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 fo