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| Biochem J, 1999 Nov 1, 343 Pt 3, 669 - 72 Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase; Hutter B et al.; The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M . tuberculosis, but not in the vaccine strain Mycobacterium bovis BCG . It is a functional L-alanine dehydrogenase (EC 1.4.1.1) and hence one of the few antigens possessing an enzymic activity . This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions . Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme . In the oxidative-deamination reaction, the enzyme showed K(m) values of 13 . 8 mM and 0.31 mM for L-alanine and NAD(+), respectively, in a random-ordered mechanism . K(m, app) values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 microM for ammonium, pyruvate and NADH, respectively . The enzyme is highly specific for all of its substrates in both directions . The pH profile indicates that oxidative deamination virtually may not occur at physiological pH . Hence L-alanine most likely is the product of the reaction catalysed in vivo . The enzyme is heat-stable, losing practically no activity at 60 degrees C for several hours. Biochem J, 1999 Nov 1, 343 Pt 3, 563 - 70 An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates; Buchanan CL et al.; Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C . It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde . Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S . solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme . The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S . solfataricus cells . The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde . It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4). J Struct Biol, 1999 Sep, 127(2), 120 - 34 Gold-ATP; Hainfeld JF et al.; The synthesis, purification, and chemical analysis of two covalent conjugates between ATP and undecagold are described, one in which gold is attached to the ribose moiety of ATP and the other in which it is attached to the N-6 position of the adenine base . The former probe was then used to bind to two ATP binding proteins, the helicase DnaB and the chaperone DnaK . After purification from unbound gold by column chromatography, binding was measured by UV-Vis spectroscopy, then the protein and gold were visualized by scanning transmission electron microscopy . Binding was observed with the conjugates, and virtually no binding occurred in the control of undecagold without the ATP attached . This new probe may be useful for studying nucleotide binding sites on proteins or for labeling nucleic acids or oligonucleotides directly . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 287 - 90 Residues within the HFRIGC sequence of HIV-1 vpr involved in growth arrest activities; Berglez JM et al.; HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death . Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities . Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important . Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects . In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 225 - 9 Fragments from alpha-actinin insert into reconstituted lipid bilayers; Goldmann WH et al.; Recent experiments have indicated that alpha-actinin interacts with phospholipid membranes . Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281-300 and 720-739 of the primary amino acid sequence on smooth muscle alpha-actinin . Having expressed these regions as fusion proteins with schistosomal GST (glutathione S-transferase), we used differential scanning calorimetry (DSC) to investigate their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers . Calorimetric measurements showed that as fusion protein concentration increased, the main chain transition enthalpy decreased and chain melting temperatures shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membranes . Centrifugation assay and subsequent SDS/Page chromatography confirmed this finding . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 196 - 200 Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase; Carvajal N et al.; Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal . Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis . Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition . The mutant was insensitive to DEPC and dye-sensitized inactivations . We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 181 - 5 Multiple domains of DFF45 bind synergistically to DFF40: roles of caspase cleavage and sequestration of activator domain of DFF40; McCarty JS et al.; CAD/DFF40, the nuclease responsible for DNA fragmentation during apoptosis, exists as a heterodimeric complex with DFF45/ICAD . The study presented here augments the accompanying inhibition and chaperone study with an analysis of specific binding strengths and locations of DFF45 binding sites within DFF40 . This allows us to show that DFF40/45 interaction is mediated by binding of three functional domains (D1, D2, and D3) of DFF45 to two domains (activator and catalytic) of DFF40 . D1 binds exclusively to the activator domain and D2 binds to the catalytic domain of DFF40 . Inhibition of DFF40 nuclease activity arises independently from D1 functional sequestration of the activator domain and D2 blockage of the catalytic domain of DFF40 . The mechanism of caspase activation of DFF40 is the disruption of the synergistic binding activity of DFF45 domains to DFF40 after caspase recognition and cleavage of DFF45 in the context of a DFF45/40 complex . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 69 - 75 Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin; Lu FM et al.; The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7 . Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively . Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins . Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin . In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8 . This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 51 - 4 Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit; Sayed A et al.; Era is an essential G-protein in Escherichia coli identified originally as a homologue protein to Ras (E . coli Ras-like protein) . It binds to GTP/GDP and contains a low intrinsic GTPase activity . Its function remains elusive, although it has been suggested that Era is associated with the cytoplasmic membrane, cell division, energy metabolism, and cell-cycle check point . Recently, a cold-sensitive phenotype was found to be suppressed by the overexpression of 16S rRNA methyltransferase, suggesting Era association with the ribosome . Here we demonstrate that Era specifically binds to 16S rRNA and the 30S ribosomal subunit . Both GTP and GDP, but not GMP, inhibit Era binding to ribosomal component . Involvement of Era in protein synthesis is suggested by the fact that Era depletion results in the translation defect both in vitro and in vivo . Anal Biochem, 1999 Oct 15, 274(2), 235 - 40 Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis; Schmidt T et al.; Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates . Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution . Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE) . The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found . The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks . Additionally, the different isoforms can now be quantified by CGE . Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification . CGE also offers high resolution and the amount of DNA required is very low . Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation . Anal Biochem, 1999 Oct 15, 274(2), 181 - 7 The efficient removal of endotoxins from insulin using quaternized polyethyleneimine-coated porous zirconia; McNeff C et al.; The synthesis and use of a zirconia-based, alkali-stable strong anion-exchange stationary phase are described for the removal of pyrogenic lipopolysaccharides (LPS) from insulin . The strong anion-exchange material is produced by deposition of polyethyleneimine (PEI) onto porous zirconia particles, followed by cross-linking with a novel reagent, 1,2-bis-(2-iodoethoxy) ethane, and quaternization with iodomethane . Physical characterization of the chromatographic support shows that it has an ion-exchange capacity of 0.6 mmol/g, and 82% of the amine sites on the surface are in quaternized form . Isocratic elution of small benzoic acid derivatives shows good column efficiency . The two primary virtues of this material are its chemical stability under alkali conditions (up to pH 13) and its lower hydrophobicity compared to previously described alkali-stable PEI-coated zirconia supports cross-linked with 1,10-diiododecane . Using this new zirconia-based phase, a purification protocol is developed for the efficient removal of Escherichia coli 0111:B4 LPS from bovine insulin samples . An endotoxin clearance rate of up to 1.3 x 10(8) was attained . Endotoxin levels were reduced to less than 5 endotoxin units/ml even at initial contamination levels as high as 5.0 x 10(6) endotoxin units/ml . Furthermore, endotoxin adsorbed to the porous zirconia column may be easily removed (depyrogenated) using alkali for repeated purification cycles . Anal Biochem, 1999 Oct 1, 274(1), 40 - 9 Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes; Layfield R et al.; We have used solid-phase chemistry to synthesize proteins equivalent to a human ubiquitin precursor (ubiquitin-52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin-protein conjugates {ubiquitin-(epsilonN)-lysine}; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D . Along with the previously synthesized ubiquitin-(alphaN)-valine, these synthetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mouse Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p . Distinct specificities of these enzymes were detected; notably, in addition to UCH-D, isopeptidase activity {ubiquitin-(epsilonN)-lysine cleavage} was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p . Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin-(epsilonN)-lysine, suggesting that 26S proteasome-associated DUBs are class II-like . This work demonstrates that the synthetic approach offers an alternative to recombinant methods for the production of small proteins in vitro . Plant Mol Biol, 1999 Aug, 40(6), 1063 - 71 Cloning and characterization of a class II DNA photolyase from Chlamydomonas; Petersen JL et al.; Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases . Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast . Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase . The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency . The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene . Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence . This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase. Plant Mol Biol, 1999 Aug, 40(6), 1009 - 18 Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains; Zhao KJ et al.; We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase . BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin: however there is only 36.9% identity between them . We propose that BjCHI1 should be classified under a new class, Chia7 . The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain . Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyljasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA) . This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA . Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B . juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge . Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E . coli JM109 expressing recombinant BjCHI1 showed chitinase activity . Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B . juncea. Plant Mol Biol, 1999 Aug, 40(6), 969 - 76 A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein; Akad F et al.; We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca . 23 kDa protein that inhibits replication of several plant viruses . This protein, named 'inhibitor of virus replication' (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv . Samsun NN . IVR was shown to be present also in induced-resistant leaf tissue of N . tabacum cv . Samsun NN . We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein . A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated . The NC330 clone hybridized with RNA from induced-resistant tissue from N . tabacum cv . Samsun NN but not with RNA from non-induced tissue . Likewise, it did not hybridize with RNA from infected or uninfected tissue of N . tabacum cv . Samsun nn . Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only . In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N . tabacum cv . Samsun NN and Samsun nn . The size of the DNA fragments differed in Samsun NN and Samsun nn . We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N . tabacum genotypes, but is expressed only in NN . We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody . This protein greatly reduced replication of TMV in N . tabacum cv . Samsun nn leaf disk assays. Plant Mol Biol, 1999 Aug, 40(6), 921 - 33 Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat; Takezawa D; Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments . Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase . The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) . The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens . BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA . A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein . Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation . The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract . These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat . Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts. J Crit Care, 1999 Sep, 14(3), 125 - 32 Myocardial oxygen consumption during dobutamine infusion in endotoxemic pigs; Herbertson MJ et al.; PURPOSE: Dobutamine infusion is used to increase whole-body oxygen delivery in septic patients to satisfy unmet oxygen demand of hypoxic tissues . However, dobutamine infusion also increases myocardial work and myocardial oxygen consumption . Our goal was to determine the importance of this effect as a fraction of the increase in whole-body oxygen consumption, in a porcine model of septic shock . MATERIALS AND METHODS: Four hours after a 50 microg/kg infusion of Escherichia coli endotoxin (0111: B4, Sigma) in eight anesthetized pigs, whole-body oxygen delivery and myocardial oxygen delivery and consumption were calculated from blood flow and arterial and venous oxygen content measurements . We directly measured whole-body oxygen consumption by analysis of inhaled and exhaled gases using a metabolic cart . Then dobutamine 10 and 20 microg/kg/min was infused and measurements were repeated . RESULTS: Dobutamine infusion increased whole-body oxygen delivery but did not increase metabolic cart measured whole-body oxygen consumption . Dobutamine infusion of 10 and 20 microg/kg/min increased myocardial oxygen consumption by 7.0 +/- 0.6 (80 +/-10%) and 12.0 +/- 2.0 mL O2/min (142 +/- 30%), respectively (P < .01) . CONCLUSIONS: In this porcine model of sepsis, dobutamine infusion significantly increases myocardial oxygen consumption . Because whole-body oxygen consumption does not change, dobutamine infusion may fail to increase and may decrease oxygen consumption by other organs. Acta Biol Hung, 1998, 49(2-4), 173 - 84 Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco; Ying Z et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS) . The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed . The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco . Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance . The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site . A N-terminal truncated form of spinach SSMT was expressed and purified from E . coli cells . Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco. Proteins, 1999, Suppl 3, 133 - 40 Threading with explicit models for evolutionary conservation of structure and sequence; Panchenko A et al.; We have attempted to predict the three-dimensional structures of 19 proteins for the CASP3 experiment, each showing less than 25% sequence identity with known structures . Predictions were based on a threading method that aligns the target sequence with the conserved cores of structural templates, as identified from structure-structure alignments of the template with homologous neighbors . Alternative alignments were scored using contact potentials and a position-specific score matrix derived from sequence neighbors of the template . We find that this method identified the correct structural family for 11 of the 19 targets and predicted the remaining 8 targets to be similar to "none" of the templates, avoiding false positives . Threading alignments are relatively accurate for 10 of the 11 targets, including alignments for 6 of 7 identified at CASP3 as fold-recognition targets . These predictions were ranked "first place" by the CASP3 assessor when compared to fold-recognition predictions made by other methods . It appears that threading with family-specific models for structure and sequence conservation has improved threading prediction accuracy. Proteins, 1999, Suppl 3, 112 - 20 Sustained performance of knowledge-based potentials in fold recognition; Domingues FS et al.; We describe the results obtained using fold recognition techniques in our third participation in the CASP experiment . The approach relies on knowledge-based potentials for alignment production and fold identification . As indicated by the increase in alignment quality and fold identification reliability, the predictions improved from CASP1 to CASP3 . In particular, we identified structural relationships in which no known evolutionary link exists . Our predictions are based on single sequences rather than multiple sequence alignments . Additionally, we voluntarily submitted only a single model for each target because, in our view, submission of a single model is the most stringent test . We describe the methods used, the strategy adopted in the predictions, and the prediction results and discuss future work. Proteins, 1999, Suppl 3, 88 - 103 Structure classification-based assessment of CASP3 predictions for the fold recognition targets; Murzin AG; The sequences of at least 23 of the 43 CASP3 targets showed no significant similarity to the sequences of known structures . The experimental structures of all but three of these 23 targets revealed substantial similarities to known structures, with at least eleven of the target structures likely being distantly homologous to known structures . Nineteen of the 23 target structures were available at the time of the final CASP3 meeting in Asilomar in December 1998, whereas the experimental data on the protein folds of the remaining four targets were obtained afterwards . The predicted three-dimensional structures for each of the 23 targets were analyzed to select those predictions sharing with the experimental structures a similar overall fold and/or having correctly folded a substantial fraction of the target sequence . Initially, predicted models were numerically evaluated and the evaluation results aided the selection process . Each target structure was then classified to identify a minimal set of structural features characteristic to its protein fold and evolutionary superfamily . The predictions containing this set were assessed comparatively to find the best predictions for each target . The predictions of new folds were assessed separately . The total number of the selected 'correct' predictions and the quality of these predictions were used to compare the performance of different predictor teams and different prediction methods in the fold prediction/recognition category. Proteins, 1999, Suppl 3, 22 - 9 Processing and analysis of CASP3 protein structure predictions; Zemla A et al.; Livermore Prediction Center provides basic infrastructure for the CASP (Critical Assessment of Structure Prediction) experiments, including prediction processing and verification servers, a system of prediction evaluation tools, and interactive numerical and graphical displays . Here we outline the essentials of our approach, with discussion of the superposition procedures, definitions of basic measures, and descriptions of new methods developed to analyze predictions . Our primary focus is on the evaluation of three-dimensional models and secondary structure predictions . To put the results of the three prediction experiments held to date on the same footing, the latest CASP3 evaluation criteria were retrospectively applied to both CASP1 and CASP2 predictions . Finally, we give an overview of our website PredictionCenter.llnl.gov), which makes the target structures, predictions, and the evaluation system accessible to the community. Proteins, 1999, Suppl 3, 7 - 14 Automated large scale evaluation of protein structure predictions; Lackner P et al.; Evaluation and assessment are critical issues in CASP experiments . Automated procedures are necessary to compare a large number of predictions with the target folds . The evaluation has to reveal the maximum extent of similarity between predictions and targets, it should be applicable across prediction categories, and it should be transparent and accessible to a wide community . Here we present an automated evaluation scheme which is an attempt to meet these requirements . In the implementation and execution of this scheme we had to solve or circumvent problems of convergence, where algorithms fail to find optimum solutions, problems of ambiguity where no unique optimum solution exists, and problems in ranking and interpretation . Key features of this implementation are (1) the root mean square deviation of structure superimposition is kept close to a constant value throughout the evaluation and (2) all structural matches found between two folds are taken into account . We discuss these points in detail and describe the numerical criteria used in the CASP3 evaluation. Genes Cells, 1999 Sep, 4(9), 501 - 15 Intragenic suppression of trans-dominant lethal substitutions in the conserved GEME motif of the beta subunit of RNA polymerase: evidence for functional cooperativity within the C-terminus; Malik T et al.; BACKGROUND: The ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the beta subunit has been implicated in all three stages of transcription . We have previously described a genetic system involving random, PCR-mediated mutagenesis of the region of rpoB encoding the C-terminal 116 amino acids of the beta subunit of Escherichia coli RNA polymerase and the characterization of dominant-negative mutations . This study identified the invariant motif GEME (residues 1271-->1274; Cromie et al . 1999) . Starting with three of these GEME-motif lethal mutations (G1271E, G1271V, M1273V), we have selected for intragenic suppressors, located within the same 3'-region, that prevent expression of the trans-dominant phenotype . RESULTS: We isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C-terminal deletions of the beta subunit) and studied the effect of the missense suppressors in vivo and in vitro . The majority of the second-site substitutions pinpoint highly conserved residues and were allele-specific . In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif-specific suppression . Two missense substitutions were found to perturb assembly of the beta subunit (M1232T and L1233P) and define a small conserved region (1228-->1233) adjacent to one of the active-site residues identified by affinity-labelling, H1237 . The majority of primary mutations were located in three main clusters within the 116 amino acid region . CONCLUSIONS: The importance and functional co-operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C-terminal of this residue) . Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as alpha-helices in holoenzyme, and that functional co-operativity is through one particular face of each helix. Genes Cells, 1999 Aug, 4(8), 475 - 85 Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase; Honda A et al.; BACKGROUND: Influenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome . Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase-associated capped RNA endonuclease . To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32P only at the cap-1 structure . RESULTS: After SDS-PAGE of UV-crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues) . The labelled PB2 was subjected, together with PB2 expressed in E . coli, to limited digestion with V8 protease . Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap-1 indicated that two separate sequences within the PB2 were involved in RNA cap-1 binding, one (N-site) at the N-terminal proximal region approximately between amino acid residues 242-282 downstream from the PB1 subunit-binding site and the other (C-site) between residues 538-577 including the cap-binding motifs . Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap-binding: (i) cross-linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N-site or the C-site; and (ii) competition of capped RNA-binding to PB2 by both of the N- and C-terminal PB2 fragments . Taking together, we propose that two separate sequences within PB2 constitute the capped RNA-binding site of the RNA polymerase . CONCLUSION: Two separate sequences, one N-(242-282) and the other C-terminal (538-577) proximal segments of PB2 subunit, constitute the RNA cap-binding site of the influenza virus RNA polymerase. Mutat Res, 1999 Sep 13, 435(1), 77 - 80 A UmuD,C-dependent pathway for spontaneous G:C to C:G transversions in stationary phase Escherichia coli mut Y; Timms AR et al.; In Escherichia coli trpA23 bacteria lacking the MutY glycosylase and incubated on plates in the absence of tryptophan, tryptophan-independent mutants continue to arise during incubation over many days . Their appearance is enhanced in umuD+,C+ strains in comparison with strains carrying a deletion through the umu operon and the umuD,C-dependent mutants were greater in number in uvrA bacteria (lacking nucleotide excision repair) than in uvr+ bacteria . Sequencing of mutations occurring in uvrA bacteria revealed the presence of G:C to C:G transversions but only in umuD+,C+ strains . There is thus a pathway in starved bacteria that generates G:C to C:G transversions and requires the inducible UmuD,C proteins . The data are consistent with the occurrence of a lesion, probably 8-oxoguanine, against which guanine may be incorporated during DNA synthesis by "dNTP stabilised" misalignment against the downstream template base . Upon realignment the configuration is substrate for MutY glycosylase which can remove the unmodified guanine . It is hypothesised that UmuD,C proteins are required for primer extension from the mismatch once formed. Mutat Res, 1999 Oct 19, 429(2), 199 - 208 Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines; Josephy PD et al.; The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions . Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins . Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101 . Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309 . This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens . We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo{4,5-f}quinoline (MeIQ), a typical heterocyclic amine mutagen . Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis . Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene. FEBS Lett, 1999 Oct 15, 459(3), 433 - 7 Cloning and characterization of two novel aldo-keto reductases (AKR1C12 and AKR1C13) from mouse stomach; Ikeda S et al.; In contrast to hepatic hydrosteroid dehydrogenases (HSDs) of the aldo-keto reductase family (AKR1C), little is known about a stomach one . From a mouse stomach cDNA library, we isolated two clones encoding proteins of 323 amino acid residues . They exhibited 93.2% amino acid sequence identity and 64-68% with any known HSDs . Recombinant proteins expressed in Escherichia coli reduced 9,10-phenanthraquinone with NAD(P)H as cofactor . The mRNAs were exclusively expressed in stomach, liver and ileum . The present study demonstrates that these proteins are new members of the HSD subfamily and they are named AKR1C12 and AKR1C13 . Immunohistochemical analysis suggests that they are involved in detoxification of xenobiotics in the stomach. FEBS Lett, 1999 Oct 15, 459(3), 411 - 4 Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between {2Fe-2S}(2+) and {4Fe-4S}(2+) clusters; Tse Sum Bui B et al.; The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by Mossbauer spectroscopy . Our data clearly demonstrate that in the as-prepared sample, the cluster is present as {2Fe-2S}(2+) with isomer shift, delta = 0.29 mm/s and quadrupole splitting, DeltaE(Q) = 0.53 mm/s, indicating incomplete cysteinyl-S coordination . Anaerobic reduction by dithionite in the presence of 55% (v/v) glycerol converts this form to {4Fe-4S}(2+) (delta = 0.45 mm/s and DeltaE(Q) = 1.11 mm/s) and is accompanied by some destruction to Fe(2+) . This cluster conversion is reversible and when exposed to air, the {4Fe-4S}(2+) cluster is quantitatively reconverted to the {2Fe-2S}(2+) cluster without any further cluster degradation. Brain Res, 1999 Sep 18, 842(1), 139 - 47 Lipopolysaccharide evokes the modulation of brain cytochrome P4501A in the rat; Renton KW et al.; The cytochrome P450 enzyme system is a multigene family of enzymes that is modulated in the liver during systemic inflammatory responses or during infection Several forms of the enzyme are expressed in discrete areas of the brain and likely play a critical role in the metabolism of drugs and endogenous chemicals in the central nervous system (CNS) . Even though the brain responds to inflammation in a manner different from most tissues, we examined the possible modification of a major cytochrome P450 form (CYP1A) in the brain during inflammation confined to that organ . Total brain CYP1A activity, as measured by ethoxyresorufin dealkylase (EROD), was downregulated 24 and 48 h following the administration of a single dose of lipopolysaccharide (LPS) . Regionally, a similar effect was determined in the cortex, hippocampus and the mid-brain but the activity in the cerebellum was unaffected . The examination of coronal brain sections using an antibody directed against CYP1A indicated that the enzyme was distributed in discrete cells of the hippocampus, thalamus and cortex and in the tanycytes surrounding the third ventricle . In each of these areas, the immunoreactivity was diminished in animals receiving LPS as compared to saline-treated animals . LPS also evoked the expression of the small molecular weight heat shock protein hsp27 throughout the brain indicating the development of an inflammatory response . These studies indicate that inflammation localized to the CNS causes an alteration in the levels and activity of a major cytochrome P450 form in the brain . This could have implications to the metabolism or activation of drugs and endogenous chemicals in the CNS during a disease state that features an inflammatory component. Arch Microbiol, 1999 Oct, 172(4), 233 - 9 The correlation of the gene csoS2 of the carboxysome operon with two polypeptides of the carboxysome in thiobacillus neapolitanus Baker SH, Lorbach SC, Rodriguez-Buey M, Williams DS, Aldrich HC, Shively JM. The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing . The first 17 amino acids of the two peptides were identical . The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon . The gene was subsequently named csoS2 . Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa . Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively . A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation . CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies . The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa. Diabetologia, 1999 Oct, 42(10), 1175 - 86 Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis; Davis EA et al.; AIMS/HYPOTHESIS: Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia . A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking . Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release . METHODS: Recombinant human glucokinase was generated in E . coli . The k(cat), glucose S(0.5), ATP K(m), and Hill number of glucokinase were determined . Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase . A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed . This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database . It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose . RESULTS: Large changes, varying greatly between mutants were found in nearly all variables . Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes . Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model . The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases . CONCLUSION/INTERPRETATION: A kinetics database of wild-type and 20 mutants of glucokinase was developed . Many kinetic differences were found for the mutants . The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations . {Diabetologia 42: 1175-1186} Methods, 1999 Sep, 19(1), 148 - 55 Mucosal delivery of vaccines; Del Giudice G et al.; Oral delivery represents one of the most pursued approaches for large-scale human vaccination . Due to the different characteristics of mucosal immune response, as compared with systemic response, oral immunization requires particular methods of antigen preparation and selective strategies of adjuvanticity . In this paper, we describe the preparation and use of genetically detoxified bacterial toxins as mucosal adjuvants and envisage the possibility of their future exploitation for human oral vaccines . J Mol Biol, 1999 Oct 1, 292(4), 931 - 44 Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation; Cole KB et al.; The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms . In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4 . 5S RNA . In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E . coli RNase P) in vitro for cleavage of a DNA substrate . In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation . A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA . This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates . This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E . coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates . M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding . The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule . This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available. J Mol Biol, 1999 Oct 1, 292(4), 855 - 69 Insight into odorant perception: the crystal structure and binding characteristics of antibody fragments directed against the musk odorant traseolide; Langedijk AC et al.; Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance . Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated . The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93 . M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala) . A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance . The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide . To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution . The crystal structure showed that only van der Waals interactions are involved in binding . The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket . This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives . The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved. J Mol Biol, 1999 Oct 1, 292(4), 787 - 96 The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI; Davies GP et al.; Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities . One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction . These activities involve ATP-dependent DNA translocation and DNA cleavage . Mutations that change amino acids within recognisable motifs in HsdR impair restriction . We have used an in vivo assay to monitor the effect of these mutations on DNA translocation . The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell . Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes . We find that the mutations abolish DNA translocation in vivo . This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation . Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity . When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels . It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems . Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo. J Mol Biol, 1999 Oct 1, 292(4), 771 - 8 Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA; Pugh GE et al.; The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA . We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity . Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted . These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA . We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested. Arch Biochem Biophys, 1999 Nov 1, 371(1), 70 - 82 Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes; Zhang XQ et al.; Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues . In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli . Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized . We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro . Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100 . In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK . This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping . Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties . However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta . Arch Biochem Biophys, 1999 Nov 1, 371(1), 35 - 40 Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy; Chae YK et al.; The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, l-tryptophan (Trp), at their intracellular concentrations . The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions . TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA . We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR . For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR . The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer . This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression . Arch Biochem Biophys, 1999 Nov 1, 371(1), 15 - 23 Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase:Fructose 2,6-bisphosphatase: effects on regulation; Wu RF et al.; Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase . The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner . To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined . Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase . These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation . In general, the effect was greater with amino acid residues located more distant from the N-terminus . The resulting changes were not as large as observed with the phosphorylated enzyme . Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities . These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities . Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase . Chem Res Toxicol, 1999 Oct, 12(10), 917 - 23 Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases; Sagi J et al.; We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases {Hang, B., Sagi, J., and Singer, B . (1998) J . Biol . Chem . 273, 33406-33413} . This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes . In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability . The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate . When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency . However, the difference in the cleavage rates between different sequences was much less than with the 15-mers . Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site . These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo. Nature, 1999 Oct 7, 401(6753), 610 - 3 Phytochrome signalling is mediated through nucleoside diphosphate kinase 2; Choi G et al.; Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light . Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths . Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E) . However, very little is known about how light signals are transduced to elicit responses in plants . Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana . In animal and human cells, NDPK acts as a tumour suppressor . We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2 . Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening . These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 356 - 64 Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1); Li D et al.; Genes encoding two manganese peroxidases from the white-rot basidiomycete Dichomitus squalens were cloned and sequenced . The mnp1 and mnp2 genes encode mature proteins of 369 and 365 amino acids, respectively . The amino acids involved in peroxidase function, those forming the Mn(II) binding site, and those forming the five disulfide bonds in other Mn peroxidases are conserved in these sequences . Both predicted D . squalens proteins contain multiple acidic residues in their C-terminal sequences, which may be involved in additional metal binding . Both genes contain seven small introns, the locations of which align with each other . The promoters of both D . squalens genes contain putative AP-2 sites, which may be involved in their regulation by nutrient nitrogen . Southern blot analysis of genomic PCR fragments suggests that these sequences represent separate genes rather than allelic variants. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 317 - 30 Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme; Kowlessur D et al.; The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan . A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli . This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue {1} . This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme . Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase . The recombinant tryptophan hydroxylase exists in solution as tetramers . The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan . The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates . Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3 . Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 284 - 95 cDNA cloning, overproduction and characterization of rat adrenodoxin reductase; Sagara Y et al.; We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR) . The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region . The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region . The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture . The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm . The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm . The absorbance ratio at 270 nm/450 nm was 7.1 . From the θ(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30% . In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM) . The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM) . The K(m) values showed a reverse correlation to the difference of pI values between the redox partners . These results indicate that AdR binds to Ad mainly by ionic interaction. Microbiol Immunol, 1999, 43(8), 765 - 71 Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1; Bayry J et al.; Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli . As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture . A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval . The sera were collected at intervals of 21, 42 and 63 days after the booster . The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT) . The guinea pig sera showed high titers both in ELISA and SNT, which could be protective . Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%) . The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization. Microbiol Immunol, 1999, 43(8), 743 - 9 Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase; Nguyen SV et al.; The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced . The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42% . The gene was expressed in E . coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever . The study results suggest that the C . burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever. Biotechniques, 1999 Oct, 27(4), 834 - 6, 838, 840 passim RecA-mediated affinity capture: a method for full-length cDNA cloning; Zhumabayeva B et al.; We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries . This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA . Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads . Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria . Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library . We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library. Biotechniques, 1999 Oct, 27(4), 790 - 6 Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA); Bouffard P et al.; We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs) . To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs . hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La) . Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled . Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA . The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones . RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA. Biotechniques, 1999 Oct, 27(4), 734 - 8 Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion; Li F et al.; DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact . Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion . A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containing dsDNA templates were used . This method compares favorably to the most efficient current methods, but is simpler and does not require the use of single-stranded templates or phage vectors. Mol Cell Biol, 1999 Nov, 19(11), 7828 - 40 The Gal3p-Gal80p-Gal4p transcription switch of yeast: Gal3p destabilizes the Gal80p-Gal4p complex in response to galactose and ATP; Sil AK et al.; The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation of GAL regulon genes . In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p . We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP . We have also found that retention of Gal80p by GSTG4AD (amino acids {aa} 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP . Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose . These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex . The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p . Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881. Mol Cell Biol, 1999 Nov, 19(11), 7461 - 72 Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA; Zebarjadian Y et al.; In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA . Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase . Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases . To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p . Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes . Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures . Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA . Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA . In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects . There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA . A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction . Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes. J Clin Microbiol, 1999 Nov, 37(11), 3475 - 80 Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay; Ikadai H et al.; A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B . caballi merozoite . A cDNA encoding a 48-kDa protein of B . caballi was cloned and designated BC48 . The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron . Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B . caballi genome . Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa . The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B . caballi 48-kDa protein . Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA) . The ELISA was able to differentiate very clearly between B . caballi-infected horse sera and B . equi-infected horse sera or noninfected normal horse sera . These results suggest that this simple and highly sensitive test might be applicable to the detection of B . caballi-infected horses in the field. J Basic Microbiol, 1999, 39(4), 237 - 42 Transcription level of operon ftsYEX and activity of promoter P1 of rpoH during the cell cycle in Escherichia coli; Gomez-Eichelmann MC et al.; In Escherichia coli, the genes of the main heat-shock proteins are under the control of the product of gene rpoH, protein sigma 32 . The distal promoter P1 of rpoH is located within the terminator of the division operon ftsYEX which could imply some coupling between ftsYEX transcription termination, P1 transcription and cell division . To study the possibility of this coupling, the level of transcription of ftsYEX and the activity of promoter P1 of rpoH were examined in synchronous cultures . Results indicate that transcription of ftsYEX and of rpoH from P1 is continuous, suggesting that ftsYEX transcription termination and P1 activity are not coupled to the cell cycle. EMBO J, 1999 Oct 15, 18(20), 5724 - 34 FtsK-dependent and -independent pathways of Xer site-specific recombination; Recchia GD et al.; Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division . Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers . Two recombinases, XerC and XerD, act at the E . coli chromosomal recombination site, dif, and at related sites in plasmids . We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors . We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif . The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers . Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates. Microbiol Res, 1999 Sep, 154(2), 179 - 83 The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way; Oh TJ et al.; The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and gamma-radiation . The induction levels of polB::lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, H2O2 or ethanol . Pentachlorophenol did not significantly induce any din genes . gamma-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ . Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD . We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner . The polB gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and H2O2 or nalidixic acid and ethanol . In addition, dinB and dinD in the presence of both nalidixic acid and H2O2 at the same time showed no synergistic responses. Biochemistry (Mosc), 1999 Sep, 64(9), 1079 - 88 Comparative structural-functional characterization of recombinant and natural adrenodoxin . Interaction with cytochrome P450scc; Lepesheva GI et al.; The conditions for heterologous expression of recombinant bovine adrenodoxin in E . coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium . A highly efficient method for purification of this recombinant ferredoxin from the E . coli cells has been developed . The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria . In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues . The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc . The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin . The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed. Biochemistry (Mosc), 1999 Sep, 64(9), 1021 - 9 Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro; Kalinin AE et al.; Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion . To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis . We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro . We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer. Mutat Res, 1999 Sep 15, 445(1), 127 - 35 Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line; Ryu J et al.; This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line . There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied . Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival . Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold . The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations . In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent . However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions . These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved. Mutat Res, 1999 Sep 15, 445(1), 93 - 8 Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain; Takimoto K et al.; We have previously reported that the majority of base substitution mutations of the Escherichia coli supF gene induced by riboflavin mediated photosensitization were G:C to C:G changes, in addition to G:C to T:A changes which were probably caused by 8-hydroxyguanine (oh(8)Gua), in wild type and mutM mutator mutant strains . This implies that lesions other than oh(8)Gua are produced by riboflavin-photosensitization . G:C to C:G base substitutions have been found in the mutations induced by ionizing radiation and reactive oxygen species, as well as spontaneous mutation . To characterize the G:C to C:G mutation, riboflavin- photosensitized plasmid DNA carrying the supF gene was left at room temperature for 5 h in the dark before transfection . The delayed transfection gave a mutational spectrum different from that for immediate transfection . G:C to C:G transversions significantly increased in mutY mutator strain, in which the transversion was not detected in the immediate transfection . Lesions causing G:C to C:G changes increased during 5-h holding after photosensitization and MutY protein presumably takes part in this type of base change mutation. Mutat Res, 1999 Aug 18, 444(2), 405 - 11 Impact of dimethyl sulfoxide and examples of combined genotoxicity in the SOS chromotest; Gebel T et al.; The bacterial SOS chromotest with Escherichia coli PQ37 was used for the assessment of genotoxicity of combined xenobiotic treatments . The modulation of test compound genotoxicity by dimethyl sulfoxide (DMSO), a common solvent for test compounds, was assessed as well . It was shown that DMSO modulated SOS chromotest genotoxicity of several xenobiotics: in comparison to test compound dissolution in water, the commonly used addition of 3.2% (v/v) DMSO as solvent lead to a significant increase in the genotoxicity of K(2)RhCl(5) and beta-propiolactone (BPL) . However, the effects of cisplatin decreased significantly when DMSO was added . Thus, albeit DMSO is not genotoxic in this test itself, it can interfere with SOS chromotest responses . Further experiments were performed in the absence of DMSO . BPL and cisplatin in combination showed an over-additive synergism in SOS genotoxicity as well as K(2)RhCl(5) and cisplatin did . Addition of Pd(NH(3))(4)Cl(2) and NaAsO(2), which are non-genotoxic in the SOS chromotest, did not enhance the K(2)RhCl(5)- or BPL-mediated SOS sfiA induction . Nevertheless, at the highest subcytotoxic dose of NaAsO(2) tested (200 microM), a slight yet significant suppression of BPL-mediated SOS genotoxicity was observed . These results confirm that the SOS chromotest is a useful tool for the rapid evaluation of the combined genotoxicity of compound mixtures . However, the use of DMSO as test solvent has to be taken with caution. Gene, 1999 Sep 17, 237(2), 333 - 42 pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens; Pandjaitan B et al.; We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity . Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus . As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins . All three recombinant allergens were readily detected in nitrocellulose-blotted E . coli extracts by the Bp36/51-specific moAb 4A6 . We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting . A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients . Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera . We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g . IgE), even when they occur in minute concentrations. J Biol Chem, 1999 Oct 22, 274(43), 31094 - 101 The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase; Revington M et al.; In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation . The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation . b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence . Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure . A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants . These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface. J Biol Chem, 1999 Oct 22, 274(43), 31034 - 8 The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA; Lutsenko E et al.; The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U.G mismatches (Gallinari, P., and Jiricny, J . (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug) . It has also been described to remove 3,N(4)-ethenocytosine (epsilonC) from epsilonC.G mismatches (Saparbaev, M., and Laval, J . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 8508-8513) . We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance . We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E . coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations . Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations . Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U.G and T.G mismatches, respectively, we conclude that Mug does not repair U.G or T.G mismatches in vivo . A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E . coli . Cell-free extracts from mug(+) ung cells show very little ability to remove uracil from DNA, but can excise epsilonC . The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E . coli that can remove this mutagenic adduct . Thus, the principal role of Mug in E . coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde. J Biol Chem, 1999 Oct 22, 274(43), 31020 - 4 Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant; Suzuki H et al.; Both the secAcsR11 and DeltasecG::kan mutations cause cold-sensitive growth, although the growth defect due to the latter mutation occurs in a strain-specific manner . Overexpression of pgsA encoding phosphatidylglycerophosphate synthase suppresses the growth defects of the two mutants . We investigated the mechanism underlying the pgsA-dependent suppression of the two mutations using purified mutant SecA and inverted membrane vesicles (IMVs) prepared from pgsA-overexpressing cells . The acidic phospholipid content increased by about 10% upon pgsA overexpression . This increase resulted in the stimulation of proOmpA translocation only when mutant SecA or SecG-depleted IMVs were used . The translocation-coupled ATPase activity of SecA was significantly defective with the mutant SecA or SecG-depleted IMVs, but it recovered to a near normal level when the acidic phospholipid level was increased . The stimulation of ATPase activity was observed only at low temperature . The steady-state level of membrane-inserted SecA was low with the mutant SecA or SecG-depleted IMVs, and it decreased further upon the increase in the acidic phospholipid content . However, the level of SecA insertion markedly increased upon the inhibition of SecA deinsertion by the addition of beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP), especially with IMVs containing increased levels of acidic phospholipids . These results indicate that the increase in the level of acidic phospholipids stimulates the SecA cycle in the two mutants by facilitating both the insertion and deinsertion of SecA. J Biol Chem, 1999 Oct 22, 274(43), 30995 - 9 Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli; Yokota N et al.; The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane . We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins . All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA . Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent . This LolA-independent release was also strictly dependent on the outer membrane sorting signal . A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events . The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient . The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane . The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane . From these results, we conclude that lipoproteins in E . coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane. J Biol Chem, 1999 Oct 22, 274(43), 30950 - 6 Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes; Wojtasek H et al.; The pheromone-binding protein (PBP) from Bombyx mori was expressed in Escherichia coli periplasm . It specifically bound radiolabeled bombykol, the natural pheromone for this species . It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems . However, in ion-exchange chromatography, multiple forms sometimes appeared . Attempts to separate them revealed that they could be converted into one another . Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0 . This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea . There was also no significant change in CD spectra in the presence of the pheromone . The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system . This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles . Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons . This pH-linked structural flexibility also explains the heterogeneity reported previously for B . mori PBP and other members of this class of proteins. J Biol Chem, 1999 Oct 22, 274(43), 30849 - 57 Glycine insertion in the hinge region of lactose repressor protein alters DNA binding; Falcon CM et al.; Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI) . Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly . All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns . In contrast, significant differences were observed in DNA binding properties . Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines . Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins . Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence. J Biol Chem, 1999 Oct 22, 274(43), 30534 - 9 Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ; Suh WC et al.; Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding . Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70 . We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ . Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis . In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK. J Biol Chem, 1999 Oct 22, 274(43), 30451 - 8 Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family; Knoops B et al.; Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa . Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidant enzyme B166 . Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes . Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identified Saccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase . Moreover, recombinant AOEB166 expressed in E . coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O(2) oxidation . The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body . Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences . Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles . Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes. J Biol Chem, 1999 Oct 22, 274(43), 30447 - 50 Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii; Gogos A et al.; A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized . The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence . In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG . mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone . Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity. Genes Dev, 1999 Oct 1, 13(19), 2594 - 603 Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase; Coburn GA et al.; The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs . We have developed a method for reconstituting a minimal degradosome from purified proteins . Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components . Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells . The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required . Rather, Rne coordinates the activation of RhlB dependent on a 3' single-stranded extension on RNA substrates . A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli. Biochemistry, 1999 Oct 12, 38(41), 13725 - 35 tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase; Bovee ML et al.; Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs . Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments . Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-{N-(L-histidyl)sulfamoyl}adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM) . By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA . Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS {Yan, W., Augustine, J., and Francklyn, C . (1996) Biochemistry 35, 6559}, which possesses a single amino acid change in the C-terminal anticodon binding domain . Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe) . Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions . Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA. Biochemistry, 1999 Oct 12, 38(41), 13530 - 41 Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex; Eubanks S et al.; Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor . To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented . The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor . Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex . Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10 . Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein. Biochemistry, 1999 Oct 12, 38(41), 13523 - 9 Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli; Quick M et al.; The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters . To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis . Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type) . The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered . Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions . Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions . These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport . The observations confirm the functional importance of TM II described in earlier studies {M . Quick and H . Jung (1997) Biochemistry 36, 4631-4636} . In contrast to Arg40, Arg117 is apparently not important for function of the mature protein . The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane . However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments . These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i . e., by stabilizing a folding intermediate. Biochemistry, 1999 Oct 12, 38(41), 13502 - 11 Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking; Williams NL et al.; Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling . Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented . In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked . These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling . Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation . This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction . The mechanistic implications of these results are discussed. DNA Seq, 1998, 9(3), 183 - 8 The tdcE gene in Escherichia coli strain W3110 is separated from the rest of the tdc operon by insertion of IS5 elements; Hesslinger C et al.; Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5 . Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes . The nucleotide sequence and insertion site of one of these elements, IS5P, was determined . It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene . In other E . coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon . Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy. Curr Eye Res, 1999 Oct, 19(4), 313 - 22 Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers; Conley CA et al.; PURPOSE . To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy . METHODS . Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits . Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue . RESULTS . Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle . Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle . The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits . CONCLUSIONS . The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism. J Mol Biol, 1999 Sep 3, 291(5), 1191 - 206 An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils; Alexandrescu AT et al.; At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils . 1H-15N HSQC, 3D HSQC-NOESY, and 15N T2 NMR experiments have been used to characterize the soluble components of this reaction . The kinetics of self-assembly show a lag phase followed by an exponential increase in polymerization . A single set of 1H-15N HSQC cross-peaks, corresponding to acid-denatured monomers, is observed during the entire course of the reaction . Under lag phase conditions, 15N resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration . The dependence of 15N T2 values on spin echo period duration demonstrates that line broadening is due to fast NMR exchange between acid-denatured monomers and soluble aggregates . Exchange contributions to T2 relaxation correlate with the squares of the chemical shift differences between native and acid-denatured CspA, and point to a stabilization of native-like structure upon aggregation . Time-dependent changes in 15N T2 relaxation accompanying the exponential phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggregate growth . CspA serves as a useful model system for exploring the conformational determinants of denatured protein misassembly. J Mol Biol, 1999 Sep 3, 291(5), 1181 - 90 Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography; Ringler P et al.; Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules . Aquaporins are present in animals, plants, algae, fungi and bacteria . Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli . A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity . Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a = b = 95 A, gamma = 90 degrees when reconstituted by dialysis in the presence of lipids . Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1) . The 8 A projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins. J Mol Biol, 1999 Sep 3, 291(5), 1129 - 34 Intrabody construction and expression . II . A synthetic catalytic Fv fragment; Ohage EC et al.; In general, proteins with structural disulfides cannot be expressed in the reducing environment of the cellular cytoplasm . To overcome this folding problem, we have previously engineered stabilizing mutations, predicted from a consensus sequence analysis, into isolated immunoglobulin VL domains . Here we show that such domains can be used as a framework in the construction of a functional heterodimeric Fv fragment, which was expressed solubly, with high yield in the cytoplasm of Escherichia coli . This designed catalytic intrabody, obtained from grafting the combining site of the esterolytic antibody 17E8, is active in the oxidized and the reduced state . Its construction required no special features on the part of the immunoglobulin, no single-chain linker and introduced no non-natural sequence motifs . The potential to design intrabodies with the recognition sequences of arbitrary immunoglobulins opens novel opportunities for gene therapy, cell biology, metabolic engineering and antibody biotechnology. J Mol Biol, 1999 Sep 3, 291(5), 1119 - 28 Intrabody construction and expression . I . The critical role of VL domain stability; Ohage E et al.; We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations . These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced . This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies) . The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield . Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue . Transcription from the strong T7 promoter does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained . Elevated temperature shifts protein from a productive folding pathway to aggregation . The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro . In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains. J Mol Biol, 1999 Sep 3, 291(5), 1105 - 18 The Cfr10I restriction enzyme is functional as a tetramer; Siksnys V et al.; It is thought that most of the type II restriction endonucleases interact with DNA as homodimers . Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow . Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation . The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown . Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer . To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed . Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer . In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme . Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule . Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites . These data are consistent with the tetramer being a functionally important form of Cfr10I. J Mol Biol, 1999 Sep 3, 291(5), 1017 - 23 Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120); Fucini P et al.; The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold . To define the architecture of the rod domain of gelation factor, we have expressed in E . coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy . Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric . Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation . Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6 . None of these polypeptides formed dimers after chemical crosslinking . Stable dimerisation therefore appears to require repeats 5 and 6 . Based on these data a model of gelation factor architecture is presented . We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor. FEBS Lett, 1999 Sep 10, 458(1), 60 - 4 Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms; Perez-Branguli F et al.; Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis . Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings . Previous morphological studies have revealed a characteristic co-localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known . In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms . The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination . The addition of SNAP-25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions. Eur J Biochem, 1999 Nov, 265(3), 1098 - 107 Pyruvate dehydrogenase from Azotobacter vinelandii . Properties of the N-terminally truncated enzyme; Hengeveld AF et al.; The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA . Previously, limited proteolysis experiments indicated that the N-terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) {Hengeveld, A . F., Westphal, A . H . & de Kok, A . (1997) Eur J . Biochem . 250, 260-268} . To further investigate this hypothesis N-terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized . Up to nine N-terminal amino acids could be removed from E1p without effecting the properties of the enzyme . Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p . The 48 amino acid deletion mutant (E1pdelta48) is catalytically fully functional: it has a Vmax value identical to that of wild-type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule . In contrast, the S0.5 for pyruvate is decreased . A heterodimer was constructed containing one subunit of wild-type E1p and one subunit of E1pdelta48 . From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N-terminal domains are needed for the binding of E1p to E2p . The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N-terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p. Eur J Biochem, 1999 Nov, 265(3), 1056 - 60 Cloning and expression of succinic semialdehyde reductase from human brain . Identity with aflatoxin B1 aldehyde reductase; Schaller M et al.; The neuromodulator gamma-hydroxybutyrate is synthesized in vivo from gamma-aminobutyrate by transamination to succinic semialdehyde and subsequent reduction of the aldehyde group . In human brain, succinic semialdehyde reductase is thought to be responsible for the conversion of succinic semialdehyde to gamma-hydroxybutyrate . In the present work, we cloned the cDNA coding for succinic semialdehyde reductase and expressed it in Escherichia coli . A data bank search indicated that the enzyme is identical with aflatoxin B1-aldehyde reductase, an enzyme implicated in the detoxification of xenobiotic carbonyl compounds . Structurally, succinic semialdehyde reductase thus belongs to the aldo-keto reductase superfamily . The recombinant protein was indistinguishable from native human brain succinic semialdehyde reductase by SDS/PAGE . In addition to succinic semialdehyde, it readily catalyzed the reduction 9,10-phenanthrene quinone, phenylglyoxal and 4-nitrobenzaldehyde, typical substrates of aflatoxin B1 aldehyde reductase . The results suggest multiple functions of succinic semialdehyde reductase/aflatoxin B1 aldehyde reductase in the biosynthesis of gamma-hydroxybutyrate and the detoxification of xenobiotic carbonyl compounds, respectively. Eur J Biochem, 1999 Nov, 265(3), 1022 - 31 Autoantibodies interacting with purified native thyrotropin receptor; Atger M et al.; Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells . An ELISA test was devised to study anti-TSHR autoantibodies directly . Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor . The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain . Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers . The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases . Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups . Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells . Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test . TSAb were only found in individuals with Graves' disease . The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation . Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects . Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease . What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation. Eur J Biochem, 1999 Nov, 265(3), 1015 - 21 Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi; Carret C et al.; In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA was cloned and sequenced . Protein sequence database searches suggested that the 41-kDa protein belongs to the phosphofructokinase B type family (PFK-B) . However, because of the low level sequence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further investigated using molecular modelling and enzymatic assays . The sequence/structure comparison of the protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding site . However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds rather than sugar ones . Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nM, whereas it was inactive on ribose or other carbohydrates . As enzymatic assays confirmed the results of the structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of the PFK-B family corresponds to an adenosine kinase from B . canis rossi . It was named BcrAK. Eur J Biochem, 1999 Nov, 265(3), 982 - 9 Postsynaptic short-chain neurotoxins from Pseudonaja textilis . cDNA cloning, expression and protein characterization; Gong N et al.; Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia . The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short-chain neurotoxins from other elapids . Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT-PCR of the venom gland mRNAs . The sequences of the corresponding proteins consist of 57-58 amino acid residues and display several unique features when compared with all known short-chain neurotoxins . Accordingly, they grouped separately in phylogenetic analysis . The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized . They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom . Thus, a new group of short-chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized. Eur J Biochem, 1999 Nov, 265(3), 950 - 6 Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli; Sawada N et al.; We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells . Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively . The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase . These results are quite similar to those of mouse 1alpha-hydroxylase . To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E . coli cells . The recombinant E . coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E . coli cells . Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined . Although no significant reduction in expression of these mutants was observed, none showed detectable activity . These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue. Eur J Biochem, 1999 Nov, 265(3), 936 - 43 Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha; Kukimoto I et al.; Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase . We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively . A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons . One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells . Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line . Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha . The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7. Eur J Biochem, 1999 Nov, 265(3), 919 - 28 Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences; Jeeves M et al.; The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity . Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial . In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants . Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies . Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA . The mtr and aroL operators have not been studied previously in vitro . For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes . We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants . This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes . In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex . Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed . This shows that the binding of the second dimer is sequence selective . We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences . This allows it to co-ordinately control promoters of different intrinsic strength . This mechanism may be of importance in a number of promoters that bind multiple effector molecules. FEMS Microbiol Lett, 1999 Oct 15, 179(2), 453 - 9 Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli; Takagi H et al.; Two cDNAs encoding feedback inhibition-insensitive serine acetyltransferases of Arabidopsis thaliana were expressed in the chromosomal serine acetyltransferase-deficient and L-cysteine non-utilizing Escherichia coli strain JM39-8 . The transformants produced 1600 to 1700 mg l(-1) of L-cysteine and L-cystine from glucose . The amount of these amino acids produced per cell was 30 to 60% higher than that of an E . coli strain carrying mutant serine acetyltransferase less sensitive to feedback inhibition. FEMS Microbiol Lett, 1999 Oct 15, 179(2), 367 - 73 Effect of specific production rate of recombinant protein on multimerization of plasmid vector and gene expression level; Saraswat V et al.; In fed-batch cultures of recombinant Escherichia coli BL21(DE3){pT7-G3IL2} at high cell concentration, the post-induction specific growth rate was carefully regulated by controlled medium feed to maximize the synthesis level of recombinant fusion interleukin-2, G3.IL-2 . A maximum concentration of G3.IL-2 (11.25 g l(-1)) was achieved in the induced recombinant culture growing at the rate of 0.056 h(-1) . A steep decrease in the expression level of G3.IL-2 was observed at the post-induction specific growth rates higher than its optimal value (0.056 h(-1)) . In the induced recombinant cultures, plasmid multimerization was observed and highly dependent on specific growth and production rate: a higher post-induction specific growth rate and an increased specific production rate tended to significantly promote it much further . Moreover, plasmid stability was found to decrease rapidly in a faster growing culture. Nucleic Acids Res, 1999 Nov 1, 27(21), 4235 - 40 RPA subunit arrangement near the 3'-end of the primer is modulated by the length of the template strand and cooperative protein interactions; Lavrik OI et al.; To analyze the interaction of human replication protein A (RPA) and its subunits with the DNA template-primer junction in the DNA replication fork, we designed several template-primer systems differing in the size of the single-stranded template tail (4, 9, 13, 14, 19 and 31 nt) . Base substituted photoreactive dNTP analogs-5-{ N -(2-nitro-5-azidobenzoyl)- trans -3-amino-propenyl-1}-2'-deoxyuridine-5'-triphosphate (NAB-4-dUTP) and 5-{ N -{ N -(2-nitro-5-azidobenzoyl)glycyl}- trans -3-aminopropenyl-1}-2'-deoxyuridine-5'-triphosphate (NAB-7-dUTP)-were used as substrates for elongation of radiolabeled primer-template by DNA polymerases in the presence or absence of RPA . Subsequent UV crosslinking showed that the pattern of p32 and p70 RPA subunit labeling, and consequently their interaction with the template-primer junction, is strongly dependent on the template extension length at a particular RPA concentration, as well as on the ratio of RPA to template concentration . Our results suggest a model of changes in the RPA configuration modulating by the length of the template extension in the course of nascent DNA synthesis. Nucleic Acids Res, 1999 Nov 1, 27(21), 4200 - 7 Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli; Zhang XJ et al.; The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in previous work to have two domains: an N-terminal 100 kDa domain with ATP-dependent helicase activity, and a C-terminal 30 kDa domain . The 30 kDa domain had nuclease activity when linked to a heterologous DNA binding protein, but by itself it appeared unable to bind DNA and lacked detectable nuclease activity . We have expressed and isolated this 30 kDa domain, called RecB(N), and show that it does have nuclease activity detectable at high protein concentration in the presence of polyethylene glycol, added as a molecular crowding agent . The activity is undetectable in a mutant RecB(N)protein in which an aspartate residue has been changed to alanine . Structural analysis of the wild-type and mutant RecB(N)proteins by second derivative absorbance and circular dichroism spectroscopy indicates that both are folded proteins with very similar secondary and tertiary structures . The results show that the Asp-->Ala mutation has not caused a significant structural change in the isolated domain and they support the conclusion that the C-terminal domain of RecB has the sole nuclease active site of RecBCD. Nucleic Acids Res, 1999 Nov 1, 27(21), 4167 - 74 PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus . I . Purification and identification of the homing-type endonuclease activities; Komori K et al.; We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction . The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning . These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively . The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases . The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II . They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins . The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined . The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein. Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 12156 - 61 Mimicry of erythropoietin by a nonpeptide molecule; Qureshi SA et al.; Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells . EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells . To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR . We first identified compound 1 (N-3-{2-(4-biphenyl)-6-chloro-5-methyl}indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core . Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR . Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells . Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage . These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO. Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11922 - 7 Human and mouse homologs of Escherichia coli DinB (DNA polymerase IV), members of the UmuC/DinB superfamily; Gerlach VL et al.; To understand the mechanisms underlying mutagenesis in eukaryotes better, we have cloned mouse and human homologs of the Escherichia coli dinB gene . E . coli dinB encodes DNA polymerase IV and greatly increases spontaneous mutations when overexpressed . The mouse and human DinB1 amino acid sequences share significant identity with E . coli DinB, including distinct motifs implicated in catalysis, suggesting conservation of the polymerase function . These proteins are members of a large superfamily of DNA damage-bypass replication proteins, including the E . coli proteins UmuC and DinB and the Saccharomyces cerevisiae proteins Rev1 and Rad30 . In a phylogenetic tree, the mouse and human DinB1 proteins specifically group with E . coli DinB, suggesting a mitochondrial origin for these genes . The human DINB1 gene is localized to chromosome 5q13 and is widely expressed. Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11764 - 9 Correlation of deformability at a tRNA recognition site and aminoacylation specificity; Chang KY et al.; The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA . These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA . In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA . We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition . Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells . A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations . Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid . The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase . Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity. FEBS Lett, 1999 Oct 8, 459(2), 233 - 7 Production of fungal fructosyl amino acid oxidase useful for diabetic diagnosis in the peroxisome of Candida boidinii; Sakai Y et al.; A high-level production of fructosyl amino acid oxidase (FAOD), whose production was toxic in Escherichia coli, was investigated through attempts to utilize the peroxisome of Candida boidinii as the place for protein accumulation . The alcohol oxidase-depleted strain (strain aod1Delta) produced FAOD at a four to five times higher level than the wild type strain in terms of protein amount and enzyme activity, although the transcriptional level was similar . As a result of this study, we could improve FAOD productivity approximately 47-fold from the original transformant, and FAOD accumulated within membrane-bound peroxisomes up to 18% of the total soluble proteins. FEBS Lett, 1999 Oct 8, 459(2), 223 - 6 Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway(1); Collard F et al.; We report the sequence of a human cDNA encoding a protein homologous to devB (a bacterial gene often found in proximity to the gene encoding glucose-6-phosphate dehydrogenase in bacterial genomes) and to the C-terminal part of human hexose-6-phosphate dehydrogenase . The protein was expressed in Escherichia coli, purified and shown to be 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway . Sequence analysis indicates that bacterial devB proteins, the C-terminal part of hexose-6-phosphate dehydrogenase and yeast Sol1-4 proteins are most likely also 6-phosphogluconolactonases and that these proteins are related to glucosamine-6-phosphate isomerases. FEBS Lett, 1999 Oct 8, 459(2), 186 - 90 Association of the proto-oncogene product dbl with G protein betagamma subunits; Nishida K et al.; The Rho family of GTP-binding proteins has been implicated in the regulation of various cellular functions including actin cytoskeleton-dependent morphological change . Its activity is directed by intracellular signals mediated by various types of receptors such as G protein-coupled receptors . However, the mechanisms underlying receptor-dependent regulation of Rho family members remain incompletely understood . The guanine nucleotide exchange factor (GEF) Dbl targets Rho family proteins thereby stimulating their GDP/GTP exchange, and thus is believed to be involved in receptor-mediated regulation of the proteins . Here, we show the association of Dbl with G protein betagamma subunits (Gbetagamma) in transient co-expression and cell-free systems . An amino-terminal portion conserved among a subset of Dbl family proteins is sufficient for the binding of Gbetagamma . In fact, Ost and Kalirin, which contain this Gbetagamma-binding motif, also associate with Gbetagamma . c-Jun N-terminal kinase was synergistically activated upon co-expression of Dbl and Gbeta in a dominant-negative Rho-sensitive manner . However, GEF activity of Dbl toward Rho as measured by in vitro GDP binding assays remained unaffected following Gbetagamma binding, suggesting that additional signals may be required for the regulation of Dbl. Mutat Res, 1999 Jul 16, 428(1-2), 115 - 24 Inter-individual differences in the metabolism of environmental toxicants: cytochrome P450 1A2 as a prototype; Guengerich FP et al.; Cytochrome P450 (P450) 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens . The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals . Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities . The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines . Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein . Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines . A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy . A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions . These results may be of relevance in consideration of genetic polymorphisms . Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others . Some dinitropyrenes are P450 1A2 substrates but others are not . 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead. Plant Physiol, 1999 Oct, 121(2), 589 - 97 Plant succinic semialdehyde dehydrogenase . Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides; Busch KB et al.; Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the gamma-aminobutyric acid shunt . We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1 . SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH from Escherichia coli and human (>59% identity) . A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD) . The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD . Fractionation of plant mitochondria revealed its localization in the matrix . The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K(0.5) = 15 microM), and exclusively used NAD+ as a cofactor (Km = 130 +/- 77 microM) . NADH was a competitive inhibitor with respect to NAD+ (Ki = 122 +/- 86 microM) . AMP, ADP, and ATP inhibited the activity of SSADH (Ki = 2.5-8 mM) . The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+ . Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of gamma-aminobutyric acid. Plant Physiol, 1999 Oct, 121(2), 373 - 82 Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon; Kang HG et al.; To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases . The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs . RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds . The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues . In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds . The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene . In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds. Microbiology, 1999 Sep, 145 ( Pt 9), 2543 - 8 Thermoprotection by glycine betaine and choline; Caldas T et al.; Glycine betaine is mostly known as an osmoprotectant . It is involved in the osmotic adaptation of eukaryotic and bacterial cells, and accumulates up to 1 M inside cells subjected to an osmotic upshock . Since, like other osmolytes, it can act as a protein stabilizer, its thermoprotectant properties were investigated . In vitro, like protein chaperones such as DnaK, glycine betaine and choline protect citrate synthase against thermodenaturation, and stimulate its renaturation after urea denaturation . In vivo, the internal concentration of glycine betaine is neither increased nor decreased after heat shock (this contrasts with a massive increase after osmotic upshock) . However, even in exponential-phase bacteria grown in usual minimal salts media, the internal glycine betaine concentration attains levels (around 50 mM) which can protect proteins against thermodenaturation in vitro . Furthermore, glycine betaine and choline restore the viability of a dnaK deletion mutant at 42 degrees C, suggesting that glycine betaine not only acts as a thermoprotectant in vitro, but also acts as a thermoprotectant for Escherichia coli cells in vivo. Microbiology, 1999 Sep, 145 ( Pt 9), 2507 - 18 The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator; Mulder MA et al.; An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter . A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E . coli and Mycobacterium smegmatis . A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies . Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon . Putative promoters associated with these show similarity to previously identified mycobacterial promoters . Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E . coli, and is required for optimal activity in M . smegmatis . The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E . coli and 12-fold in M . smegmatis . An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent . Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase . The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures . The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M . smegmatis. Microbiology, 1999 Sep, 145 ( Pt 9), 2485 - 95 Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1); Marolda CL et al.; In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases . In this work the complete DNA sequence of a 6.9 kb intervening region is presented . Seven new ORFs were identified . All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria . Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases . The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster . O7-specific DNA primers were designed and tested for serotyping of O7 E . coli strains. Microbiology, 1999 Sep, 145 ( Pt 9), 2453 - 61 Disruption of tonB in Bordetella bronchiseptica and Bordetella pertussis prevents utilization of ferric siderophores, haemin and haemoglobin as iron sources; Nicholson ML et al.; The Bordetella bronchiseptica tonB gene was cloned by detection of a chromosomal restriction fragment hybridizing with each of two degenerate oligonucleotides that corresponded to Pro-Glu and Pro-Lys repeats characteristic of known TonB proteins . The tonB(Bb) gene was situated upstream of exbB and exbD homologues and downstream of a putative Fur-regulated promoter . Hybridization results indicated that the tonB operon and flanking regions were highly conserved between B . bronchiseptica, Bordetella pertussis and Bordetella parapertussis . Disruption of tonB in B . bronchiseptica resulted in inability to grow in iron-limiting media, and inability to utilize alcaligin, enterobactin, ferrichrome, desferroxamine B, haemin and haemoglobin . Although it was not possible to inactivate tonB in a clinical B . pertussis isolate, tonB was disrupted in a laboratory B . pertussis strain previously selected for the ability to grow on Luria-Bertani medium . This B . pertussis tonB mutant shared a similar iron complex utilization deficient phenotype with the B . bronchiseptica tonB mutant . The B . bronchiseptica tonB operon present on a plasmid did not complement an Escherichia coli tonB mutant, but inefficient reconstitution of enterobactin utilization was observed in one fepA mutant harbouring plasmid copies of the B . pertussis fepA homologue and tonB(Bb) operon. Microbiology, 1999 Sep, 145 ( Pt 9), 2385 - 91 RheA, the repressor of hsp18 in Streptomyces albus G; Servant P et al.; In Streptomyces albus, Hsp18, a protein belonging to the family of small heat-shock proteins, can be detected only at high temperature . Disruption of orfY, located upstream and in the opposite orientation to hsp18, resulted in an elevated level of hsp18 mRNA at low temperature . Genetic and biochemical experiments indicated that the product of orfY, now called RheA (Repressor of hsp eighteen), directly represses hsp18 . In Escherichia coli, an hsp18'-bgaB transcriptional fusion was repressed in a strain expressing S . albus RheA . DNA-binding experiments with crude extracts of E . coli overproducing RheA indicated that RheA interacts specifically with the hsp18 promoter . Transcription analysis of rheA in the S . albus wild-type and in rheA mutant strains suggested that RheA represses transcription not only of hsp18 but also of rheA itself. Microbiology, 1999 Sep, 145 ( Pt 9), 2293 - 301 Evidence that a single EF-Ts suffices for the recycling of multiple and divergent EF-Tu species in Streptomyces coelicolor A3(2) and Streptomyces ramocissimus; Hoogvliet G et al.; The tsf genes from Streptomyces coelicolor A3(2) and Streptomyces ramocissimus, encoding the guanine-nucleotide exchange factor EF-Ts, were cloned and sequenced . Streptomycetes have multiple and highly divergent EF-Tu species, with EF-Tu1 and EF-Tu3 showing only about 65% amino acid sequence identity, and yet these can apparently interact with a single EF-Ts species . tsf lies in an operon with rpsB, which encodes ribosomal protein S2 . The amino acid sequence of S2 from S . coelicolor differs from most other bacterial S2 homologues in having a C-terminal extension of 70 aa residues with a highly repetitive organization, the function of which is unknown . Transcription analysis of the rpsB-tsf operon of S . coelicolor by promoter probing, nuclease S1 mapping and Northern blotting revealed that the genes give rise to a bicistronic transcript from a single promoter upstream of rpsB . An attenuator was identified in the rpsB-tsf intergenic region; it results in an approximately 2:1 ratio of rpsB vs tsf transcripts . Although tuf1, encoding the major EF-Tu, is located in the rpsL ribosomal protein operon, an additional promoter in the fus-tuf1 intergenic region leads to a significant excess of EF-Tu over ribosomes . Most amino acid residues known from the Escherichia coli crystal structure of the EF-Tu-EF-Ts complex to be directly involved in interaction between the two elongation factors are conserved between E . coli and Streptomyces . However, whenever interaction residues in the EF-Tu moiety show divergence among Streptomyces EF-Tu1, EF-Tu2 and EF-Tu3, the single Streptomyces EF-Ts exhibits compensatory substitutions of the corresponding residues . These apparently enable productive interaction to occur with all three EF-Tus. Microbiology, 1999 Sep, 145 ( Pt 9), 2255 - 63 Four genes encoding different type I signal peptidases are organized in a cluster in Streptomyces lividans TK21; Parro V et al.; Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21 . Three of the sip genes constitute an operon and the fourth is the first gene of another operon encompassing three additional, unrelated genes . A DNA fragment containing the four sip genes complemented an Escherichia coli type I signal peptidase mutant when cloned in a multicopy plasmid . Clustering of four different type I signal peptidase genes seems, so far, to be a unique feature of Streptomyces. Microbiology, 1999 Sep, 145 ( Pt 9), 2221 - 7 Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2); Sun J et al.; The enhanced green fluorescent protein (EGFP) gene is a modified version of the green fluorescent protein gene of the jellyfish Aequorea victoria with a codon usage that corresponds well to that found in many GC-rich streptomycete genes . Here the use of EGFP as a reporter for the analysis of spatially and temporally regulated gene expression in Streptomyces coelicolor A3(2) is demonstrated . The EGFP gene was inserted into plasmids that can replicate in Escherichia coli, greatly facilitating the construction of EGFP gene fusions . The plasmids can be transferred readily to S . coelicolor by conjugation, whereupon two of them (pIJ8630 and pIJ8660) integrate at the chromosomal attachment site for the temperate phage phiC31 . These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a sigma factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by S . coelicolor . While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redD occurred only in ageing substrate mycelium . A further plasmid derivative (pIJ8668) was made that lacks the phiC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations. Mol Gen Genet, 1999 Sep, 262(2), 342 - 50 The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress; Skorko-Glonek J et al.; The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock . The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress . We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous {Fe(II)} ions, which are known to enhance oxidation of proteins . Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm . Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells . Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (40H-TEMPO) that acts mainly in the soluble fraction of the cell . Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol . Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA . Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes. Mol Gen Genet, 1999 Sep, 262(2), 302 - 9 The SOS response is induced by replication fork blockage at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites; Taki K et al.; A new model system for the study of the SOS response has been developed . In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids . Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter . SOS induction can, therefore, be controlled by arabinose . The extent of the SOS response was monitored by measuring the activity of beta-galactosidase, expressed from a lacZ gene fused to the 5' region of the sfiA gene, a typical SOS-responsive gene . Expression of the fusion gene is completely dependent on recA+ and lexA+ genes . Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction . The properties of this system are discussed. Anal Chem, 1999 Oct 1, 71(19), 4423 - 6 Detection of low dose radiation induced DNA damage using temperature differential fluorescence assay; Rogers KR et al.; A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported . Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation . Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses . The double-strand, sensitive dye PicoGreen was used as an indicator of DNA denaturation . Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng of double stranded DNA (10(6) copies for plasmid puc 19) are detected by this method. Anal Chem, 1999 Oct 1, 71(19), 4321 - 7 Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants; Lewis JC et al.; A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample . The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates . Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired . The newly constructed plasmids were transformed into E . coli for expression of the fusion proteins . By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced . These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins . The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format . To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay. Mol Plant Microbe Interact, 1999 Oct, 12(10), 894 - 900 Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA; Liu H et al.; Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus . To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector . Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells . Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein . We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA . To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells . Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E . coli proteins alone . These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus. Ren Fail, 1999 Sep, 21(5), 477 - 82 An animal model of septicemia-induced hypercatabolic acute renal failure; Wu ZL et al.; A rat model of hypercatabolic acute renal failure (ARF) was developed in order to further investigate the mechanism of this condition . Sprague Dawley rats were separated into three groups: a septicemic group, an ischemic ARF group, and a hypercatabolic ARF group . Septicemia was produced by the i.p . injection of 1 x 10(7) colony-forming units/mL of Escherichia coli . Ischemic ARF was induced by 60 minutes clamping of the left renal artery following a contralateral nephrectomy . Hypercatabolic ARF was produced by combining ischemic ARF with the i.p . injection of 1 x 10(7) colony-forming units/mL of Escherichia coli . The hypercatabolic ARF group exhibited septic clinical features after the surgical procedures . The blood urea nitrogen and the serum creatinine, potassium and carbon dioxide combining power of hypercatabolic ARF were significantly higher than other two groups 24 hours after surgery . In addition, the rats wit hypercatabolic ARF had a greater loss of body weight and a higher mortality rate compared to the other two groups . The features of this form of experimental ARF are similar to the clinical characteristics of hypercatabolic ARF . Consequently, this appears to be a useful model of hypercatabolic ARF. Am J Physiol, 1999 Oct, 277(4 Pt 2), F599 - 610 Ksp-cadherin gene promoter . II . Kidney-specific activity in transgenic mice; Igarashi P et al.; Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney . To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice . Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection . Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice . Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin . In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin . Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site . However, heterocellular expression was observed consistent with repeat-induced gene silencing . We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo . Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site. J Virol, 1999 Nov, 73(11), 9098 - 109 Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus; Vazquez MI et al.; The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process . A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells . We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I . Vazquez, G . Rivas, D . Cregut, L . Serrano, and M . Esteban, J . Virol . 72:10126-10137, 1998) . To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter . Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV) . Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region . Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate . Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread. J Bacteriol, 1999 Oct, 181(20), 6547 - 51 Escherichia coli Lrp (leucine-responsive regulatory protein) does not directly regulate expression of the leu operon promoter; Landgraf JR et al.; Studies by R . Lin et al . (J . Bacteriol . 174:1948-1955, 1992) suggested that the Escherichia coli leu operon might be a member of the Lrp regulon . Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in leu operon expression between lrp(+) and lrp strains . Furthermore, when leuP-lacZ transcriptional fusions that lacked the leu attenuator were used, expression from the leu promoter varied less than twofold between lrp(+) and lrp strains, irrespective of whether or not excess leucine was added to the medium . The simplest explanation of the observations of Lin et al . is that the known elevated leucine transport capacity of lrp strains (S . A . Haney et al., J . Bacteriol . 174:108-115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of leu operon expression. J Bacteriol, 1999 Oct, 181(20), 6524 - 9 Purification of P(II) and P(II)-UMP and in vitro studies of regulation of glutamine synthetase in Rhodospirillum rubrum; Johansson M et al.; The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography . The uridylylated form of the P(II) protein could be generated in E . coli . The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R . rubrum grown under different conditions . P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate . Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E . coli system . In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated. J Bacteriol, 1999 Oct, 181(20), 6419 - 24 MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli; Raskin DM et al.; By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in Escherichia coli . MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell . We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell's middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle . Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ . The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion. J Bacteriol, 1999 Oct, 181(20), 6411 - 8 Mechanism of repression of the aroP P2 promoter by the TyrR protein of Escherichia coli; Yang J et al.; Previously, we have shown that expression of the Escherichia coli aroP P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P . Wang, J . Yang, and A . J . Pittard, J . Bacteriol . 179:4206-4212, 1997) . Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the aroP P2 promoter . In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes . We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression . The TyrR binding site of aroP, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2 . Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression. J Bacteriol, 1999 Oct, 181(20), 6377 - 86 Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge; Franco PJ et al.; Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V) . While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na(+)-coupled transport . Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier . Eight revertant substitutions were isolated from the R52S strain: Asp-19-->Gly, Asp-55-->Asn, Pro-60-->Gln, Trp-116-->Arg, Asn-244-->Ser, Ser-247-->Arg, Asn-248-->Lys, and Ile-352-->Val . Two revertants were also isolated from the R52V strain: Trp-116-->Arg and Thr-338-->Arg revertants . The R52Q strain yielded an Asp-55-->Asn substitution and a first-site revertant, Lys-52 (R52K) . The R52K strain had transport properties similar to those of the wild type . Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116-->Arg, Ser-247-->Arg, and Asn-248-->Lys revertants regained significant Na(+)-coupled accumulation . In general, downhill melibiose transport in the presence of Na(+) was better in the revertant strains than in the parental mutants . Three revertant strains, Asp-19-->Gly, Asp-55-->Asn, and Thr-338-->Arg strains, required a high Na(+) concentration (100 mM) for maximal activity . Kinetic measurements showed that the N248K and W116R revertants lowered the K(m) for melibiose, while other revertants restored transport velocity . We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII . We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I). J Bacteriol, 1999 Oct, 181(20), 6361 - 70 Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid; Ali Azam T et al.; The genome DNA of Escherichia coli is associated with about 10 DNA-binding structural proteins, altogether forming the nucleoid . The nucleoid proteins play some functional roles, besides their structural roles, in the global regulation of such essential DNA functions as replication, recombination, and transcription . Using a quantitative Western blot method, we have performed for the first time a systematic determination of the intracellular concentrations of 12 species of the nucleoid protein in E . coli W3110, including CbpA (curved DNA-binding protein A), CbpB (curved DNA-binding protein B, also known as Rob {right origin binding protein}), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td mutant phenotype A) . Intracellular protein levels reach a maximum at the growing phase for nine proteins, CbpB (Rob), DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp, and StpA, which may play regulatory roles in DNA replication and/or transcription of the growth-related genes . In descending order, the level of accumulation, calculated in monomers, in growing E . coli cells is Fis, Hfq, HU, StpA, H-NS, IHF*, CbpB (Rob), Dps*, Lrp, DnaA, IciA, and CbpA* (stars represent the stationary-phase proteins) . The order of abundance, in descending order, in the early stationary phase is Dps*, IHF*, HU, Hfq, H-NS, StpA, CbpB (Rob), DnaA, Lrp, IciA, CbpA, and Fis, while that in the late stationary phase is Dps*, IHF*, Hfq, HU, CbpA*, StpA, H-NS, CbpB (Rob), DnaA, Lrp, IciA, and Fis . Thus, the major protein components of the nucleoid change from Fis and HU in the growing phase to Dps in the stationary phase . The curved DNA-binding protein, CbpA, appears only in the late stationary phase . These changes in the composition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the genome functions. J Bacteriol, 1999 Oct, 181(20), 6306 - 11 In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions; Bouveret E et al.; The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity . PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan . All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan . Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan . We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation . Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan . A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan . Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive . Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan . This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan. J Bacteriol, 1999 Oct, 181(20), 6292 - 9 Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli; Bechtloff D et al.; The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in Escherichia coli . An intR1 strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated oriC, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins . Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins . The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the intR1 strain . The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication . These three proteins were analyzed by peptide mass mapping and appeared to be the products of the dps, gapA, and pyrI genes . Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear. J Bacteriol, 1999 Oct, 181(20), 6284 - 91 Mutation analysis of the 5' untranslated region of the cold shock cspA mRNA of Escherichia coli; Yamanaka K et al.; The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA . The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock . In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression . It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C . However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation . A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase . Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase . In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct . The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence . The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence . The relationship between the mRNA secondary structure and translation efficiency is discussed. J Bacteriol, 1999 Oct, 181(20), 6278 - 83 rpoS function is essential for bgl silencing caused by C-terminally truncated H-NS in Escherichia coli; Ohta T et al.; From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions . H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing . However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability . Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS . To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon . These isolated mutations were mapped to five loci on the chromosome . Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel . Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively . The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein . It was found that sigma(S) is defective in both types of mutant cells . These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study . We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed . Our results did not support the idea that StpA has an adapter function in the genetic background used. Mol Cells, 1999 Aug 31, 9(4), 422 - 8 Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli; Kim TS et al.; A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E . coli selenophosphate synthetase gene . Unlike the E . coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA . Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells . To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E . coli selD deficient mutant, MB08 . Like the E . coli wild type selD gene, the mutant sps2 gene complemented the selD mutation . However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation . The SPS2-CYS protein expressed in E . coli was purified and its catalytic activity was determined . The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein . These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread. Mol Cells, 1999 Aug 31, 9(4), 358 - 64 A new autoantigen reactive with prediabetic nonobese diabetic mice sera; Kang Y et al.; The identification and characterization of new autoantigens would widen the knowledge of the pathogenic mechanism of insulin dependent diabetes mellitus . Screening of lambda gt11 mouse insulinoma (MIN6N8a) cell cDNA library with prediabetic nonobese diabetic (NOD) mice sera resulted in the isolation of a strong positive clone, named the clone 3-5, of 1579 nucleotides without a poly A region . After 5'-rapid amplification of the cDNA end (RACE), complete nucleotide sequence of the clone 3-5 gene consisting of 2231 nucleotides showed that the 3-5 gene had the theoretical open reading frame of 634 amino acids . However, the real antigenic protein of the clone 3-5 was only 21 amino acids long encoded by only 63 nucleotides . The 21 amino acids were expressed as a fusion protein in E . coli and purified by affinity chromatography . The purified 3-5 recombinant protein was examined for its reactivity with prediabetic NOD mice sera by immunoblotting . The only non-denatured form of the 3-5 protein showed a binding reactivity with NOD mice sera, demonstrating that the conformational epitope of 3-5 protein was important for antibody recognition . The prevalence of autoantibody reactive to the 3-5 protein was about 78% (14/18) and 46% (11/24) in prediabetic and acute diabetic NOD mice sera, respectively . However, the sera from other mouse strains such as BALB/c, ICR, C57BL/6, SJL/J, and NOD/SCID did not show a positive reactivity to the 3-5 protein, which indicated that immune reactivity against the 3-5 protein was autoimmune diabetic mouse-specific. Free Radic Biol Med, 1999 Oct, 27(7-8), 764 - 72 Possible role of RAC-GTPase-activating protein in the termination of superoxide production in phagocytic cells; Szaszi K et al.; The mechanism leading to the termination of superoxide production of phagocytes is poorly understood . The aim of the present study was to investigate the involvement of the active (GTP-bound) form of the GTP-binding proteins in maintaining continuous electron transport through the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex . Activation of the enzyme was carried out under in vitro conditions and a shift from the active to the inactive form of the GTP-binding protein was attained (i) by addition of an excess of GDP to the assembled enzyme complex or (ii) by variation of the Rac-GTPase activating (Rac-GAP) capacity of the constituents of the cell-free system . Significant inhibition of O2*- production was observed when guanine dinucleotides were added after the assembly of the active enzyme complex . The effect was specific for GDP and GDP,S whereas ADP, CDP and UDP were ineffective . GTP was significantly less efficient in inducing superoxide production in a cell-free system containing endogenous GAP activity than in a system devoid of GAP activity . It is suggested that the active, GTP-bound form of Rac is required for sustained catalytic function and Rac-GAP proteins are involved in the downregulation of the oxidase. Hum Gene Ther, 1999 Sep 20, 10(14), 2365 - 72 Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits; Malecaze F et al.; The most common complication of cataract surgery is the development of posterior capsule opacification (PCO) . Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule . Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment . To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO . The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo . In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001) . These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO. Poult Sci, 1999 Sep, 78(9), 1275 - 82 Effects of fumonisin B1 on selected immune responses in broiler chicks; Li YC et al.; Three experiments were conducted to evaluate immune responses in chicks fed fumonisin B1 (FB1) . Day-old male chicks were randomly allotted to dietary treatments: 0, 50, 100, or 200 mg FB1/kg diet . In Experiment 1, chicks were fed diets for 3 wk and were injected intravenously with 4.6x10(6) Escherichia coli on Day 21 . Blood samples were collected at 60, 120, and 180 min postinjection, and liver, spleen, and lung were collected after 180 min . Chicks fed 200 mg FB1/kg diet had significantly higher numbers of bacterial colonies in blood, spleen, and liver (P<0.05) than control chicks . In Experiment 2, chicks were placed on the diets for 4 wk and were injected with 0.5 mL inactivated Newcastle Disease virus vaccine on Weeks 2 and 3 of the experiment, and primary and secondary antibody titers were measured 7 d after each injection . The secondary antibody response in chicks fed 200 mg FB1/kg diet was significantly lower (P<0.05) than that of control chicks . In Experiment 3, lymphocyte proliferation in chicks exposed to FB1 in vivo or in vitro was determined . Results of the in vivo study showed that cell proliferation in response to mitogens was lower (P<0.05) in chicks fed 200 mg FB1/kg diet than in control chicks . For the in vitro study, cell proliferation was lower (P<0.05) when cells were exposed to > or = 2.5 microg FB1/mL . Data of the current study suggested that FB1 is immunosuppressive in chicks when present in the ration at 200 mg FB1/kg diet. Eur J Oral Sci, 1999 Oct, 107(5), 352 - 9 Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides; Li W et al.; Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin . The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP) . MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli . The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography . The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments . The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme . Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20 . Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP . These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin. J Chromatogr A, 1999 Sep 3, 855(1), 203 - 13 Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography; Muller C et al.; A procedure for renaturation of heterodimeric platelet-derived growth factor (PDGF-AB) from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography is described . Either prepurified or crude PDGF-AB inclusion bodies solubilized with guanidinium hydrochloride were subjected to buffer exchange from denaturing to renaturing conditions during chromatography . Renaturation of PDGF-AB involves folding of the solubilized and unfolded molecules into dimerization competent monomers during size-exclusion chromatography and subsequent dimerization of folded monomers into the biologically active heterodimeric growth factor . Optimized conditions result in an overall yield of 75% active PDGF-AB with respect to size-exclusion chromatography and subsequent dimerization . The described approach allows renaturation at high protein concentrations and circumvents aggregation which is observed when refolding is carried out by dilution. J Biol Chem, 1999 Oct 15, 274(42), 30019 - 22 Substrate gating confers steroid specificity to estrogen sulfotransferase; Petrotchenko EV et al.; Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA) . To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST . Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities . Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant . The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased . The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side . The present mutagenesis study indicates that the 3beta-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C-19 methyl group of DHEA . Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST. J Biol Chem, 1999 Oct 15, 274(42), 29912 - 20 An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum . Identification of a MAPK signature; Dorin D et al.; The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli . The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms . The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts . Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity . Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector . Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity . Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location . This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2 . The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting. J Biol Chem, 1999 Oct 15, 274(42), 29883 - 8 SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein; Scotti PA et al.; In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described . In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP . This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY . Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP . We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion . Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids . These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes. J Biol Chem, 1999 Oct 15, 274(42), 29705 - 11 Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane; van Geest M et al.; The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K . pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments . A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, while three segments, Vb, VIII and IX, were predicted to have a periplasmic location (Van Geest, M., and Lolkema, J . S . (1996) J . Biol . Chem . 271, 25582-25589) . A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes revealed that the protein traverses the endoplasmic reticulum membrane 11 times . In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J . S . (1999) J . Biol . Chem . 274, 2816-2823) . In the present study, using site-directed Cys labeling, the topology of segments VIII and IX in the full-length CitS protein was determined in the E . coli membrane . Engineered cysteine residues in the loop between the two segments were accessible to a membrane-impermeable thiol reagent exclusively from the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and IX are both membrane-spanning . It follows that the folding of CitS in the E . coli and endoplasmic reticulum membrane is the same . Cysteine accessibility studies of CitS-PhoA fusion molecules demonstrated that in the E . coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the PhoA fusions do not correctly predict the topology . An engineered cysteine residue downstream of TMS VIII moved from a periplasmic to a cytoplasmic location when the fusion protein containing TMSs I-VIII was extended with segment IX . Thus, downstream segment IX is both essential and sufficient for the insertion of segment VIII of CitS in the E . coli membrane. Mol Biochem Parasitol, 1999 Sep 20, 103(1), 15 - 23 Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase; Darling JA et al.; Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite . T . gondii AK thus represents a promising target for rational design of antiparasitic compounds . In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T . gondii was overexpressed in E . coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques . Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1) . Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs . Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T . gondii AK . Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo. J Antibiot (Tokyo), 1999 Jul, 52(7), 643 - 8 Convergent syntheses of oral THF 1beta-methylcarbapenems; Bitha P et al.; Convergent syntheses of oral THF 1beta-methylcarbapenems 4 (OCA-983) and 5 starting from M2-phosphate 1 were developed . Reaction of the M2-phosphate 1 with THF thiols containing a requisite prodrug side chain, 9 and 10, gave the desired oral THF 1beta-methylcarbapenems 4 and 5, respectively, in 46% and 42% overall yields. Biol Pharm Bull, 1999 Sep, 22(9), 947 - 50 Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase; Hayashi H et al.; Two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase have been isolated from licorice, Glycyrrhiza glabra L., and characterized . The deduced amino acid sequence of GgSQS1 was 88%, 81%, 78%, 45-44%, and 45-41% identical to those of GgSQS2, Nicotiana, Arabidopsis, mammal and yeast squalene synthases, respectively . Squalene synthase activity was found in the cell-free extracts of Escherichia coli transformed with the recombinant plasmids for GgSQS1 and GgSQS2, respectively . Genomic Southern blot hybridization indicated that there are three squalene synthase genes in the licorice genome . Northern blot analysis showed that GgSQS2 mRNA is mainly expressed during the exponential growth phase of the cultured licorice cells. Biol Pharm Bull, 1999 Sep, 22(9), 932 - 5 Phagocytic activity of ethyl alcohol fraction of deer antler in murine peritoneal macrophage; Suh JS et al.; The mechanism of phagocytic activity of the ethyl alcohol fraction of Cervus nippon (CN-E) was investigated in vivo . The administration of CN-E (100 mg/kg, p.o.) enhanced lucigenin chemiluminescence and the engulfment of fluorescein-conjugated E . coli particles in murine peritoneal macrophages . Phagocytic activity was suppressed by the treatment of S-nitrosoglutathione (GSNO) which is an exogenous nitric oxide donor depending on the concentration of dose . CN-E suppressed the production of nitric oxide and enhanced the concentration in {Ca2+}i . The enhancement in {Ca2+}i was diminished by the treatment of EGTA . These results indicate that CN-E enhances the phagocytic activity of murine peritoneal macrophage via a suppression of nitric oxide production and an increase in {Ca2+}i. Biol Pharm Bull, 1999 Sep, 22(9), 904 - 9 Identification of a high-copy-number plasmid suppressor of a lethal phenotype caused by mutant DnaA protein which has decreased intrinsic ATPase activity; Makise M et al.; The induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells causes a lethal phenotype . Based on our results that the decreased ATPase activity of DnaA E204Q is activated to the level of a wild-type protein in the presence of partially purified stimulating factors for DnaA ATPase, we tested the hypothesis that genes encoding the stimulating factors can be cloned as high-copy-number plasmid suppressors for the lethality caused by DnaA E204Q . We isolated a number of high copy-number plasmids that suppress the lethal phenotype . The genes responsible for the suppression of the lethal phenotype were revealed to be dnaN, relA, pcnB and cyaA by subcloning, and a site-directed mutational analysis . The mechanisms by which these genes suppressed the lethal phenotype were examined in vivo and in vitro. Biophys J, 1999 Oct, 77(4), 2284 - 94 Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase; Guthold M et al.; The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer . To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface . In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk . Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding . These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location . Sequential images of single, transcribing RNAP molecules were also investigated . Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation . Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution . This assay also showed that the pattern of pause sites and the termination site were affected by the surface . By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed. Biophys J, 1999 Oct, 77(4), 1960 - 72 Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity; Yoshimura K et al.; Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock . To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids . Gly(22) was highlighted in random mutagenesis screens of E . coli MscL (, Proc . Nat . Acad . Sci . USA . 95:11471-11475) . By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science . 282:2220-2226), Gly(22) is buried within the constriction that closes the pore . Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state . In contrast, hydrophobic substitutions increased the threshold pressure . Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation . These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation. Biotechnol Appl Biochem, 1999 Oct, 30 ( Pt 2), 163 - 70 Evaluation of refolding conditions for a recombinant human interleukin-3 variant (daniplestim); Boyle DM et al.; The refolding of daniplestim, a human interleukin-3 variant (SC-55494) from Escherichia coli inclusion bodies, was optimized using a reversed-phase HPLC method developed to permit quantification of the reduced and oxidized forms of daniplestim . The presence of cysteine or dithiothreitol accelerated refolding of daniplestim from E . coli inclusion body slurries dissolved in urea or guanidine solutions and was complete in 4-6 h . Regardless of the dissolution and refolding protocol used to renature daniplestim, equivalently bioactive protein was produced . Under refolding conditions, no covalent modification of daniplestim by cysteine or cyanate was observed . The folding process was characterized further by following the unfolding of purified daniplestim by far-UV CD and fluorescence spectroscopies under both oxidizing and reducing conditions at pH values between 7 and 11 . Formation of the single disulphide bond had a large stabilizing effect on daniplestim structure ( approximately 4-5 kCal at pH 9.5) . This thermodynamic stabilization drove the refolding process towards the native form, even under conditions where the reduced protein was largely unfolded . From these data, scaleable refolding conditions for daniplestim were established. Biochem Biophys Res Commun, 1999 Oct 5, 263(3), 678 - 80 N-terminal truncated cytochrome P450 2B4: catalytic activities and reduction with alternative electron sources; Shumyantseva VV et al.; It was shown that riboflavin binds to the truncated cytochrome P450 2B4 and forms a complex with the K(d) = 26 microM . Noncovalent complex of truncated (Delta2-27) cytochrome P450 2B4 with riboflavin was essential for electron transfer realization and catalyzed the NADH-dependent and hydrogen peroxide-supported monooxygenase reactions of aminopyrine N-demethylation and aniline p-hydroxylation . Flavocytochrome molecular maquette was capable of supporting photoactivatable electron transfer and could be photoreduced and electroreduced quantitatively in the absence of pyridine nucleotides . Biochem Biophys Res Commun, 1999 Oct 5, 263(3), 652 - 6 Expression and mutagenesis studies of cobrotoxin from Taiwan cobra; Chang LS et al.; The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra) . The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain . Expressed cobrotoxin was isolated from inclusion bodies of E . coli and subjected to refolding into its folded structure . The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions . Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin . An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47 . These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule . J Mol Biol, 1999 Oct 15, 293(1), 125 - 37 Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES; Makio T et al.; We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin . The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence . The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method . When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites . The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity . A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity . When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin . The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone . The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place . These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity . J Mol Biol, 1999 Oct 15, 293(1), 57 - 66 Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure; Maeda N et al.; We previously reported the presence of a highly active, carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 . In this study, structural analysis of Pk -Rubisco has been performed . Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure . In order to investigate the existence of small subunits in native Pk -Rubisco, immunoprecipitation and native-PAGE experiments were performed . No specific protein other than the expected large subunit of Pk -Rubisco was detected when the cell-free extracts of P . kodakaraensis KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme . Furthermore, native and recombinant Pk -Rubiscos exhibited identical mobilities on native-PAGE . These results indicated that native Pk -Rubisco consisted solely of large subunits . Electron micrographs of purified recombinant Pk -Rubisco displayed pentagonal ring-like assemblies of the molecules . Crystals of Pk -Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution . The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other . These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk -Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure . This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos . J Mol Biol, 1999 Oct 15, 293(1), 41 - 56 Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics; Kipriyanov SM et al.; To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent . It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct . After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts . The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions . Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells . They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL . Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week . In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications . J Mol Biol, 1999 Oct 8, 292(5), 1149 - 60 Large-scale effects of transcriptional DNA supercoiling in vivo; Krasilnikov AS et al.; The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter . An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitatively measured by chemical probing of intracellular DNA . By placing a cruciform-forming sequence at varying distances from the promoter, we found that the half-dissociation length of transcription supercoiling wave is approximately 800 bp . This is the first proof that transcription can affect DNA structure on such a remarkably large scale in vivo . Moreover, cooperative binding of the cI repressor to the upstream promoter DNA did not preclude efficient diffusion of transcriptional supercoiling . Finally, our plasmids appeared to contain discrete domains of DNA supercoiling, defined by the features and relative orientation of different promoters . J Mol Biol, 1999 Oct 8, 292(5), 973 - 86 Separate contributions of UhpA and CAP to activation of transcription of the uhpT promoter of Escherichia coli; Olekhnovich IN et al.; Activation of promoters by multiple transcription factors might occur through favorable contacts of the activators with themselves or RNA polymerase, or by changes in DNA geometry that enhance formation of the transcription complex . Transcription of the Escherichia coli uhpT gene, encoding the organophosphate transporter, requires the response regulator UhpA and is stimulated by the global regulator protein CAP . CAP binds to the uhpT promoter at a single site, centered at -103.5 bp relative to the start of transcription, and UhpA binds to multiple sites between positions -80 and -32 . Overexpression of UhpA did not reduce the degree of CAP stimulation of uhpT-lacZ expression, showing that CAP action is more complex than enhancement of the binding of UhpA . Footprinting experiments demonstrated that UhpA and CAP modestly stimulated each other's binding to the uhpT promoter, but did not affect the positioning of the binding sites . An in vitro transcription system was used to examine the contribution of each transcription factor at the uhpT promoter . Action of UhpA and CAP proteins was not affected by template supercoiling . Kinetic analyses of productive and abortive initiation showed that CAP acted both to stabilize by fivefold the open promoter complexes formed in the presence of UhpA and to enhance by twofold the rate of their formation . These results indicate that open complex formation requires UhpA and that CAP stabilizes the open complex . J Mol Biol, 1999 Oct 8, 292(5), 965 - 72 Single molecular observation of the interaction of GroEL with substrate proteins; Yamasaki R et al.; To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy . GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine . As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5 . The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography . The recovered complexes were then observed by fluorescence microscopy . For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level . Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese . The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins . J Mol Biol, 1999 Oct 8, 292(5), 949 - 56 Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency; Shusta EV et al.; Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins . Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae . While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants . Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast . Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency . Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties . Biochemistry, 1999 Sep 7, 38(36), 11693 - 9 Self-processing of FtsH and its implication for the cleavage specificity of this protease; Akiyama Y; FtsH, a membrane-bound and ATP-dependent protease of Escherichia coli, is involved in degradation of some of uncomplexed integral membrane proteins and short-lived cytoplasmic proteins . It is composed of an N-terminal membrane-spanning region and a following large cytoplasmic domain that contains ATPase and protease active sites . In the present study, it was found that FtsH undergoes C-terminal processing in vivo . The processing was blocked by loss of function mutations of FtsH . Purified FtsH-His(6)-Myc, a C-terminally tagged derivative of FtsH, was self-processed in vitro . This in vitro processing was observed only in the presence of ATP and not in the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) . Moreover, such processing did not occur in the case of the ATPase motif mutant protein . These results indicated that this processing is a self-catalyzed reaction that needs ATP hydrolysis . Mutations in the hflKC genes that encode a possible modulator of FtsH, and the growth phase of the cells as well, affected the processing . Complementation experiments with genetically constructed variants suggested that both the processed and the unprocessed forms of FtsH are functional . The cleavage was found to occur between Met-640 and Ser-641, removing a heptapeptide from the C-terminus of FtsH . Systematic mutational analyses of Met-640 and Ser-641 revealed preferences for positively charged and hydrophobic amino acid residues at these positions for processing . This cleavage specificity may be shared by the self-cleavage and the substrate-cleavage reactions of this protease. Biochemistry, 1999 Sep 7, 38(36), 11643 - 50 Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine; Witkowski A et al.; beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction . Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude . On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction . The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer . These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate . Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor . These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases. Biochemistry, 1999 Sep 7, 38(36), 11604 - 12 Core-directed protein design . I . An experimental method for selecting stable proteins from combinatorial libraries; Finucane MD et al.; The design of proteins represents a significant challenge to modern-day structural biology . A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target . Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins . Here we present the experimental counterpart of this approach . We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows . Hexahistidine-tagged proteins were displayed on the surface of phage . These protein-phage were immobilized onto Ni-coated surfaces . The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins . Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression . Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads . SPR had an advantage in that the selection process could be monitored directly . This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads . We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper {Finucane, M . D., and Woolfson, D . N . (1999) Biochemistry 38, XXXXX-XXXXX} . As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure. Biospectroscopy, 1999, 5(5 Suppl), S3 - 18 EPR spectroscopy: a powerful technique for the structural and functional investigation of metalloproteins; More C et al.; Numerous metal centers in proteins can be prepared in a redox state in which their ground state is paramagnetic . Complementary data provided by EPR, Mossbauer, electron nuclear double resonance, magnetic circular dichroism, and NMR spectroscopies have therefore played a major role in the elucidation of the structure and function of these centers . Among those techniques the most commonly used is certainly EPR spectroscopy . In this article various aspects of the current applications of EPR to the structural and functional study of metalloproteins are presented . They are illustrated by recent studies carried out in our laboratory in the field of metalloenzymes and electron transfer systems . The power of numerical simulation techniques is emphasized throughout this work. Dev Comp Immunol, 1999 Sep, 23(6), 451 - 7 Degranulation of eosinophilic granular cells with possible involvement in neutrophil migration to site of inflammation in tilapia; Matsuyama T et al.; Observations were made on stretched preparations from the swim bladder and peritoneum obtained from tilapia (Oreochromis niloticus) after injection of formalin-killed Escherichia coli, proteose peptone, compound 48/80 or HBSS into the swim bladder . The eosinophilic granular cells (EGCs) in the swim bladder degranulated rapidly after inoculation . However, the peritoneal EGCs did not degranulate, indicating that degranulation occurs only at the injected site . There was also a correlation between the ratio of the degranulated EGCs and number of the exudate neutrophils . Killed E . coli (1 mg/fish) produced the greatest degranulation response of EGCs and migration of neutrophils into the inflammatory site . Additionally, the rate of the degranulation and number of the neutrophils were lowest when HBSS was injected . The results of this study verify that degranulation of EGCs is involved in the neutrophil migration and suggest that fish EGCs are analogous to mammalian mast cells. Am J Epidemiol, 1999 Oct 1, 150(7), 770 - 7 Epidemiology of rotavirus diarrhea in Egyptian children and implications for disease control; Naficy AB et al.; Reliable epidemiologic data are essential for formulating effective policy to control rotavirus disease through immunization . The objective of this study was to describe the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, in 1995-1996 . Rotavirus diarrhea incidence rates (episodes per person-year) were 0.13 for infants aged <6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months . Fifty-six percent of children with rotavirus diarrhea had clinical dehydration; 90% of rotavirus diarrheal episodes occurred between July and November . In infants under 1 year of age, receipt of breast milk was associated with a lower incidence of rotavirus diarrhea . No other sociodemographic or environmental factor was found to be significantly associated with rotavirus diarrhea . Of 46 rotavirus isolates with strains identified, 41 (89%) were G serotypes 1 and 2 . Rotavirus diarrhea was a major cause of morbidity in this cohort . Promotion of breastfeeding may exert a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures . The use of effective immunization against rotavirus in early infancy should be considered a public health priorityPIP: This study describes the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, during 1995-96 . Samples consisted of a cohort of children under the age of 24 months assembled from two villages in the vicinity of Abu Homos . The age-specific incidence rates of rotavirus diarrheal episodes per person-year were 0.13 for infants aged 6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months . No rotavirus diarrheal incidence occurred in infants under 20 weeks of age . The monthly incidence rates of rotavirus diarrhea demonstrate that 90% of the disease episodes occurred during the warmer months of July-November, with a peak incidence in August . In infants under 1 year of age, breast-feeding was associated with a lower incidence of rotavirus diarrhea . Promotion of breast-feeding may employ a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures . Cell Mol Biol (Noisy-le-grand), 1999 Jul, 45(5), 555 - 65 Upstream element of the sea urchin arylsulfatase gene serves as an insulator; Akasaka K et al.; Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters . Here we report that a DNA fragment located in the upstream region of sea urchin, H . pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner . The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter . In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter . The insulation mechanism operates also unidirectionally in Drosophila . We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells . These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution. Vet Microbiol, 1999 Sep 15, 69(3), 207 - 16 Seroprevalence of F4+ enterotoxigenic Escherichia coli in regions with different pig farm densities; Van den Broeck W et al.; Sera of young sows from 135 closed Belgian pig breeding farms were examined for the presence of serum antibodies specific for the F4 fimbrial antigen of enterotoxigenic Escherichia coli (ETEC) . Since 80% of all pig farms in Belgium are located in the provinces West-Vlaanderen (44%, approximately 18 farms/10 km2), Oost-Vlaanderen (20%, approximately 9 farms/10 km2), Antwerpen (10%, approximately 5 farms/10 km2) and Vlaams-Brabant (6%, approximately 3 farms/10 km2), the farms examined were randomly selected in these four regions . On 68% of all tested farms, sows were not vaccinated against enteric colibacillosis . In general, 65% of these non-vaccinated farms were F4-seropositive (mean optical density {OD405} >0.5) of which 38% were weakly (mean OD405 = 0.5-1.0), 21% moderately (mean OD405 = 1.0-2.0) and 6% strongly positive (mean OD405 > or =2.0) . These percentages showed major differences between the four provinces . In Vlaams-Brabant, only 31% of non-vaccinated farms were seropositive, of which most were regarded as weakly positive . In Antwerpen, Oost- and West-Vlaanderen however, this percentage was clearly higher and respectively 68, 71, and 79% of non-vaccinated farms were seropositive, of which most were weakly positive . These observations indicate that F4+ ETEC is widely spread and highly prevalent on non-vaccinated pig breeding farms in Belgium . Moreover, an increasing F4-seropositivity with increasing pig farm density suggests a possible influence of the swine density on the prevalence of F4+ ETEC infections. Genetics, 1999 Oct, 153(2), 753 - 62 Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster; Roth GE et al.; The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae . Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff . We cloned Sgs-1 from a YAC covering 25B2-3 . Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region . Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein . The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated . By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site . Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant . FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4 . Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA . This has implications for the model of how Sgs transcripts accumulate in late third instar larvae. Genetics, 1999 Oct, 153(2), 643 - 52 Mutational analysis of yeast TFIIB . A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB; Wu WH et al.; TFIIB is an essential component of the RNA polymerase II core transcriptional machinery . Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro . In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo . A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects . Twenty-two single amino acid replacements in TFIIB were defined and characterized . These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions . Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability . In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy . This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2mu) or enhancers (sub1Delta, ssu72-1) of sua7 mutations . We show that the interactions of ssu72-1 and sub1Delta with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection . These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II. Genetics, 1999 Oct, 153(2), 539 - 54 Molecular evolution of the Escherichia coli chromosome . V . Recombination patterns among strains of diverse origin; Milkman R et al.; Incorporation patterns of donor DNA into recipient chromosomes following transduction or conjugation have been studied in the progeny of a variety of Escherichia coli crosses in which donor and recipient nucleotide sequences differ by 1-3% . Series of contiguous or variously spaced PCR fragments have been amplified from each recombinant chromosome and digested with a commercial restriction endonuclease previously shown to distinguish the respective parents in a given fragment . We conclude that entering donor DNA fragments are frequently abridged (cut and shortened) before incorporation, the cutting being due to restriction systems, and the shortening presumably due to exonuclease activity . Analysis of several backcrosses confirms, and extends to conjugation, the importance of restriction in E . coli recombination in nature . The transmission patterns in conjugation are similar to those of transduction, but (as expected) on a much larger scale . Asymmetric results of reciprocal crosses imply that mismatch frequency is not a major factor . Marked differences among the results of simple crosses according to parental strain combinations are consistent with observations that E . coli strains in nature vary dramatically in their restriction-modification systems. Bone, 1999 Oct, 25(4), 431 - 7 Anabolic effect of aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone; Kuroda S et al.; Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis . Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration . We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4 . Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes . Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5' end using the ExoIII system . Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector . Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified . The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay . Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks . Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT) . The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect . Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4 . DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis . These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone. Antonie Van Leeuwenhoek, 1999 May, 75(4), 293 - 7 A ColE1-compatible expression vector for the production of His-tagged fusion proteins; Barriault D et al.; Plasmid pDB31 is a ColE1-compatible expression vector based on the p15A origin of replication . It is designed to express His-tagged fusion proteins in cells co-hosting a compatible expression vector . It was constructed by assembling the operator/promoter region plus the 6xHis and the multiple cloning site of pQE31 (QIAGEN) with the p15A origin of replication plus KanR of pGP1-2 . The plasmid was found to be stable in Escherichia coli strains BL21 and DH11S . It was used to produce and purify the ferredoxin reductase component of Comamonas testosteroni B-356 biphenyl dioxygenase inside a clone hosting the remaining dioxygenase genes on a compatible plasmid. Biochem J, 1999 Oct 15, 343 Pt 2, 487 - 504 Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members; O'connor T et al.; Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli . These members of the aldo-keto reductase (AKR) superfamily have been purified from E . coli as recombinant proteins . The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde . Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone . In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid . Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone . Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied . Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls . However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids . Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease . Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues . These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine . The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone . As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further . Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting . Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules . Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions. Biochem J, 1999 Oct 15, 343 Pt 2, 397 - 402 Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase; Stoughton DM et al.; Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid . The active site of the 418 amino acid E . coli enzyme was localized to its N-terminal half . The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini . Several basic residues have been identified at or near the active site of the E . coli enzyme by chemical modification and site-directed mutagenesis . Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity . Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme . Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence . Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity . The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site . Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding. Arch Biochem Biophys, 1999 Oct 15, 370(2), 190 - 200 Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies; Soucek P; Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines . In this paper expression of CYP2A6 in Escherichia coli is reported . In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis . The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture . Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized . Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein . Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked . The N-terminal formylmethionine block was removed by mild acid treatment . Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP) . Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione . CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed . Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies . Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme . Arch Biochem Biophys, 1999 Oct 15, 370(2), 143 - 50 Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain; Rapisarda VA et al.; Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides . The aim of this work was to characterize the NADH-linked cupric reductase activity from the E . coli respiratory chain . We have used E . coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III) . We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III) . The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD . The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively . The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II) . The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein . To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties . Mol Microbiol, 1999 Sep, 33(6), 1190 - 9 Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells; Nakagawa I et al.; Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure . The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface . However, the mechanisms by which Stxs kill target cells remain unclear . Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells . The Stx1B gene-transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene-transfected cells were found to be necrotic and no DNA fragmentation occurred . The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline-inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline . These apoptotic changes were abrogated by pretreatment with Z-VAD-fmk . These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm. Mol Microbiol, 1999 Sep, 33(6), 1162 - 75 Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion; Abe A et al.; Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E . coli-secreted proteins) that are required for full virulence . Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation . An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure . In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin) . A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm . In contrast, this mutation did not affect the secretion of the Esp proteins . The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level . The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant . Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins . Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted . Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion . Gel filtration studies showed that Tir-CesT forms a large multimeric complex . Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifically binding to its amino-terminus, forming a multimeric stabilized complex. Mol Microbiol, 1999 Sep, 33(6), 1141 - 51 DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli; Heitman J et al.; Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate . Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo . Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli . In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome . EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain . The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E . coli DNA ligase gene . These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair. J Exp Med, 1999 Oct 4, 190(7), 915 - 22 Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice; Johansen FE et al.; Mucosal surfaces are protected specifically by secretory immunoglobulin A (SIgA) and SIgM generated through external translocation of locally produced dimeric IgA and pentameric IgM . Their active transport is mediated by the epithelial polymeric Ig receptor (pIgR), also called the transmembrane secretory component . Paracellular passive external transfer of systemic and locally produced antibodies also provides mucosal protection, making the biological importance of secretory immunity difficult to assess . Here we report complete lack of active external IgA and IgM translocation in pIgR knockout mice, indicating no redundancy in epithelial transport mechanisms . The knockout mice were of normal size and fertility but had increased serum IgG levels, including antibodies to Escherichia coli, suggesting undue triggering of systemic immunity . Deterioration of their epithelial barrier function in the absence of SIgA (and SIgM) was further attested to by elevated levels of albumin in their saliva and feces, reflecting leakage of serum proteins . Thus, SIgA did not appear to be essential for health under the antigen exposure conditions of these experimental animals . Nevertheless, our results showed that SIgA contributes to maintenance of mucosal homeostasis . Production of SIgA might therefore be a variable in the initiation of human immunopathology such as inflammatory bowel disease or gluten-sensitive enteropathy. Vet Microbiol, 1999 Aug 31, 68(3-4), 255 - 63 Receptor-specific binding of purified F4 to isolated villi; Van den Broeck W et al.; Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT+Sta+STb+) were performed and the purity and yield of F4ac were compared . Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10(11) bacteria/ml) . The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e . 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10(12) bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10(12) bacteria, i.e . 26.2%) and the relative contamination higher (1.0 mg per 10(12) bacteria, i.e . 73.8%) . A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels . The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained . The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay . Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb. Vet Microbiol, 1999 Aug 31, 68(3-4), 209 - 17 Prevalence of virulence factors of Escherichia coli strains isolated from diarrheic and healthy piglets after weaning; Osek J; This study determined the prevalence of F4, F5, F6, F17 and F41 fimbriae and the genes for FedA (F18 fimbriae), LT and ST enterotoxins, and Shiga toxins Stx1, Stx2 and Stx2e among E . coli isolated from 372 weaned pigs with diarrhea and 46 healthy pigs of the same age . Agglutination tests showed that most isolates were negative for all five fimbrial antigens . The F4 antigen was found in 71 (19.1%) and the F5, F6, or F41 antigen was detected in 6.4% of isolates from diseased pigs . Genes for the F18 fimbriae were detected in 10 (2.7%) strains from diarrheic pigs and in 1 of 46 isolates from healthy pigs . Most isolates (213, 57.3%) from pigs with diarrhea were positive for LTI only or for LTI and STI or Stx2e toxin genes . Fifteen strains (13.7%) possessed only the STI or STII toxin genes . All F4-positive bacteria had genes for LTI or LTI and STI, whereas F18-positive isolates had genes for LTI, LTI/STI, or LTI/Stx2e . Of the strains isolated from diseased pigs, 264 (71.0%) were negative for the fimbrial antigens (genes) examined in this study . The fimbria-negative isolates frequently possessed genetic determinants for LTI (118, 31.7%) or for STII (16, 4.3%) enterotoxins. Ann Thorac Surg, 1999 Sep, 68(3), 830 - 6; discussion 836-7 Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease; Symes JF et al.; BACKGROUND: Patients presenting with medically intractable angina who have undergone previous coronary bypass (CABG) and/or percutaneous revascularization procedures are frequently deemed "inoperable" based on angiographic findings of diffuse distal disease or a lack of available conduits . We initiated a phase I clinical trial to assess the safety and bioactivity of intramyocardial transfection of plasmid DNA encoding for the angiogenic mitogen vascular endothelial growth factor (ph-VEGF165) in such patients . METHODS: phVEGF165 (125 microg, n = 10; 250 microg, n = 10) was injected directly into the myocardium through a mini left anterior thoracotomy as sole therapy in 20 patients (15 male, 5 female, age 48 to 74 years) with class III or IV angina, reversible ischemia on stress sestamibi scans, and "inoperable" coronary artery disease . RESULTS: All patients tolerated surgery uneventfully and were extubated on the table . No perioperative myocardial infarction, hemodynamic instability, or change in ventricular function occurred . Mean hospital stay was 3.9 days . There was one late death (4 months) . Plasma VEGF protein level increased from 30.6+/-4.1 pg/mL pretreatment to 73.7+/-10.1 pg/mL 14 days posttreatment (p = 0.0002) and returned to baseline by day 90 . All 16 patients followed to day 90 reported a reduction in angina (nitroglycerin use/week = 60.2+/-4.9 preop vs 3.5+/-1.6 at 90 days; p<0.0001) . Seventy percent (7 of 10) patients were completely angina free at 6 months . A reduction in ischemic defects on single photon emission computerized tomography sestamibi scans was observed in 13 of 17 patients at 60 days (7 of 8 in the 250-microg group) . Stress perfusion score decreased from 19.4+/-3.7 at baseline to 15.9+/-3.4 at 60 days (p = 0.025) . Angiographic evidence of improved collateral filling of at least one occluded vessel was observed in all patients evaluated at day 60 . CONCLUSIONS: Direct myocardial gene transfer with phVEGF165 via a mini-thoracotomy can be performed safely and may result in significant symptomatic improvement in patients with "inoperable" coronary artery disease. Mol Immunol, 1999 Jul, 36(10), 669 - 83 Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments; Kuttner G et al.; Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies . The scFv fragments were solubly expressed in E . coli . Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments) . The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants . The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region) . For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region) . Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions . On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions. Mol Immunol, 1999 Jul, 36(10), 639 - 45 New Bet v 1 isoforms including a naturally occurring truncated form of the protein derived from Austrian birch pollen; Friedl-Hajek R et al.; Bet v 1, the major pollen allergen from white birch, displays a considerable degree of heterogeneity . Until now, all molecular and immunological characterization studies of Bet v 1 isoforms have been performed with commercially available pollen of Swedish origin . In regard to clinical studies with Austrian birch pollen allergic individuals, knowledge about the isoform repertoire in Austrian birch pollen was necessary . cDNAs coding for Bet v 1 isoforms from Austrian birch pollen were cloned by PCR amplification and sequenced . Besides the Austrian variants of the Swedish isoforms Bet v 1a (62% of the clones), ALK167 (4%), and Bet v 1d/h, Bet v 1g, and Bet v 11 (24%), three sequences with a significantly lower homology to known isoforms and two Bet v 1a-homologous sequences with a 7 bp insertion coding for a truncated protein were detected . No Austrian variants of the majority of the Swedish isoforms were found . The isoforms coding for truncated proteins were expressed in Escherichia coli and tested by immunoblotting . They bound a polyclonal anti-Bet v 1 antibody but did not recognize birch pollen allergic patients' serum IgE and two Bet v 1-specific monoclonal antibodies . The similarity of the Bet v 1 isoform patterns of Swedish and Austrian birch pollen justifies the use of Bet v 1 derived from Swedish pollen for clinical studies with birch pollen allergic individuals from outside Northern Europe. Virus Res, 1999 Sep, 63(1-2), 53 - 63 Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha; Yu YX et al.; Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years . Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand . Up to now, the molecular biology of these important pathogens has been poorly documented . To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV) . Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing . Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length . Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined . Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins . Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases . But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined. J Dairy Sci, 1999 Sep, 82(9), 1904 - 9 1,25-dihydroxyvitamin D3 enhances milk antibody titers to Escherichia coli J5 vaccine; Reinhardt TA et al.; Recent evidence in cattle and mice has suggested that 1,25-dihydroxyvitamin D3 may stimulate T-lymphocyte differentiation pathways responsible for humoral immunity . The use of 1,25-dihydroxyvitamin D3 as an adjuvant for an Escherichia coli J5 vaccine was tested . Ten midlactation cows received J5 vaccine and were revaccinated 6 wk after the first vaccine administration . Five of these cows were given 200 micrograms of 1,25-dihydroxyvitamin D3, in addition to the primary vaccination, and they received an additional 200 micrograms of 1,25-dihydroxyvitamin D3 1 wk after the primary vaccination . No 1,25-dihydroxyvitamin D3 was administered with the wk 6 J5 vaccine booster . Milk immunoglobin (Ig)M, IgG, and IgA antibodies to E . coli J5 were significantly increased in cows receiving 1,25-dihydroxyvitamin D3 + E . coli J5 vaccine compared with cows that received only E . coli J5 vaccine . Serum IgG and IgG1 antibodies to E . coli J5 were also significantly enhanced by 1,25-dihydroxyvitamin D3 treatment compared with cows receiving vaccine alone . In contrast, serum IgG2 titers tended to decline in cows receiving 1,25-dihydroxyvitamin D3 . Taken together, these data suggest that 1,25-dihydroxyvitamin D3 has potential usefulness in boosting humoral responses to vaccines such as J5 and may further enhance the protective qualities of vaccines. FEBS Lett, 1999 Oct 1, 459(1), 139 - 42 Biological activities of granzyme K are conserved in the mouse and account for residual Z-Lys-SBzl activity in granzyme A-deficient mice; Wilharm E et al.; Tryptase-like activities of T and NK cells contribute to the induction of target cell apoptosis, but only granzyme A (GzmA) has been shown to exhibit Z-Lys-SBzl esterase activity in murine T cells . GzmA-deficient mice exhibit residual Z-Lys-SBzl hydrolyzing activity and almost normal levels of lymphocyte-mediated cytotoxicity . Here we report the cloning and biochemical characterization of recombinant mouse granzyme K (GzmK) . The purified murine protein shows Z-Lys-SBzl hydrolyzing activity and is inhibited by bikunin, the light chain of inter-alpha-trypsin inhibitor, like the human homolog . We conclude that GzmK expressed by GzmA-deficient T cells accounts for the remaining Z-Lys-SBzl activity . Functional similarities between GzmA and GzmK may explain the subtle immunological deficits observed in GzmA-deficient mice. Am J Respir Crit Care Med, 1999 Oct, 160(4), 1347 - 53 Ileal VO(2)-O(2) alterations induced by endotoxin correlate with severity of mitochondrial injury; Crouser ED et al.; Sepsis is usually associated with altered O(2) metabolism in systemic organs . Until recently, inadequate O(2) delivery was thought to be the putative mechanism underlying these metabolic alterations . However, current investigations suggest that impaired O(2) consumption due to disrupted O(2) use by mitochondria may be the culprit . Therefore, we hypothesized that endotoxin (LPS)-induced V O(2)- O(2) alterations would correlate with the severity of mitochondrial injury in a systemic organ (i.e., the ileum) . Using an in situ autoperfused feline ileum preparation, we assessed V O(2)- O(2) relationships and mitochondrial ultrastructure after 2 h in LPS-treated (3 mg/kg, intravenous; n = 11) and time-matched control (n = 5) animals . Mitochondrial injury was graded in a blinded fashion on the basis of characteristics associated with established stages of cell injury . LPS-treated animals developed severe mitochondrial injury in the ileal mucosa despite unchanged regional tissue perfusion and ileal oxyhemoglobin levels compared with controls . Worsening of mitochondrial injury correlated with increases in the critical O(2) delivery (r = 0.85; p < 0.002) and decreases in the maximum O(2) extraction (r = -0.61; p < 0.02) in the ileum . These results suggest that mitochondrial injury, leading to impaired O(2) utilization, may be primarily responsible for altered V O(2)- O(2) relationships in systemic organs during sepsis. Am J Respir Crit Care Med, 1999 Oct, 160(4), 1196 - 204 Sequential changes in autonomic regulation of cardiac myocytes after in vivo endotoxin injection in rat; Abi-Gerges N et al.; We report that in vivo injection of endotoxin (EDTX, 6 mg . kg(-)(1)) induces cardiovascular alterations in rats that closely mimic the clinical situation, as assessed by in vivo hemodynamic measurements in anesthetized and conscious, chronically instrumented animals . The patch-clamp technique was used to characterize the L-type calcium current (I(Ca)) and its autonomic regulation in isolated cardiac myocytes . The density of I(Ca) progressively decreased at 12 and 36 h after EDTX injection . However, the dihydropyridine (+/-)Bay K 8644 (100 nM) enhanced I(Ca) to levels similar to those in control and EDTX-treated myocytes . In addition, the net stimulatory effect of a beta-adrenergic agonist (isoproterenol) on I(Ca) was increased 12 h after EDTX injection . This change in the beta-adrenergic effect declined 24 h later . The potentiation in the beta-adrenergic stimulation of I(Ca) was mimicked by L858051 (10 microM), a direct activator of adenylyl cyclase, but not by IBMX (200 microM), a phosphodiesterase inhibitor . Besides, the antiadrenergic effect of acetylcholine on I(Ca) was unchanged 12 h after EDTX injection, but increased 36 h after EDTX injection . These results support the hypothesis that time-dependent changes in the adenylyl cyclase pathway in cardiac myocytes may contribute, via the autonomic regulation of I(Ca), to the severity of myocardial dysfunction during sepsis. Am J Respir Crit Care Med, 1999 Oct, 160(4), 1165 - 70 Role of cyclooxygenase-2 in oleic acid-induced acute lung injury; Gust R et al.; Eicosanoid production appears to be important to both edemagenesis and the pattern of pulmonary perfusion in experimental acute lung injury (ALI) . We hypothesized that these effects could be mediated by the inducible form of cyclooxygenase (COX-2) . We used positron emission tomography to evaluate the pulmonary perfusion pattern in dogs given oleic acid (OA) only (n = 6), the novel COX-2 inhibitor SC-236 50 min before OA (n = 3), and SC-236 given 20 min before endotoxin (Etx), followed by OA given 30 min after Etx (n = 5) . Thromboxane B(2) (TXB(2)) and prostacyclin (6-keto prostaglandin F(1alpha); 6-keto PGF(1alpha)) metabolite concentrations in plasma and lung tissue were measured in these groups and in another group given Etx + OA (n = 4) . Inhibition of COX-2 before administration of OA alone or before administration of Etx and OA did not have any significant effect on plasma or lung tissue concentrations of TXB(2) . However, inhibition of COX-2 prior to Etx and OA significantly reduced the plasma and lung tissue concentrations of 6-keto PGF(1alpha) as compared with those in the group given only Etx + OA . Moreover, SC-236 prevented the expected loss of perfusion redistribution associated with Etx + OA only . The effect of endotoxin on pulmonary perfusion in ALI is therefore the result of a COX-2-mediated increase in prostacyclin production in lung tissue. Structure Fold Des, 1999 Sep 15, 7(9), 1155 - 66 X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution; Li C et al.; BACKGROUND: The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP . Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen . AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i) . RESULTS: The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit . The final model of PurM consists of two crystallographically independent dimers and four sulfates . The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4% . The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers . A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved . CONCLUSIONS: The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed . The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves . Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains. Chem Biol, 1999 Oct, 6(10), 743 - 53 Context-dependent mutagenesis by DNA lesions; Delaney JC et al.; BACKGROUND: Detailed analyses of mutational hotspots following DNA damage provide an understanding of oncogene activation and tumor suppressor gene inactivation, and hence provide an insight into the earliest steps in the induction of cancer . A mutational hotspot might be created by preferential lesion formation, decreased lesion repair, or increased misinsertion past the lesion during DNA replication . The respective contribution of these factors might be influenced by the DNA sequence context of the hotspot . RESULTS: As a prelude to addressing the contribution of all possible nearest-neighbor contexts on the replication past O6-methylguanine (m6G) and repair of m6G in vivo, we have devised a mutation frequency (MF) detection strategy on the basis of the properties of type IIs restriction enzymes . We also report a method for constructing site-specific single-stranded viral DNA genomes that should yield identical ligation efficiencies regardless of the lesion or its surrounding sequence context . Using repair-deficient Escherichia coli, we discovered that m6G in three sequence contexts was nearly 100% mutagenic in vivo, showing that the DNA polymerase holoenzyme almost always placed a thymine base opposite m6G during replication . In partially repair-proficient cells, the Ada O6-methylguanine-DNA methyltransferase repair protein was twice as efficient on m6G when a guanine base rather than an adenine base was 5' to the lesion . CONCLUSIONS: The system allows the mutagenic potential of, theoretically, any DNA lesion that exhibits point mutations, in any varied local sequence context, to be rapidly determined . The assay demonstrates low background, high throughput, and does not require phenotypic selection, making it possible to discern the effects of sequence context on the processing of m6G. Nat Genet, 1999 Oct, 23(2), 194 - 8 Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis; Goedecke W et al.; Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved . Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) . NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref . 3) . HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence . HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref . 4), which must be repaired by HR to ensure proper chromosome segregation . How the cell controls the choice between the two repair pathways is not understood . We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref . 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6) . Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes . Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis . In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent . In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable . We propose that Ku70 acts as a switch between the two DSB repair pathways . When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway. Nat Struct Biol, 1999 Oct, 6(10), 969 - 75 Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase; Becker A et al.; Pyruvate formate-lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419) . We have determined by X-ray crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant . The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-sheets; this architecture resembles that of ribonucleotide reductases . Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Calpha-Sgamma = 3.7 A) . Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sgamma (3.3 A), which in turn is close to Cys 419Sgamma (3.7 A) . Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical . We propose a homolytic radical mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions. Nat Struct Biol, 1999 Oct, 6(10), 900 - 2 Enzymes that push DNA around; Keck JL et al.; DNA topoisomerases are proteins that regulate DNA topology in cells through selective cycles of DNA cleavage, manipulation, and religation . Two papers describe an ensemble of different protein conformations and nucleotide-protein complexes of Escherichia coli topoisomerase . These results lead to new insights about how this enzyme recognizes DNA and catalyzes supercoil relaxation. J Med Chem, 1999 Sep 23, 42(19), 3835 - 44 Design and fast synthesis of C-terminal duplicated potent C(2)-symmetric P1/P1'-modified HIV-1 protease inhibitors; Alterman M et al.; An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C(2)-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds . These were synthesized with the objective to identify suitable P1/P1' substituents to provide inhibitors with improved antiviral activity . Groups with various physical properties were attached to the para-positions of the P1/P1' benzyloxy groups in the parent inhibitor . A p-bromobenzyloxy compound, prepared in only three steps from commercially available starting materials, was utilized as a common precursor in all reactions . The subsequent coupling reactions were completed within a few minutes and relied on palladium catalysis and flash heating with microwave irradiation . All of the compounds synthesized exhibited good inhibitory potency in the protease assay, with K(i) values ranging from 0.09 to 3.8 nM . A 30-fold improvement of the antiviral effect in cell culture, compared to the parent compound, was achieved with four of the inhibitors . The differences in K(i) values were not correlated to the differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence of human serum . The poorest enzyme inhibitors in fact belong to the group with the best antiviral effect . The binding features of two structurally related inhibitors, cocrystallized with HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water phase. Biochemistry, 1999 Sep 14, 38(37), 12180 - 6 Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2; Doray B et al.; Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells . In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells . To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli . The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1 . To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1 . If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants . The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E . coli and insect cells . In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells . The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system . These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations. Biochemistry, 1999 Sep 14, 38(37), 12138 - 49 Role of His159 in yeast enolase catalysis; Vinarov DA et al.; There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle . However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously . In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor {Duquerroy et al . (1995) Biochemistry 34, 12513-12523} . To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala . The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli . The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes . In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude . The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate . These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction . We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site . This proposal is consistent with the catalytic mechanism of yeast enolase . Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA . Metal binding studies support the above proposal . Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type . The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase . The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate {Klemba, M., and Regan, L . (1995) Biochemistry 34, 10094-10100 . Regan, L . (1993) Annu . Rev . Biophys . Biomol . Struct . 22, 257-281 . Cha et al . (1994) J . Biol . Chem . 269, 2687-2694}. Biochemistry, 1999 Sep 14, 38(37), 12097 - 103 Redesigning the substrate specificity of human O(6)-alkylguanine-DNA alkyltransferase . Mutants with enhanced repair of O(4)-methylthymine; Encell LP et al.; Human O(6)-alkylguanine-DNA alkyltransferase (MGMT) repairs potentially cytotoxic and mutagenic alkylation damage at the O(6)-position of guanine and the O(4)-position of thymine in DNA . We have used random sequence mutagenesis and functional complementation to obtain human MGMT mutants that are resistant to the MGMT inhibitor, O(6)-benzylguanine {Encell, L . P., Coates, M . M., and Loeb, L . A . (1998) Cancer Res . 58, 1013-1020} . Here we describe screening of O(6)-benzylguanine-resistant mutants for altered substrate specificity, i.e., for an increased level of utilization of O(4)-methylthymine (m(4)T) relative to that of O(6)-methylguanine (m(6)G) . One mutant identified by the screen, 56-8, containing eight substitutions near the active site (C150Y, S152R, A154S, V155G, N157T, V164M, E166Q, and A170T), was purified and characterized kinetically . The second-order rate constant for repair of m(4)T by the mutant was up to 11.5-fold greater than that of WT MGMT, and the relative m(4)T specificity, k(m(4)T)/k(m(6)G), was as much as 75-fold greater . In competition experiments with both substrates present, the mutant was 277-fold more sensitive to inhibition by m(4)T than WT MGMT . This mutant, and others like it, could help elucidate the complex relationship between adduction at specific sites in DNA and the cytotoxicity and mutagenicity of alkylating agents. Biochemistry, 1999 Sep 14, 38(37), 12052 - 61 Crystallographic and kinetic evidence of a collision complex formed during halide import in haloalkane dehalogenase; Pikkemaat MG et al.; Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions . The rate-limiting step in the reaction of DhlA is the release of the halide ion . The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments . At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site . To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure . The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme . One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import . Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations . This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site . Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme . The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport. Biochemistry, 1999 Sep 14, 38(37), 11933 - 41 Intersubunit proximity of residues in the RecA protein as shown by engineered disulfide cross-links; Skiba MC et al.; Mutational studies of regions that make up the oligomeric interface within the RecA protein filament structure have shown that F217 is an important determinant of RecA function and oligomer stability . All substitutions, other than Tyr and Cys, completely inhibit RecA activities and exhibit a substantial decrease in protein filament stability {Skiba, M . C., and Knight, K . L . (1994) J . Biol . Chem . 269, 3823-3828; Logan, K . M., et al . (1997) J . Mol . Biol . 266, 306-316} . Although the RecA crystal structure exhibits no obvious constraints that explain this mutational stringency, the structure does reveal a hydrophobic pocket in the neighboring monomer that may accommodate the F217 side chain . Together with the F217C mutation, we have introduced a series of Cys substitutions within the interacting surface on the neighboring monomer and have tested for disulfide formation under various conditions, e.g., with or without ATP and ssDNA . We show that the location of F217 in the crystal structure is in general agreement with its position in the catalytically active RecA-ATP-DNA complex . Functional studies with the mutant proteins support the idea that ATP-induced movement of the wild-type F217 side chain toward this hydrophobic pocket is important in mediating allosteric changes in the RecA protein structure. Biochemistry, 1999 Sep 14, 38(37), 11876 - 86 Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187; Drohat AC et al.; The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy . The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively . The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2 . This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes . The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5) . These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187 . The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187 . Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect . Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187. Biochemistry, 1999 Sep 14, 38(37), 11866 - 75 Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase; Drohat AC et al.; Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix {Stivers, J . T., et al . (1999) Biochemistry 38, 952} . A general acid-base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1' of the deoxyribose . Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding . The pH dependence of k(cat) for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for catalysis . This unprotonated group has a small DeltaH of ionization (-0.4 +/- 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64 . The pH dependence of k(cat) for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5-10 . Hence, the pK(a) of His187 must be outside this pH range if it serves as an electrophilic catalyst . These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state . In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG. EMBO J, 1999 Oct 1, 18(19), 5423 - 33 Resuming translation on tmRNA: a unique mode of determining a reading frame; Williams KP et al.; The bacterial ribosome switches from an mRNA lacking an in-frame stop codon and resumes translation on a specialized RNA known as tmRNA, SsrA or 10Sa RNA . We find that the ribosome can reach and use the extreme 3' terminal codon of the defective mRNA prior to switching . The first triplet to be translated in tmRNA (the resume codon) is determined at two levels: distant elements in tmRNA restrict resume codon choice to a narrow window and local upstream elements provide precision . Insights from a randomization-selection experiment secure the alignment of tmRNA sequences from diverse species . The triplet UA(A/G) (normally recognized as a stop codon by release factor-1) is strongly conserved two nucleotides upstream of the resume codon . The central adenosine of this triplet is essential for tmRNA activity . The reading frame of tmRNA is determined differently from all other known reading frames in that the first translated codon is not specified by a particular tRNA anticodon. Antimicrob Agents Chemother, 1999 Oct, 43(10), 2493 - 6 Sparfloxacin selects gyrase mutations in first-step Mycoplasma hominis mutants, whereas ofloxacin selects topoisomerase IV mutations; Kenny GE et al.; The role of mutations in the genes for GyrA and ParC in quinolone resistance in Mycoplasma hominis was studied . Selection with sparfloxacin gave mutations at GyrA83 (Ser-->Leu; Escherichia coli numbering) or GyrA87 (Glu-->Lys), and mutants had increased levels of resistance to sparfloxacin (8- to 16-fold) but not to ofloxacin . Selection with ofloxacin gave changes at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys), and mutants were four- to eightfold more resistant to ofloxacin but not to sparfloxacin . Selection of second-step mutants from strains with ParC mutations with either quinolone yielded double mutants with additional mutations at GyrA83 (Ser-->Trp or Ser-->Leu) or GyrA87 (Glu-->Lys) . Second-step selection of GyrA mutants gave additional mutations at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys) . Two-step mutants showed high levels of resistance to ofloxacin (MICs, 64 to 128 microg/ml) and moderate levels of resistance to sparfloxacin (MICs, 2 to 8 microg/ml) . The primary target of ofloxacin in first-step mutants of Mycoplasma hominis was ParC, whereas that for sparfloxacin was GyrA. Vet Immunol Immunopathol, 1999 Sep 20, 70(3-4), 203 - 21 Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins; Sestak K et al.; The intraperitoneal inoculation of pigs with baculovirus-expressed transmissible gastroenteritis virus (TGEV) structural proteins (S, N, M) in conjunction with thermolabile Escherichia coli mutant toxin (LT-R192G) in incomplete Freund's adjuvant (IFA) was tested in an attempt to elicit active immunity to TGEV in gut-associated lymphoid tissues (GALT) . Four groups of 63 (1-5-week-old) suckling, TGEV-seronegative pigs were used to assess the efficacy of the recombinant protein vaccine (group 3) in comparison with sham (group 1), commercial vaccine (group 2), and virulent TGEV Miller-strain-inoculated pigs (group 4) . The TGEV-specific mucosal and systemic immune responses were measured after in vivo and in vitro stimulation with TGEV-antigens . The major T-cell subset distribution was analyzed in vivo and in vitro after stimulation of mononuclear cells with TGEV (from mesenteric lymph nodes of group 3 inoculated with TGEV-recombinant proteins) . Induction of active immunity was assessed by challenge of pigs with virulent TGEV at 27 days of age . Baculovirus-expressed TGEV proteins coadministered with LT-R192G in IFA induced mesenteric lymph node immune responses associated with IgA-antibodies to TGEV and partial protection against TGEV-challenge . The high titers of serum IgG- and virus-neutralizing-antibodies to TGEV in group 3 pigs most likely reflected the dose of TGEV S-protein administered . At the day of TGEV-challenge, the in vitro stimulation of mononuclear cells from the mesenteric lymph nodes of group 3 pigs with inactivated TGEV resulted in an increase in double positive (CD4+CD8+), natural killer (CD2+CD4-CD8+dim) and cytotoxic (CD2+CD4-CD8+bright) T-cell phenotypes, accompanied by increased expression of interleukin-2 receptor and a decrease of the null (CD2-CD4-CD8-/SW6+) cell phenotype. Biotechnol Bioeng, 1999 Nov 20, 65(4), 382 - 8 New fusion protein systems designed to give soluble expression in Escherichia coli; Davis GD et al.; Three native E . coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E . coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein . These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E . coli . Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble) . Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C . Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction . Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E . coli at 37 degrees C and again showed that the fusion protein was almost completely soluble . Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography . Vaccine, 1999 Oct 1, 17 Suppl 2, S44 - 52 Genetically derived toxoids for use as vaccines and adjuvants; Del Giudice G et al.; Until very recently, development of vaccines has been based on an empirical approach . For example, bacterial toxins have been detoxified using empirical chemical treatment . Progress in biotechnology and molecular biology has allowed the fine knowledge of the structure-function relationship of several bacterial toxins . Thanks to this, the genetic attenuation of bacterial toxins has been made possible . Following this approach, a genetically detoxified pertussis toxin has been produced . This molecule is now the component of an acellular pertussis vaccine, which has been shown to be highly immunogenic and efficacious in infants . The same strategy of molecular detoxification of bacterial toxins has been applied to cholera toxin and to the Escherichia coli heat-labile enterotoxin . Toxin mutants devoid of any toxic activity have been produced and shown in animals to be highly immunogenic and to exhibit strong adjuvanticity when administered at mucosal sites in conjunction with several antigens . These successful results show that rational design of stronger and safer vaccines is feasible. Protein Eng, 1999 Sep, 12(9), 797 - 806 The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides; Christmann A et al.; The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin . Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot . In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein . EETI-II and derived variants were produced via fusion to maltose binding protein MalE . By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield . EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein . Gene expression was under control of the strong and tightly regulated tetA promoter/operator . Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein . To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host . Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold . Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting . These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules. Protein Eng, 1999 Sep, 12(9), 771 - 8 Design, engineering and production of functional single-chain T cell receptor ligands; Burrows GG et al.; Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen presenting cells (APCs) that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells . The peptide binding/T cell recognition domains of rat MHC class II (alpha-1 and beta-1 domains) were expressed as a single exon for structural and functional characterization . These recombinant single-chain T cell receptor ligands (termed 'beta1alpha1' molecules) of approximately 200 amino acid residues were designed using the structural backbone of MHC class II molecules as template, and have been produced in Escherichia coli with and without N-terminal extensions containing antigenic peptides . Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native MHC class II heterodimer . The proteins exhibited a cooperative two-state thermal folding-unfolding transition . Beta1alpha1 molecules with a covalently linked MBP-72-89 peptide showed increased stability to thermal unfolding relative to the empty beta1alpha1 molecules . This new class of small soluble polypeptide provides a template for designing and refining human homologues useful in detecting and regulating pathogenic T cells. J Biol Chem, 1999 Oct 8, 274(41), 29358 - 65 The Escherichia coli ssuEADCB gene cluster is required for the utilization of sulfur from aliphatic sulfonates and is regulated by the transcriptional activator Cbl; van Der Ploeg JR et al.; The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates . Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH(2)-dependent monooxygenase and an NAD(P)H-dependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J . R., and Leisinger, T . (1999) J . Biol . Chem . 274, 26639-26646) . Synthesis of beta-galactosidase from a transcriptional chromosomal ssuE'-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysR-type transcriptional regulator . Electrophoretic mobility shift assays with the ssu promoter region and measurements of beta-galactosidase from plasmid-encoded ssuE'-'lacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the -35 region . CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE'-lacZ fusion although the ssu promoter region contained three CysB-binding sites . Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE'-lacZ fusion. J Biol Chem, 1999 Oct 8, 274(41), 29149 - 55 Bipartite modular structure of intrinsic, RNA hairpin-independent termination signal for phage RNA polymerases; Kwon YS et al.; The phage SP6 RNA and T7 RNA polymerases, which are closely related to each other, intrinsically stop at two signals in the Escherichia coli rrnB terminator t1 through different mechanisms . The downstream signal functioned without an RNA secondary structure formation, in which the signal was still active when separated from the upstream, hairpin-forming signal, and IMP incorporation enhanced its efficiency . The sequence from -15 to -1 was essential for the downstream, hairpin-independent termination (at -1) . The results of SP6 transcription with heteroduplex templates and ribonucleotide analogs suggested that the downstream signal consists of two functionally different modules . The effects of iodo-CMP or IMP incorporation into RNA on termination efficiency were not sensitive to incorporation at -9 and upstream, but they were reactive to incorporation at -6 and -2, as reflected by strong iodo-rC:dG and weak rI:dC base pairing . Thus, the downstream module (from -8 approximately -6 to -1) appears to facilitate the release of RNA . Mismatches in the templates at -6 to +1 allowed for efficient termination, unlike those upstream of the sequence . The upstream module (from -15 to -9 approximately -7) functions as a duplex . Pausing of the SP6 elongation complex at the termination site was detected when RNA release was suppressed by the incorporation of 5-bromo-UMP, and it was dependent on the upstream module . Results of single-round SP6 transcriptions using 3'-deoxynucleotides and immobilized templates indicated that RNA was not released from the elongation complexes halted at the termination site on the template variants carrying mutations in the upstream or downstream module, whereas such complexes on the wild type template were dissociated . Thus, halting or simple pausing was not sufficient for termination even when the downstream module was intact . The upstream module appears to mediate such conformation change necessary for termination. J Biol Chem, 1999 Oct 8, 274(41), 29011 - 8 The active site of the Escherichia coli MutY DNA adenine glycosylase; Wright PM et al.; Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches . Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial . To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined . The glycosylase activity was completely abolished in the D138N MutY mutant . The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches . Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate . Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates . However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity . Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA . Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA . With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation. J Biol Chem, 1999 Oct 8, 274(41), 28999 - 9004 Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1); Lerm M et al.; The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac) . In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines . Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates . The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1 . Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells . The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1 . A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1 . Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1 . In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)) . Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF . The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements. J Biol Chem, 1999 Oct 8, 274(41), 28893 - 9 The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding; Renoult C et al.; The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end . Here, we characterize the cofilin-actin interface . We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding . In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H . G., Suck, D., Pai, E . F., and Holmes, K . C . (1990) Nature 347, 37-44) . We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A . (1997) J . Cell Biol . 138, 771-781) . The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F . (1996) Cell 85, 1047-1055). Cancer Gene Ther, 1999 Sep-Oct, 6(5), 456 - 64 In vivo expression of prostate-specific adenoviral vectors in a canine model; Steiner MS et al.; The activity and expression of transgene beta-galactosidase (lacZ) by replication-deficient adenoviral vectors (Ad-lacZ) containing prostate-specific promoters were compared using an in vivo canine model . The prostate tissue-specific promoters were prostate-specific antigen, probasin, and mouse mammary tumor virus long-terminal repeat, which were fused separately to an Escherichia coli lacZ gene . Dogs underwent laparotomy, and adenoviral vectors were delivered by direct intraprostatic injection . At 72 hours postinjection, the prostate and various other organs were harvested to evaluate the degree of prostate expression and dissemination of adenoviral vectors . Expression of lacZ in tissues was determined by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining, beta-galactosidase assay, and E . coli lacZ reverse transcriptase-polymerase chain reaction (PCR) . The presence of adenoviral DNA sequences in canine tissues was determined by PCR using primers specific for the type 5 adenoviral genome . All three of the prostate-specific adenoviruses tested effectively expressed the lacZ gene in the canine prostate, but expression levels were lower than that of the control viral vector AdRSVlacZ following intraprostatic injection . By PCR, adenoviral vector DNA was detected in other organs and tissues, including the bladder and vas deferens . However, reverse transcriptase-PCR analysis revealed that prostate-specific Ad-lacZ vectors only transcribed lacZ mRNA in the prostate and not in nonprostatic tissues . Thus, these novel prostate-specific adenoviral vectors each have equal in vivo expression exclusively in the prostate and may potentially be used for prostate cancer gene therapy. Biochem Cell Biol, 1999, 77(3), 229 - 36 Tyr-503 of beta-galactosidase (Escherichia coli) plays an important role in degalactosylation; Penner RM et al.; Substitutions for Tyr-503 of beta-galactosidase caused large decreases of the activity . Both the galactosylation (k2) and degalactosylation (k3) rates were decreased . Substitutions by residues without transferable protons, caused k3 to decrease much more than k2 while substitutions with residues having transferable protons, caused approximately equal decreases of k2 and k3 . Several lines of evidence showed this . The Km values of the substituted enzymes were much smaller than those for the wild type if the substituted amino acid residues did not have transferable protons; this was not the case when the substituted residues had transferable protons . Inhibition studies showed that the Km values were not small because of small Ks values but were small because of relatively small k3 values (compared with the k2 values) . The conclusion that the k3 values are small relative to k2 upon substitution with residues without transferable protons is also based upon other studies: studies indicating that the reaction rates were similar with different substrates, studies in the presence of alcohol acceptors, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2-deoxy-2-F-beta-D-galactopyranoside decreased much less than the rate of reactivation; studies on burst kinetics, and pH studies . The data suggest that Tyr-503 may be important for the degalactosylation reaction because of its ability to transfer protons and thereby facilitate cleavage of the transient covalent bond between galactose and Glu-537. Perit Dial Int, 1998 Jul-Aug, 18(4), 429 - 32 CAPD in patients with autosomal dominant polycystic kidney disease; Hadimeri H et al.; OBJECTIVE: To investigate whether there are specific complications to continuous ambulatory peritoneal dialysis (CAPD) in patients with autosomal dominant polycystic kidney disease (ADPKD) due to defects in various wall structures--causing hernia and diverticulitis--and to enlarged kidneys . DESIGN: The clinical experience of CAPD in 26 patients with ADPKD, treated for 11+/-6 months, was studied in retrospect and compared with that of 26 contemporary controls . Medical records were reviewed with respect to survival in this treatment form and any complication . Peritoneal dialysis capacity (PDC), as measured in 21 ADPKD patients and 20 controls, was also evaluated . SETTING: University Hospital . RESULTS: Before initiation of CAPD, enlarged kidneys necessitated nephrectomy in 2 of 26 ADPKD patients; both cases were registered as preparation for transplantation, not for CAPD . Survival in CAPD was similar in ADPKD patients and controls . Hernia was present in 4 ADPKD patients and 2 controls, and required transfer to hemodialysis in 1 patient from each group, temporarily . The incidence of peritonitis was 1 per 20 months in ADPKD patients versus 1 in 27 months in the controls, not significantly different . Peritonitis was caused by colonic bacteria in similar numbers . Residual renal function was 1.9 2.1 mL/min per 1.73 m2 in ADPKD patients versus 1.9+/-1.4 mL/min per 1.73 m2 in the controls . No difference was detected in any of the variables measured by PDC . CONCLUSION: There were no specific problems related to ADPKD. Gene Ther, 1999 May, 6(5), 893 - 903 CpG DNA and LPS induce distinct patterns of activation in human monocytes; Hartmann G et al.; A specific set of immune functions is switched on in response to DNA containing unmethylated CpG dinucleotides in particular base contexts ('CpG motifs') . Plasmids, viral vectors and antisense oligodeoxynucleotides used for DNA vaccination, gene replacement or gene blockade contain immunostimulatory CpG motifs which may have independent biological activity . Although the immune stimulatory effects of CpG motifs on murine cells are well established, the evaluation of their possible effects on human cells is complicated by the higher LPS sensitivity of human leukocytes compared with those in mice . To address this issue, we analyzed CpG- and LPS-mediated immune activation of human PBMC . The biologic activity of LPS could be detected within 4 h using intracellular TNF staining of monocytes with flow cytometry at concentrations just one-twentieth (0.0014 Eu/ml) of the lower detection limit for the routinely used LAL assay (0.03 EU/ml) . In contrast to the rapid LPS response, CpG DNA-stimulated TNF and IL-6 synthesis in human monocytes was not detectable until 18 h . E . coli DNA induced IL-6 synthesis in a concentration-dependent manner (30 micrograms/ml E . coli DNA; 409 pg/ml +/- 75 pg/ml, n = 7, IL-6 ELISA), but calf thymus DNA did not (< 10 pg/ml) . Likewise, the CpG oligodeoxynucleotides 1760 (phosphorothioate) and 2059 (unmodified) induced IL-6 synthesis, but the corresponding control oligonucleotides 1908 and 2077 did not CpG DNA and LPS enhanced IL-6 synthesis synergistically . ICAM-1-expression of monocytes was increased 4.6-fold by E . coli DNA, 3.5-fold by 1760 and three-fold by 2059, compared with 3.6-fold by a maximal LPS stimulus and no change with non-CpG DNA . In conclusion, CpG-motifs induce TNF, IL-6 and ICAM-1 expression in human monocytes, but the kinetics of this differ from that induced by LPS, which makes it possible to distinguish immune activation by these agents . These results have important implications for the clinical development of therapeutic DNA in humans. Gene Ther, 1999 May, 6(5), 816 - 22 Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation; Shinozaki K et al.; Interleukin-10 (IL-10) is an ideal candidate cytokine for suppressing the alloimmune response in transplantation . To determine whether genetic modulation of the hepatic graft with IL-10 could prolong survival following orthotopic liver transplantation, we constructed a replication-deficient adenovirus vector expressing human IL-10 (AdCMVhIL-10) . Intraportal injection of this vector into a donor rat 24-48 h before grafting resulted in efficient release of IL-10 into the circulation of a recipient rat after transplantation . Moreover, levels of hIL-10 from the suprahepatic vena cava were significantly (1.48-fold) higher than those from the infrahepatic vena cava (P = 0.013), indicating local IL-10 production within the transduced hepatic graft . AdCMVhIL-10 induced a prolongation of median survival to more than 87 days, with two of five transduced grafts showing more than 100 days of ongoing survival, when compared with 11 days for grafts transduced with a control adenovirus vector carrying the E . coli beta-galactosidase gene (P = 0.0021) and 11 days for untreated grafts (P = 0.0021) . Pathological findings occurring in the AdCMVhIL-10-transduced hepatic grafts revealed no evidence of progressive rejection reaction resulting in graft failure . These results demonstrate that hepatic grafts modulated by IL-10 gene transfer make local and effective immunosuppression feasible in the transplantation setting. Plant J, 1999 Sep, 19(5), 543 - 54 Protein dynamics, activity and cellular localization of soybean lipoxygenases indicate distinct functional roles for individual isoforms; Fischer AM et al.; Vegetative lipoxygenases (VLXs) in soybean are hypothesized to function in nitrogen storage and partitioning . Isoform-specific antibodies for four of the five known VLX isoenzymes were used to investigate the influence of source-sink status on protein levels, as well as to analyze the tissue and subcellular localization of the different isoforms . VLXD responded most strongly to sink limitation, although the levels of VLXA, B and C increased as well . After sink limitation, VLXD and the vegetative storage protein, VSPalpha, accumulated in the vacuoles of bundle sheath and paraveinal mesophyll cells, while VLXA, B and C localized to the cytosol of these cells . All five known VLX isoenzymes were active with both linoleic and linolenic acid substrates after expression in Escherichia coli . The strong upregulation of VLXD levels after sink limitation as well as the localization of this isoform to the vacuoles of paraveinal mesophyll and bundle sheath cells (where VSPs are found) strongly suggest that VLXD should be considered as a major storage protein in soybean leaves . Furthermore, since VLXA, B and C also accumulate in sink-limited soybean leaves, are located in the cytosol of paraveinal mesophyll cells and are active at pH values typically found in this compartment, their activities may well contribute to lipid metabolism in this tissue . This multi-gene family is thus ideally poised to play a pivotal role in the balance of N deposition relative to lipid-based storage, defense or signaling, by modulating contributions to these processes in the transient storage cells of the paraveinal mesophyll. Eur J Biochem, 1999 Oct, 265(2), 788 - 97 Characterization of recombinant and plant-derived mistletoe lectin and their B-chains; Eck J et al.; Mistletoe lectin I (pML) and its isoforms ML II and III constitute the active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy . The heterodimeric disulfide-linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP) . Recently, the sequence coding for the mistletoe lectin prepro-protein was identified and the existence of a single intron-free gene was shown {Eck, J., Langer, M., Mockel, B., Baur, A., Rothe, M., Zinke, H . & Lentzen, H . (1999) Eur . J . Biochem . 