIVE: To explore an efficient approach to enhance antitumor effect of suicide gene therapy by improving in vivo antigen presenting function and their immunological mechanisms . METHODS: The CT26 colon adenocarcinoma was treated with CD/5FC system combined with the mGM-CSF or/and mSCF gene transfection, and the antitumor effects were evaluated by the tumor growth, tumor-free mouse rate, splenic CTL activity and cytokine expression of tumor milieu . RESULTS: The tumor burden was reduced significantly and higher survival rate was obtained after the combined treatment . It was found that some cytokines(including mIL-4, mIL-2, mIFN-gamma and mTNF-alpha) were expressed in the tumor milieu after mGM-CSF or/and mSCF gene transfection following CD/5FC treatment but not in the control groups . The cytotoxic activity against CT26 cells of spleen cells of the treated mice was significantly incressed . CONCLUSION: The combination of mSCF or/and mGM-CSF gene transfection enhances the antitumor effect of the suicide gene therapy . Such an enhancement is associated with the induction of specific antitumor immune response and secretion of cytokines in tumor milieu.

Zhonghua Zhong Liu Za Zhi, 1998 Mar, 20(2), 108 - 11
{Antitumor effect of combined therapy with adenovirus-mediated CD suicide gene and interleukin 2 gene transfer and its immunological mechanism}; Ju D et al.; OBJECTIVE: Adenovirus harboring E . coli cytosine deaminase gene(AdCD) and adenovirus harboring interleukin 2 gene (AdIL-2) were used for the combined treatment of established tumors in vivo . METHODS: C57BL/6 mice were inoculated with B16F10 melanoma cells and 3 days later received at the tumor site injections of AdCD and/or AdIL-2 followed by i.p . injection of 5-fluorocytosine (5-Fc) 300 mg/kg per day for 10 days . RESULTS: Mice received combined therapy developed tumors more slowly and survived much longer when compared with mice treated with AdCD/5-Fc, AdIL-2, control adenovirus AdlacZ/5-FC, or PBS . To investigate the immunological mechanism of the antitumor effects of the combined treatment it was found to induce enhanced natural killer cell activity and specific cytotoxic T cell activity . FACS analysis demonstrated that AdIL-2/AdCD/5-Fc therapy increased the expression of H-2Kb and B7-1 on freshly isolated tumor cells . The CD4+ and CD8+ T cell infiltration in the tumor increased significantly . CONCLUSION: Transfer of CD suicide gene plus 5-Fc combined with transfer of IL-2 gene synergistically inhibits the growth of melanoma in mice . Besides the cytotoxic effect of 5-Fc, specific and non-specific antitumor immunity might be responsible for the therapeutic effect . The combined therapy might have therapeutic potentials for human cancer.

Zhonghua Zhong Liu Za Zhi, 1998 Mar, 20(2), 85 - 7
{Construction and expression of ScFv from anti-human gastric cancer McAb 3H11}; Li J et al.; OBJECTIVE: To construct single chain antibody (ScFv) from anti-gastric cancer McAb 3H11 . METHODS: Phage display technology was used to construct ScFv library from 3H11 hybridoma cells . Site-directed mutagenesis was used to resume the N-terminal sequences of 3H11 ScFv . RESULTS: The library was screened against human gastric cancer cells MGC803 but no positive clone was obtained . Then, a random mutated ScFv library was constructed by error-prone PCR method, and panning selection was performed . Again the identification of positive clone was failed . Finally the N-terminal sequences of ScFv variable region was resumed to 3H11 original sequences by site-directed mutagenesis via PCR, and the expressed ScFv in bacterial culture supernatant showed binding activity to gastric cancer cells . CONCLUSION: The N-terminal of the variable region introduced by PCR primers may seriously affect binding activity of the antibody.

J Biochem (Tokyo), 2000 Aug, 128(2), 337 - 44
Structural study of the N-terminal domain of the alpha subunit of Escherichia coli RNA polymerase solubilized with non-denaturing detergents; Otomo T et al.; The amino-terminal domain of the alpha subunit (alphaNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the alpha, beta, and beta' subunits into the core-enzyme . It has a tendency to form aggregates by itself at higher concentrations . For NMR structural analysis of alphaNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique . Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, alphaNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances . Analysis of the pattern of NOEs observed between the backbone amide and alpha-protons demonstrated that alphaNTD has three alpha-helices and two beta-sheets . Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two beta-sheets has two additional beta-strands . The irregular NOE patterns observed for the three positions in the beta-sheets suggest the presence of beta-bulge structures . The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly.

J Biochem (Tokyo), 2000 Aug, 128(2), 283 - 91
Studies on the structure-function relationship of the HNK-1 associated glucuronyltransferase, GlcAT-P, by computer modeling and site-directed mutagenesis; Ohtsubo K et al.; All members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain . In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling . The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins . Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity . A three-dimensional (3-D) model of GlcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template . This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five alpha-helix and beta-sheet repeats . The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomucoid . There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located . The biological significance of the second cluster is currently not clear but it may be associated with the interaction of the enzyme with modulation molecules, manganese and membrane phospholipids.

J Biochem (Tokyo), 2000 Aug, 128(2), 261 - 9
Molecular cloning and characterization of a unique 60 kDa/72 kDa antigen gene encoding enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Mycoplasma hyopneumoniae; Chung TL et al.; The recombinant clone expressing a 60 kDa (P60) antigen was isolated from Escherichia coli by screening a lambda EMBL3 genomic library using rabbit produced antiserum against Mycoplasma hyopneumoniae . Sequence analysis revealed that an interrupted (by a UGA codon) open reading frame coding for a 72 kDa protein (P72) may contain the P60 antigen gene . Western blot analysis with an anti-P60 monospecific antibody confirmed the presence of a P72 antigen from the total protein of M . hyopneumoniae, and a 72 kDa protein was also expressed in E . coli after changing the codon (UGA to UGG) by site-directed mutagenesis . BLAST (Basic Local Alignment Search Tool) comparison showed that the amino acid sequences of P72 share approximately 70% homology with the phosphotransferase enzyme I (PTSI) of bacteria and other mycoplasma species . The biological function of the P72 cytosolic protein was further confirmed by complementation using an E . coli ptsI mutant . The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) is known to mediate the uptake and phosphorylation of carbohydrates and to be involved in signal transduction . The immune responses of specific pathogen free (SPF) pigs and farm animals toward this unique antigen were observed . The transcription start positions of the PTSI gene were determined in M . hyopneumoniae and E . coli by primer extension experiments and the promoter site was also predicted.

J Biochem (Tokyo), 2000 Aug, 128(2), 245 - 50
Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118; Futami J et al.; In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies . The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118 . From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol . This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link . These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol . Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link . It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.

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