264, 775-784} . The aim of this study was to prepare pure and homogeneous rMLB-chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect . Expression in E . coli resulted in the production of insoluble protein (inclusion bodies) . A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain . Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA) . Asialofetuin was able to compete with binding of both chains (50% at 0.8 microM) . The specificity of the B-chains to lactose was more distinct with halfmaximal competition at 4.9 mM (pMLB) and > 90 mM (rMLB), respectively . Furthermore, in a coassociation process rMLA- and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML-heterodimer . The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared . Cytotoxicity was determined using MOLT-4 cells and enzymatic rRNA N-glycosidase activity was measured in a coupled transcription/translation assay . The IC50 values of the two heterodimers were similar in both assays; rMLB-chain did not show any cytotoxic effect . In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved at 1.6 mM (rML) and 1.8 mM (pML) . Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML . Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activity and the rRNA N-glycosidase activity, are prerequisites for the cytotoxic effects on target cells. Exp Eye Res, 1999 Oct, 69(4), 385 - 95 Herpes simplex virus mediated gene transfer to primate ocular tissues; Liu X et al.; We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene . To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically . Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 10(7) pfu), and the distribution of cells expressing lacZ was evaluated . In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity . In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells . Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively . We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis . This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys . Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable . Biochemistry, 1999 Sep 28, 38(39), 12805 - 13 Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy; Dhawan IK et al.; The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants . The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA . In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14 . These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA . Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins . The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme . VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K . Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA . The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation. Biochemistry, 1999 Sep 28, 38(39), 12747 - 57 The hemes of Escherichia coli nitrate reductase A (NarGHI): potentiometric effects of inhibitor binding to narI; Rothery RA et al.; We have potentiometrically characterized the two hemes of Escherichia coli nitrate reductase A (NarGHI) using EPR and optical spectroscopy . NarGHI contains two hemes, a low-potential heme b(L) (E(m,7) = 20 mV; g(z)() = 3.36) and a high-potential heme b(H) (E(m, 7) = 120 mV; g(z)() = 3.76) . Potentiometric analyses of the g(z)() features of the heme EPR spectra indicate that the E(m,7) values of both hemes are sensitive to the menaquinol analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) . This inhibitor causes a potential-inversion of the two hemes (for heme b(L), E(m,7) = 120 mV; for heme b(H), E(m,7) = 60 mV) . This effect is corroborated by optical spectroscopy of a heme b(H)-deficient mutant (NarGHI(H56R)) in which the heme b(L) undergoes a DeltaE(m,7) of 70 mV in the presence of HOQNO . Another potent inhibitor of NarGHI, stigmatellin, elicits a moderate heme b(L) DeltaE(m,7) of 30 mV, but has no detectable effect on heme b(H) . No effect is elicited by either inhibitor on the line shape or the E(m,7) values of the {3Fe-4S} cluster coordinated by NarH . When NarI is expressed in the absence of NarGH {NarI(DeltaGH)}, two hemes are detected in potentiometric titrations with E(m,7) values of 37 mV (heme b(L); g(z)() = 3.15) and -178 mV (heme b(H); g(z)() = 2.92), suggesting that heme b(H) may be exposed to the aqueous milieu in the absence of NarGH . The identity of these hemes was confirmed by recording EPR spectra of NarI(DeltaGH)(H56R) . HOQNO binding titrations followed by fluorescence spectroscopy suggest that in both NarGHI and NarI(DeltaGH), this inhibitor binds to a single high-affinity site with a K(d) of approximately 0.2 microM . These data support a functional model for NarGHI in which a single dissociable quinol binding site is associated with heme b(L) and is located toward the periplasmic side of NarI. Biochemistry, 1999 Sep 28, 38(39), 12709 - 17 A single-point mutation in the extreme heat- and pressure-resistant sso7d protein from sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core; Consonni R et al.; Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity . The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus . Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry . We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding . The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively . Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols . The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster . This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements . We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains . On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability . To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation. Parasitology, 1999 Sep, 119 ( Pt 3), 267 - 72 DNA probes for detection of the fish microsporidians Microgemma caulleryi and Tetramicra brevifilum; Leiro J et al.; DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species) . The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species . First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III . The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli . Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves) . The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself . The specificity of candidate probes was tested in dot-blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue . These assays identified a ca 1180 bp probe for M . caulleryi, denominated C38, and a ca 1363 bp probe for T . brevifilum, denominated F9 . Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T . brevifilum genomic DNA, and C38 to as little as 125 ng of M . caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e . using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used . The probes developed in this study will greatly facilitate detection and identification of M . caulleryi and T . brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species. Nat Med, 1999 Oct, 5(10), 1157 - 63 A universal influenza A vaccine based on the extracellular domain of the M2 protein; Neirynck S et al.; The antigenic variation of influenza virus represents a major health problem . However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains . We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli . Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge . The protection was mediated by antibodies, as it was transferable by serum . The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections. Virology, 1999 Sep 30, 262(2), 479 - 91 DNA contacts by protein domains of the molluscum contagiosum virus type-1B topoisomerase; Hwang Y et al.; All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils . Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains . Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker . A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified . MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme . MCV-TOP(82-323) was sensitive to sequences in the favored 5'-(T/C)CCTT-3' recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323 . Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5'-CCCTT-3' sequence at +10, +9, -2, and -3 to be important . Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step . MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5'-(T/C)CCTT-3' recognition site, consistent with a model in which the amino-terminal domain contacts this region . These findings provide insight into the division of labor within the MCV-TOP enzyme . Bioconjug Chem, 1999 Sep-Oct, 10(5), 884 - 91 RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging; Edwards DS et al.; A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands . The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex {(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)} (RP463) . RP463 was synthesized either in two steps, in which the binary ligand complex {(99m)Tc(fMLFK-HYNIC)(tricine)(2)} (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of {(99m)Tc}pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS . The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of {(99m)Tc}pertechnetate . Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h . {(99)Tc}RP463 was prepared and characterized by HPLC and electrospray mass spectrometry . In an in vitro assay, {(99)Tc}RP463 showed an IC(50) of 2 nM against binding of {(3)H}fMLF to receptors on PMNs . {(99)Tc}RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM . The localization of RP463 in the infection foci was assessed in a rabbit infection model . RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system . As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection . Visualization of the infected area could be as early as 2 h . A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit . This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes. Melanoma Res, 1999 Aug, 9(4), 369 - 74 Inhibition of B16 melanoma metastasis by overexpression of the cysteine proteinase inhibitor cystatin C; Cox JL et al.; Progression to metastasis has been correlated with increased cysteine proteinase activity for a number of tumour types . One mechanism of cysteine proteinase regulation in normal cells is by natural protease inhibitors, the cystatins . Here we further characterize a transfected cell line showing increased cystatin C transcription driven by cytomegalovirus (CMV) promoter/enhancer sequences . Properties of this cystatin C altered cell line such as growth in vitro, lung colonization after tail vein injection in mice, production of cystatin, and cysteine proteinase inhibitor activities were examined . Although there was no difference between the growth rate of the cystatin transfected cell line and that of the control, there was a substantial difference in metastatic ability . No increase was noted in cystatin C secretion into the media for the cystatin C transfected cell line compared with the control transfected cell line . There was, however, a difference in cysteine protease inhibitor activity in the cell-free extracts . These results show that alteration of cystatin C levels by overexpression in B16 melanoma alters properties associated with metastasis. Life Sci, 1999, 65(11), PL121 - 7 In vitro and in vivo TNFalpha synthesis modulation by methylguanidine, an uremic catabolyte; Autore G et al.; This study was performed in order to examine whether the uraemic toxin, methylguanidine (MG), can modulate tumor necrosis factor alpha (TNF alpha) release by activated macrophages . In this study we have evaluated the ability of MG to influence TNF alpha release in vitro, in Escherichia coli lypopolysaccharide- (LPS)-stimulated J774 cells preincubated overnight with MG, and in vivo in rats treated with MG before and after LPS challenge . Parallel experiments employing N(G)-nitro-L-arginine methyl esther (L-NAME) were also carried out for comparison . The effect of LPS (6 x 10(3) u/ml) on TNF alpha release by J774, following overnight incubation with MG or L-NAME (1 mM), was examined 3 hours after LPS challenge . LPS-stimulated J774 released 287.83+/-88 u/ml TNF alpha into the culture medium . MG (1 mM) significantly inhibited TNF alpha release by 73% (P<0.05) . L-NAME (1 mM) significantly inhibited TNF alpha release too by 72.88% (P<0.05) . The effect of MG and L-NAME have been also studied in vivo . Serum TNF alpha levels in LPS treated rats 2 h after LPS challenge were 88.33+/-31.7 u/ml as compared to the serum TNF alpha levels of control rats (undetectable) . Treatment of rats with MG (30 mg/kg, i.p.) strongly and significantly reduced TNF alpha release (98.71% inhibition; with P<0.001); in the same experimental setting L-NAME (10 mg/kg, i.p.) also significantly reduced TNF alpha serum levels (76.47% inhibition; with P<0.01) . These results could indicate that immune disfunction related to uremia may be related to the inhibitory capability of uremic catabolyte, MG, on TNF alpha synthesis and release. Chin J Biotechnol, 1998, 14(4), 227 - 32 Cloning and expression of phenylalanine ammonia lyase cDNA in Escherichia coli; Jingzhong L et al.; The phenylalanine ammonia lyase (PAL) cDNA was amplified from Petroselinum crispum RNA by using the RT-PCR technique . The amplified 2.2 kb DNA fragment was sequenced and inserted into expression vector pET23b . The resulting plasmid pET23bPAL was then transformed into E . coli JM109DE3 . The expressed PAL protein in JM109DE3 (pET23bPAL) accounted for more than 15% of total proteins in the engineering E . coli cells . The activity and specificity of the expressed PAL was identified and tested by using HPLC technique . The results indicated that the PAL activity was good enough for application to enzymatic therapy of phenylketonurea (PKU). Mol Gen Genet, 1999 Aug, 262(1), 154 - 62 Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326; Rother D et al.; Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons . Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons . The merR gene was expressed in E . coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography . Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa . Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments . No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays . The dissociation constants (K(D)) for binding of MerR were: binding site I, 8.5 x 10(-9) M; binding site II, 1.2 x 10(-8) M; and for the complete promoter/operator region 1 x 10(-8) M . The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively . The K(D) value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 x 10(-7) M. Mol Gen Genet, 1999 Aug, 262(1), 121 - 30 Regulation of divergent transcription from the uvrA-ssb promoters in Sinorhizobium meliloti; Tapias A et al.; The Sinorhizobium meliloti uvrA gene was isolated by complementation of a Rhodobacter sphaeroides UvrA- mutant . DNA sequencing of the region upstream of the S . meliloti uvrA gene reveals the presence of the ssb gene in the opposite transcriptional orientation . PCR-mediated mutagenesis demonstrated that expression of these two genes is inducible by DNA damage, and depends, in both cases, on the direct repeat GTTCN7GTTC (cited according to the direction of uvrA transcription) . Comparison of the sequences of recA and uvrA promoters from different bacterial species of the alpha group of the Proteobacteria has identified the direct repeat GTTCYYKTTTTGTTC as the SOS box in this phylogenetic group. Chin J Biotechnol, 1998, 14(3), 173 - 8 Selection of human anti-HAV McAb from a phage antibody library; Wan Z et al.; The phage displaying antibody fragments were subjected to three rounds of panning with hepatitis A virus (HAV) antigen in solid phase . The eluted phage was enriched nearly 100 fold, and the percentage of recombinant clones increased from 25% to 100% after three rounds of panning . The HAV antigen-specific monoclonal antibody (McAb) was screened by sandwich ELISA, and the specificity of these antibodies was further confirmed by competitive inhibition ELISA. Chin J Biotechnol, 1998, 14(3), 165 - 71 Expression of calmodulin-binding domain of neuronal nitric synthase and its binding activity to calmodulin; Zhu M et al.; To facilitate the study of associations of nNOS functions with its calmodulin (CaM) binding domain and to prepare nNOS specific inhibiting peptides from phage peptide library, we have amplified the coding gene of nNOS CaM-binding domain (nNOS 2544-2988 bp) and expressed it in E.coli . The recombinant product in the size of 22 kDa was purified (over 90% in pure) by His.Tag-Sepharose column and its obvious CaM-binding activity was detected with CaM overlay assay . Since it possesses the sequence specificity and effective calmodulin-binding activity, the protein was considered an ideal target for screening nNOS specific peptides from peptide library and also an antigen for marking nNOS antibody. Hum Mutat . 1999 Oct;14(4):355. Acute intermittent porphyria: characterization of two novel mutations in the porphobilinogen deaminase gene, one amino acid deletion (453-455delAGC) and one splicing aceptor site mutation (IVS8-1G>T); De Siervi A et al.; A partial deficiency of Porphobilinogen deaminase (PBG-D) is responsible for acute intermittent porphyria (AIP) . AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000 . Here, two new mutations and three previously reported were found in the PBG-D gene in 12 Argentinean AIP patients corresponding to 5 different families . To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced . The new mutations are 453-455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8-1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used . Both mutations produce amino acid deletion without frameshift effect . To further characterize the 453-455delAGC mutation, the pKK-PBGD construct for the mutant allele was expressed in E . coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele . Finally, three missense mutations, previously reported, were identified in three unrelated families . J Biochem (Tokyo), 1999 Oct, 126(4), 769 - 75 Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A; Kaji H et al.; A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor . The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues . The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level . To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2 . In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0 . 5 mg of the purified recombinant protein per 1 liter culture being produced . Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA . The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group . Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain . This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A. J Biochem (Tokyo), 1999 Oct, 126(4), 756 - 61 EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature; Kominami S et al.; Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified . Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme . Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K . In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS . The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle. J Biochem (Tokyo), 1999 Oct, 126(4), 748 - 55 Isolation and cloning of rat poly(ADP-ribose) glycohydrolase: presence of a potential nuclear export signal conserved in mammalian orthologs; Shimokawa T et al.; Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly(ADP-ribose) degradation . To understand its structure-and-function relationship, we purified Parg from rat testis 9,740-fold using an improved affinity column; the purified product was a 60 kDa protein . Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3,974 nucleotides, encoding a 109 kDa protein, was isolated . The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide . When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein . Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half . Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs . Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues . The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing. J Biochem (Tokyo), 1999 Oct, 126(4), 689 - 93 A cold-adapted protease engineered by experimental evolution system; Taguchi S et al.; A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature . The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate . This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner . Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T) . From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V . The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis . No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51 . In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points . In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred . This mutual effect may be related to the enhanced activity of the double mutant. J Biochem (Tokyo), 1999 Oct, 126(4), 639 - 42 Identification of cysteine-380 as the essential residue for the human N-acetyl-D-glucosamine 2-epimerase (renin binding protein); Takahashi S et al.; Renin binding protein (RnBP) is a proteinous renin inhibitor firstly isolated from porcine kidney . Recently, the protein was identified as the enzyme, N-acetyl-D-glucosamine (GlcNAc) 2-epimerase . The GlcNAc 2-epimerase activity of recombinant human RnBP was specifically inhibited by SH-reagents such as N-ethylmaleimide, 5, 5'-dithiobis-2-nitrobenzoate, and iodoacetic acid, indicating that the most probable reactive site is a cysteine residue . To identify the active site residue(s), we have constructed ten cysteine residue mutants (C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S, and C390S) for human GlcNAc 2-epimerase and expressed them in Escherichia coli cells . The relative specific activities of C41S, C66S, C125S, C210S, C239S, C302S, C386S, and C390S are nearly the same to that of the wild-type enzyme . The specific activity of the C104S mutant is 26% of that of the wild-type enzyme . The expression of the C380S mutant in E . coli cells was detected on Western blotting, whereas GlcNAc 2-epimerase activity was not detected in the extract . These results indicate that Cys380 is essential for the enzymatic activity of human GlcNAc 2-epimerase. Virology, 1999 Sep 30, 262(2), 398 - 407 Diarrhea induction by rotavirus NSP4 in the homologous mouse model system; Horie Y et al.; Comparison of the NSP4 amino acid sequences from 31 strains of mammalian rotaviruses revealed the presence of four distinct NSP4 alleles; i.e., the Wa, KUN, AU-1, and EW alleles . The EW allele consists only of NSP4s from murine rotavirus strains and is divergent from other NSP4 alleles from the evolutionary perspective . There have been conflicting reports regarding the enterotoxigenic activity of NSP4 in the mouse model system; heterologous simian and porcine rotavirus NSP4s function as an enterotoxin in mice, while a homologous EC NSP4 does not play a dominant role as an enterotoxin in the cystic fibrosis conductance regulator knockout mice . To further examine the enterotoxigenic activity of NSP4, we expressed in Escherichia coli a recombinant protein consisting of glutathione S-transferase and amino acid residues 86-175 of the EW NSP4 . We found that this fusion protein caused diarrhea in the majority (8/14) of 5- to 6-day-old CD1 mice . This study confirmed and extended that group A rotavirus NSP4s were able to induce diarrhea in neonatal mice and had an enterotoxigenic activity . Res Vet Sci, 1999 Oct, 67(2), 197 - 8 Genetic diversity among Escherichia coli O149:K91 strains isolated from pigs with diarrhoea determined by randomly amplified polymorphic DNA analysis; Osek J; The genetic relatedness among 72 Escherichia coli strains of serotype O149:K91 isolated from pigs with diarrhoea was investigated by randomly amplified polymorphic DNA (RAPD) analysis . Fimbrial and toxic virulence markers of the isolates were also tested . Amplification with primer 1254 resulted in three different RAPD types whereas primer 1290 generated one RAPD profile only . Based on the RAPD and fimbrial/toxin types the strains were classified into five distinct groups . Exp Parasitol, 1999 Oct, 93(2), 73 - 81 Acanthocheilonema viteae: characterization of a molt-associated excretory/secretory 18-kDa protein; Pogonka T et al.; Post-invasive third-stage larvae (pL3) of Acanthocheilonema viteae were labeled with {(35)S}-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed . The molting supernatant (MSN) contained abundant proteins of 14, 18, 29, and 36 kDa . The 14- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities . The cDNA for the most abundant protein in the MSN, an 18-kDa protein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence . The Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader sequence of nematodes, an open reading frame of 427 base pairs, and a poly(A) tail in typical distance to a polyadenylation signal . The deduced amino acid sequence encodes for a protein with a calculated size of 15.8 kDa . The N-terminus starts with a hydrophobic signal sequence and a predicted cleavage site after amino acid 20 . The Av18 protein showed homologies to the deduced amino acid sequence of the larval transcripts Bm-alt-1 and alt-2 of Brugia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to the Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2 . Av18 is present in all parasite stages within the mammalian host, as determined by immunoblot with sera against the Escherichia coli-expressed protein and RT-PCR experiments . However, it was released into culture medium only by L3 and adult female worms . In female worms Av18 was localized in the cuticular region as demonstrated by immunofluorescent antibody tests using cryosections . Curr Genet, 1999 Sep, 36(3), 153 - 8 The NIT2 nitrogen regulatory protein of Neurospora: expression and stability of nit-2 mRNA and protein; Tao Y et al.; In Neurospora crassa, NIT2 is a global transcription factor that positively regulates the expression of up to 100 genes that are related to nitrogen metabolism . NIT2 is responsible for the lifting of nitrogen catabolite repression when the cellular levels of glutamine or other favored nitrogen sources become limited . Immunoprecipitation of the NIT2 protein showed that it is constitutively expressed, although its expression level is elevated a few-fold when nitrate instead of glutamine is used as the sole nitrogen source . The NIT2 protein is very stable and its stability is not affected by the nitrogen sources . The nit-2 transcripts appear to be very stable as well . The lack of significant regulation of cellular levels of nit-2 mRNA and of NIT2 protein suggests that its interactions with other proteins, e.g., in the nitrate assimilation pathway, with NIT4 and NMR, or post-translational modification of NIT2, may play important roles in modulating the function of NIT2 in response to environmental stimuli.
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